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Li Z, Jiang S, Liu W, Yang X, Liu F, Li X, Li J, Yu M, Wei Z, Wang B, Qian D. A promising endeavor against human cytomegalovirus: Predominant epitopes-based recombinant subunit vaccine RH EcIE1/pp65/pp150. Virulence 2025; 16:2497903. [PMID: 40277436 PMCID: PMC12064061 DOI: 10.1080/21505594.2025.2497903] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2024] [Revised: 12/23/2024] [Accepted: 04/21/2025] [Indexed: 04/26/2025] Open
Abstract
Human cytomegalovirus (HCMV) is widespread in the population, typically remaining latent. However, it can cause severe morbidity and mortality in transplant patients and immunodeficient individuals. Currently, there is no approved vaccine against HCMV. This study used immunoinformatics methods to predict the predominant T and B-cell epitopes of three key HCMV proteins, including phosphoprotein 65 (pp65), pp150, and immediate-early protein 1 (IE1). Subsequently, we synthesized a recombinant subunit vaccine (RHEcIE1/pp65/pp150) from Escherichia coli, comprising RHEc-1 and RHEc-2. We observed that the RHEcIE1/pp65/pp150 vaccine exhibited high safety and immunogenicity in mice, enhancing a significant upregulation of CD80, CD86, CD40, and MHCII on dendritic cells and macrophages. Additionally, the vaccine activated innate immune responses through the NF-κB signalling pathway, triggering CD4+ and CD8+T cells to secrete tumour necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-2, directing the T-cell response towards Th1. Moreover, it stimulated CD4+T cells to secrete IL-4, IL-6, and IL-10, promoting B-cell immunity. Furthermore, the RHEcIE1/pp65/pp150 vaccine induced the formation of abundant memory cells and high levels of neutralizing antibody titres, conducive to providing long-lasting protection. Taken together, the RHEcIE1/pp65/pp150 vaccine is a promising endeavour against HCMV, and these findings contribute valuable insights to the development of HCMV vaccine candidates.
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MESH Headings
- Cytomegalovirus Vaccines/immunology
- Cytomegalovirus Vaccines/genetics
- Cytomegalovirus Vaccines/administration & dosage
- Animals
- Vaccines, Subunit/immunology
- Vaccines, Subunit/genetics
- Vaccines, Subunit/administration & dosage
- Cytomegalovirus/immunology
- Cytomegalovirus/genetics
- Mice
- Viral Matrix Proteins/immunology
- Viral Matrix Proteins/genetics
- Cytomegalovirus Infections/prevention & control
- Cytomegalovirus Infections/immunology
- Humans
- Vaccines, Synthetic/immunology
- Vaccines, Synthetic/administration & dosage
- Vaccines, Synthetic/genetics
- Phosphoproteins/immunology
- Phosphoproteins/genetics
- Epitopes, T-Lymphocyte/immunology
- Epitopes, T-Lymphocyte/genetics
- Epitopes, B-Lymphocyte/immunology
- Epitopes, B-Lymphocyte/genetics
- Female
- Immediate-Early Proteins/immunology
- Immediate-Early Proteins/genetics
- Antibodies, Viral/blood
- Mice, Inbred BALB C
- Immunogenicity, Vaccine
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Affiliation(s)
- Zonghui Li
- Department of Pathogenic Biology, Department of Special Medicine, School of Basic Medicine, Qingdao University, Qingdao, China
- Department of Clinical Laboratory, Chengdu Aerotropolis Asia Heart Hospital, Chengdu, China
| | - Shasha Jiang
- Department of Pathogenic Biology, Department of Special Medicine, School of Basic Medicine, Qingdao University, Qingdao, China
- Department of Clinical Laboratory, Honghui Hospital, Xi’an Jiaotong University, Xi’an, China
| | - Wenxuan Liu
- Department of Pathogenic Biology, Department of Special Medicine, School of Basic Medicine, Qingdao University, Qingdao, China
| | - Xiaoli Yang
- Department of Pathogenic Biology, Department of Special Medicine, School of Basic Medicine, Qingdao University, Qingdao, China
| | - Fengjun Liu
- Department of Pathogenic Biology, Department of Special Medicine, School of Basic Medicine, Qingdao University, Qingdao, China
| | - Xu Li
- Department of Pathogenic Biology, Department of Special Medicine, School of Basic Medicine, Qingdao University, Qingdao, China
| | - Jun Li
- Department of Pathogenic Biology, Department of Special Medicine, School of Basic Medicine, Qingdao University, Qingdao, China
| | - Meng Yu
- Department of Pathogenic Biology, Department of Special Medicine, School of Basic Medicine, Qingdao University, Qingdao, China
| | - Zhun Wei
- Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao, China
| | - Bin Wang
- Department of Pathogenic Biology, Department of Special Medicine, School of Basic Medicine, Qingdao University, Qingdao, China
| | - Dongmeng Qian
- Department of Pathogenic Biology, Department of Special Medicine, School of Basic Medicine, Qingdao University, Qingdao, China
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Jentzer A, Cantais A, Roblin X, Barrau M, Garcin A, Bourlet T, Pozzetto B, Pillet S. Predictive Factors of Cytomegalovirus Colonic Reactivation in Patients with Active Ulcerative Colitis. Viruses 2025; 17:555. [PMID: 40284998 PMCID: PMC12031004 DOI: 10.3390/v17040555] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2025] [Revised: 03/28/2025] [Accepted: 04/08/2025] [Indexed: 04/29/2025] Open
Abstract
Cytomegalovirus (CMV)-associated colitis reflects the adverse impact of CMV reactivation on ulcerative colitis (UC). Its diagnosis requires the detection of viral markers in intestinal biopsies sampled during endoscopy, which may constitute invasive and expensive analyses. Moreover, less than 30% of acute flare-ups in steroid refractory UC are associated with CMV colitis. This retrospective study aimed to identify non-invasive factors that are predictive of CMV reactivation, and was conducted from 2014 to 2019 in a cohort of UC patients consulting at the University Hospital of Saint-Etienne, France. Patient characteristics, disease activity, immunosuppressive treatment and tissue CMV DNA load were collected at the time of UC relapse. Factors potentially associated with CMV reactivation were analyzed through a multivariate analysis. A total of 173 UC patients providing 323 pairs of intestinal biopsies were analyzed. In the CMV seropositive subgroup, a Mayo endoscopic score ≥2 (OR 2.553, 95% CI 1.353-4.818, p = 0.004) was identified as a predictive factor of CMV colitis in the multivariate analysis; in contrast, biological parameters exhibited no predictive value. In addition, the use of anti-TNFα monoclonal antibodies was associated with a reduced risk of CMV reactivation (OR 0.384, 95% CI 0.158-0.935, p = 0.035). Intestinal biopsies appear to be unavoidable for assessing disease activity and CMV reactivation in UC patients.
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Affiliation(s)
- Alexandre Jentzer
- CIRI—Centre International de Recherche en Infectiologie (GIMAP Team, University of Lyon, Univerity of Saint-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1), Cedex 02, 42218 Saint-Etienne, France; (A.J.); (A.C.); (X.R.); (T.B.); (S.P.)
| | - Aymeric Cantais
- CIRI—Centre International de Recherche en Infectiologie (GIMAP Team, University of Lyon, Univerity of Saint-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1), Cedex 02, 42218 Saint-Etienne, France; (A.J.); (A.C.); (X.R.); (T.B.); (S.P.)
| | - Xavier Roblin
- CIRI—Centre International de Recherche en Infectiologie (GIMAP Team, University of Lyon, Univerity of Saint-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1), Cedex 02, 42218 Saint-Etienne, France; (A.J.); (A.C.); (X.R.); (T.B.); (S.P.)
- Department of Gastroenterology, University-Hospital of Saint-Etienne, Cedex 02, 42055 Saint-Etienne, France;
| | - Mathilde Barrau
- Department of Gastroenterology, University-Hospital of Saint-Etienne, Cedex 02, 42055 Saint-Etienne, France;
| | - Arnauld Garcin
- Clinical Research, Innovation and Pharmacology Unit (URCIP), SNA/EPIS, Faculty of Medicine Jacques Lisfranc, Saint-Etienne University Hospital, Cedex 02, 42023 Saint-Etienne, France;
| | - Thomas Bourlet
- CIRI—Centre International de Recherche en Infectiologie (GIMAP Team, University of Lyon, Univerity of Saint-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1), Cedex 02, 42218 Saint-Etienne, France; (A.J.); (A.C.); (X.R.); (T.B.); (S.P.)
- Laboratory of Infectious Agents and Hygiene, University-Hospital of Saint-Etienne, Cedex 02, 42055 Saint-Etienne, France
| | - Bruno Pozzetto
- CIRI—Centre International de Recherche en Infectiologie (GIMAP Team, University of Lyon, Univerity of Saint-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1), Cedex 02, 42218 Saint-Etienne, France; (A.J.); (A.C.); (X.R.); (T.B.); (S.P.)
- Laboratory of Infectious Agents and Hygiene, University-Hospital of Saint-Etienne, Cedex 02, 42055 Saint-Etienne, France
| | - Sylvie Pillet
- CIRI—Centre International de Recherche en Infectiologie (GIMAP Team, University of Lyon, Univerity of Saint-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1), Cedex 02, 42218 Saint-Etienne, France; (A.J.); (A.C.); (X.R.); (T.B.); (S.P.)
- Laboratory of Infectious Agents and Hygiene, University-Hospital of Saint-Etienne, Cedex 02, 42055 Saint-Etienne, France
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Almeida GWC, Oliveira MT, Martines IGL, Fiori GC, Nevels MM, Groves IJ, Sinclair J, Medina-Pestana J, da Silva RS, Nakamura M, Requião-Moura L, Poole E, da Silva MCC. Expression Profile of Human Cytomegalovirus UL111A cmvIL-10 and LAcmvIL-10 Transcripts in Primary Cells and Cells from Renal Transplant Recipients. Viruses 2025; 17:501. [PMID: 40284944 PMCID: PMC12031159 DOI: 10.3390/v17040501] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2025] [Revised: 03/11/2025] [Accepted: 03/25/2025] [Indexed: 04/29/2025] Open
Abstract
Human cytomegalovirus (HCMV) is a high-risk pathogen in immunocompromised individuals, especially in transplant recipients. HCMV viremia must be monitored, and frequently, patients are treated with antiviral agents. HCMV has a variety of strategies to modulate host antiviral responses, and one important player is a viral homolog of the cellular interleukin-10 (cIL-10). The viral UL111A gene produces several HCMV IL-10 transcripts and protein isoforms through alternative splicing. The cmvIL-10 (isoform A) has similar properties to cIL-10, while LAcmvIL-10 (isoform B) has more restricted biological properties. Other isoforms are produced (C to H) but have unknown functions. Here, we investigated the expression of the most abundant transcripts, cmvIL-10 and LAcmvIL-10, in productively and latently infected cells and in peripheral blood mononuclear cells from renal transplant recipients up to 60 days post-transplantation. This study investigated HCMV cmvIL-10 and LAcmvIL-10 transcription profiles in vitro, in productive and latent infection, and in vivo, in peripheral blood mononuclear cells (PBMCs) of renal transplant patients. In vitro, both cmvIL-10 and LAcmvIL-10 transcripts were detected in both types at high levels and low levels in MRC-5 and latent infected (CD14+). When PBMCs from transplant patients were analyzed, LAcmvIL-10 was detected mostly sporadically and in only a few patients, while cmvIL-10 was found in all patients at all time points. Furthermore, it was observed in PBMCs that expression of cmvIL-10 was positively associated with an increase in viral DNA detection in the subsequently collected sample, indicating that expression of cmvIL-10 might precede viral DNA replication. These results contribute to the understanding of HCMV biology in different phases of infection. In addition, our initial analysis suggests that monitoring cmvIL-10, along with viral DNA, could improve early detection of HCMV reactivation in transplant recipients.
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Affiliation(s)
- Giovana W. C. Almeida
- Centro de Ciências Naturais e Humanas (CCNH), Universidade Federal do ABC (UFABC), São Bernardo do Campo 09606-070, SP, Brazil; (G.W.C.A.); (M.T.O.); (I.G.L.M.); (G.C.F.)
| | - Martha T. Oliveira
- Centro de Ciências Naturais e Humanas (CCNH), Universidade Federal do ABC (UFABC), São Bernardo do Campo 09606-070, SP, Brazil; (G.W.C.A.); (M.T.O.); (I.G.L.M.); (G.C.F.)
| | - Isabella G. L. Martines
- Centro de Ciências Naturais e Humanas (CCNH), Universidade Federal do ABC (UFABC), São Bernardo do Campo 09606-070, SP, Brazil; (G.W.C.A.); (M.T.O.); (I.G.L.M.); (G.C.F.)
| | - Giuliano C. Fiori
- Centro de Ciências Naturais e Humanas (CCNH), Universidade Federal do ABC (UFABC), São Bernardo do Campo 09606-070, SP, Brazil; (G.W.C.A.); (M.T.O.); (I.G.L.M.); (G.C.F.)
| | - Michael M. Nevels
- School of Biology, University of St. Andrews, St. Andrews KY16 9ST, UK;
| | - Ian J. Groves
- Molecular Medicine, Cleveland Clinic, Cleveland, OH 44106, USA;
| | - John Sinclair
- Department of Medicine, University of Cambridge, Cambridge CB2 1TN, UK;
| | - José Medina-Pestana
- Hospital do Rim, Fundação Oswaldo Ramos, Vila Clementino 04038-002, SP, Brazil; (J.M.-P.); (R.S.d.S.); (M.N.); (L.R.-M.)
- Nephrology Division, Universidade Federal de São Paulo, Vila Clementino 04021-001, SP, Brazil
| | - Rayra Sampaio da Silva
- Hospital do Rim, Fundação Oswaldo Ramos, Vila Clementino 04038-002, SP, Brazil; (J.M.-P.); (R.S.d.S.); (M.N.); (L.R.-M.)
| | - Monica Nakamura
- Hospital do Rim, Fundação Oswaldo Ramos, Vila Clementino 04038-002, SP, Brazil; (J.M.-P.); (R.S.d.S.); (M.N.); (L.R.-M.)
| | - Lucio Requião-Moura
- Hospital do Rim, Fundação Oswaldo Ramos, Vila Clementino 04038-002, SP, Brazil; (J.M.-P.); (R.S.d.S.); (M.N.); (L.R.-M.)
- Nephrology Division, Universidade Federal de São Paulo, Vila Clementino 04021-001, SP, Brazil
| | - Emma Poole
- Department of Pathology, University of Cambridge, Cambridge CB2 0QQ, UK
| | - Maria C. Carlan da Silva
- Centro de Ciências Naturais e Humanas (CCNH), Universidade Federal do ABC (UFABC), São Bernardo do Campo 09606-070, SP, Brazil; (G.W.C.A.); (M.T.O.); (I.G.L.M.); (G.C.F.)
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Cimato G, Zhou X, Brune W, Frascaroli G. Human cytomegalovirus glycoprotein variants governing viral tropism and syncytium formation in epithelial cells and macrophages. J Virol 2024; 98:e0029324. [PMID: 38837351 PMCID: PMC11265420 DOI: 10.1128/jvi.00293-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2024] [Accepted: 05/02/2024] [Indexed: 06/07/2024] Open
Abstract
Human cytomegalovirus (HCMV) displays a broad cell tropism, and the infection of biologically relevant cells such as epithelial, endothelial, and hematopoietic cells supports viral transmission, systemic spread, and pathogenesis in the human host. HCMV strains differ in their ability to infect and replicate in these cell types, but the genetic basis of these differences has remained incompletely understood. In this study, we investigated HCMV strain VR1814, which is highly infectious for epithelial cells and macrophages and induces cell-cell fusion in both cell types. A VR1814-derived bacterial artificial chromosome (BAC) clone, FIX-BAC, was generated many years ago but has fallen out of favor because of its modest infectivity. By sequence comparison and genetic engineering of FIX, we demonstrate that the high infectivity of VR1814 and its ability to induce syncytium formation in epithelial cells and macrophages depends on VR1814-specific variants of the envelope glycoproteins gB, UL128, and UL130. We also show that UL130-neutralizing antibodies inhibit syncytium formation, and a FIX-specific mutation in UL130 is responsible for its low infectivity by reducing the amount of the pentameric glycoprotein complex in viral particles. Moreover, we found that a VR1814-specific mutation in US28 further increases viral infectivity in macrophages, possibly by promoting lytic rather than latent infection of these cells. Our findings show that variants of gB and the pentameric complex are major determinants of infectivity and syncytium formation in epithelial cells and macrophages. Furthermore, the VR1814-adjusted FIX strains can serve as valuable tools to study HCMV infection of myeloid cells.IMPORTANCEHuman cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading cause of congenital infections. HCMV infects various cell types, including epithelial cells and macrophages, and some strains induce the fusion of neighboring cells, leading to the formation of large multinucleated cells called syncytia. This process may limit the exposure of the virus to host immune factors and affect pathogenicity. However, the reason why some HCMV strains exhibit a broader cell tropism and why some induce cell fusion more than others is not well understood. We compared two closely related HCMV strains and provided evidence that small differences in viral envelope glycoproteins can massively increase or decrease the virus infectivity and its ability to induce syncytium formation. The results of the study suggest that natural strain variations may influence HCMV infection and pathogenesis in humans.
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Affiliation(s)
| | - Xuan Zhou
- Leibniz Institute of Virology (LIV), Hamburg, Germany
| | - Wolfram Brune
- Leibniz Institute of Virology (LIV), Hamburg, Germany
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Lawrence SM. Human cytomegalovirus and neonatal infection. CURRENT RESEARCH IN MICROBIAL SCIENCES 2024; 7:100257. [PMID: 39070527 PMCID: PMC11276932 DOI: 10.1016/j.crmicr.2024.100257] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/30/2024] Open
Abstract
Human cytomegalovirus is an ancient virus that has co-evolved with humans. It establishes a life-long infection in suspectable individuals for which there is no vaccination or cure. The virus can be transmitted to a developing fetus in seropositive pregnant women, and it is the leading cause of congenital infectious disease. While the majority of infected infants remain asymptomatic at birth, congenital cytomegalovirus infection can lead to substantial long-term neurodevelopmental impairments in survivors, resulting in considerable economic and social hardships. Recent discoveries regarding cytomegalovirus pathophysiology and viral replication cycles might enable the development of innovative diagnostics and therapeutics, including an effective vaccine. This Review will detail our understanding of human cytomegalovirus infection, with an in-depth discussion regarding the viral genome and transcriptome that contributes to its pathophysiology. The neonate's clinical course will also be highlighted, including maternal and neonatal testing, treatment recommendations, and long-term outcomes.
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Affiliation(s)
- Shelley M. Lawrence
- University of Utah, College of Medicine, Department of Pediatrics, Division of Neonatology, Salt Lake City, UT, USA
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Amjad W, Hamaad Rahman S, Schiano TD, Jafri SM. Epidemiology and Management of Infections in Liver Transplant Recipients. Surg Infect (Larchmt) 2024; 25:272-290. [PMID: 38700753 DOI: 10.1089/sur.2023.346] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/13/2024] Open
Abstract
Background: Improvements in liver transplant (LT) outcomes are attributed to advances in surgical techniques, use of potent immunosuppressants, and rigorous pre-LT testing. Despite these improvements, post-LT infections remain the most common complication in this population. Bacteria constitute the most common infectious agents, while fungal and viral infections are also frequently encountered. Multi-drug-resistant bacterial infections develop because of polymicrobial overuse and prolonged hospital stays. Immediate post-LT infections are commonly caused by viruses. Conclusions: Appropriate vaccination, screening of both donor and recipients before LT and antiviral prophylaxis in high-risk individuals are recommended. Antimicrobial drug resistance is common in high-risk LT and associated with poor outcomes; epidemiology and management of these cases is discussed. Additionally, we also discuss the effect of coronavirus disease 2019 (COVID-19) infection and monkeypox in the LT population.
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Affiliation(s)
- Waseem Amjad
- Gastroenterology and Hepatology, University of Maryland, Baltimore, Maryland, USA
| | | | - Thomas D Schiano
- Recanati-Miller Transplantation Institute, Division of Liver Diseases, Mount Sinai Medical Center, New York, New York, USA
| | - Syed-Mohammed Jafri
- Gastroenterology and Hepatology, Henry Ford Hospital, Detroit, Michigan, USA
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Xu Y, Lv Y, Lin M, Li M, Cui D, Wang Y, Shen C, Xie J. A novel multiplex real-time PCR assay for the detection of cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1/2 and strategies for application to blood screening. Diagn Microbiol Infect Dis 2024; 109:116234. [PMID: 38432126 DOI: 10.1016/j.diagmicrobio.2024.116234] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2023] [Revised: 02/11/2024] [Accepted: 02/22/2024] [Indexed: 03/05/2024]
Abstract
A multiplex real-time PCR has been developed to simultaneously detect transfusion-transmissible pathogens cytomegalovirus, Epstein-Barr virus and herpes simplex virus, as well as to provide sample quality testing, for the conserved regions of the cytomegalovirus UL123 gene, the Epstein-Barr virus BKRF1 gene, and the herpes simplex virus 1/2 UL30 gene, tested on 500 blood donors and 320 transfusion recipients. The laboratory sensitivities for all 3 pathogens were 100 copies/μL. Compared to the commercial real-time PCR reference kit, the multiplex real-time PCR assay for the detection of CMV, EBV and HSV presented 100% consistency. In 820 whole blood samples, the multiplex real-time PCR assay identified 34 (4.15%) positive for CMV DNA, 15 (1.83%) positive for EBV DNA, and 6 (0.73%) positive for HSV DNA. For blood transfusions in high-risk groups, whole blood herpes virus test should be included in the spectrum of pathogen testing for blood donors and recipients.
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Affiliation(s)
- Yushan Xu
- Department of Blood Transfusion, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, China
| | - Yan Lv
- Department of Blood Transfusion, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, China
| | - Mengjiao Lin
- Department of Blood Transfusion, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, China
| | - Miaomiao Li
- Department of Blood Transfusion, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, China
| | - Dawei Cui
- Department of Blood Transfusion, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, China
| | - Yongjun Wang
- Key Laboratory of Blood Safety Research of Zhejiang Province, Blood Center of Zhejiang Province, Hangzhou, China
| | - Cuifen Shen
- Department of Clinical Laboratory, Huzhou Central Hospital, Huzhou 313000, China
| | - Jue Xie
- Department of Blood Transfusion, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, China.
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Francois AK, Rohani A, Loftus M, Dochnal S, Hrit J, McFarlane S, Whitford A, Lewis A, Krakowiak P, Boutell C, Rothbart SB, Kashatus D, Cliffe AR. Single-genome analysis reveals a heterogeneous association of the herpes simplex virus genome with H3K27me2 and the reader PHF20L1 following infection of human fibroblasts. mBio 2024; 15:e0327823. [PMID: 38411116 PMCID: PMC11005365 DOI: 10.1128/mbio.03278-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2023] [Accepted: 02/05/2024] [Indexed: 02/28/2024] Open
Abstract
The fate of herpesvirus genomes following entry into different cell types is thought to regulate the outcome of infection. For the Herpes simplex virus 1 (HSV-1), latent infection of neurons is characterized by association with repressive heterochromatin marked with Polycomb silencing-associated lysine 27 methylation on histone H3 (H3K27me). However, whether H3K27 methylation plays a role in repressing lytic gene expression in non-neuronal cells is unclear. To address this gap in knowledge, and with consideration that the fate of the viral genome and outcome of HSV-1 infection could be heterogeneous, we developed an assay to quantify the abundance of histone modifications within single viral genome foci of infected fibroblasts. Using this approach, combined with bulk epigenetic techniques, we were unable to detect any role for H3K27me3 during HSV-1 lytic infection of fibroblasts. By contrast, we could detect the lesser studied H3K27me2 on a subpopulation of viral genomes, which was consistent with a role for H3K27 demethylases in promoting lytic gene expression. In addition, viral genomes co-localized with the H3K27me2 reader protein PHF20L1, and this association was enhanced by inhibition of the H3K27 demethylases UTX and JMJD3. Notably, targeting of H3K27me2 to viral genomes was enhanced following infection with a transcriptionally defective virus in the absence of Promyelocytic leukemia nuclear bodies. Collectively, these studies implicate a role for H3K27me2 in fibroblast-associated HSV genome silencing in a manner dependent on genome sub-nuclear localization and transcriptional activity. IMPORTANCE Investigating the potential mechanisms of gene silencing for DNA viruses in different cell types is important to understand the differential outcomes of infection, particularly for viruses like herpesviruses that can undergo distinct types of infection in different cell types. In addition, investigating chromatin association with viral genomes informs on the mechanisms of epigenetic regulation of DNA processes. However, there is a growing appreciation for heterogeneity in the outcome of infection at the single cell, and even single viral genome, level. Here we describe a novel assay for quantifying viral genome foci with chromatin proteins and show that a portion of genomes are targeted for silencing by H3K27me2 and associate with the reader protein PHF20L1. This study raises important questions regarding the mechanism of H3K27me2-specific targeting to viral genomes, the contribution of epigenetic heterogeneity to herpesvirus infection, and the role of PHF20L1 in regulating the outcome of DNA virus infection.
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Affiliation(s)
- Alison K. Francois
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA
| | - Ali Rohani
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA
| | - Matt Loftus
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA
| | - Sara Dochnal
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA
| | - Joel Hrit
- Department of Epigenetics, Van Andel Institute, Grand Rapids, USA
| | - Steven McFarlane
- MRC - University of Glasgow, Centre for Virus Research, Glasgow, United Kingdom
| | - Abigail Whitford
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA
| | - Anna Lewis
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA
| | - Patryk Krakowiak
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA
| | - Chris Boutell
- MRC - University of Glasgow, Centre for Virus Research, Glasgow, United Kingdom
| | | | - David Kashatus
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA
| | - Anna R. Cliffe
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA
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Groves IJ, Matthews SM, O’Connor CM. Host-encoded CTCF regulates human cytomegalovirus latency via chromatin looping. Proc Natl Acad Sci U S A 2024; 121:e2315860121. [PMID: 38408244 PMCID: PMC10927566 DOI: 10.1073/pnas.2315860121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2023] [Accepted: 01/09/2024] [Indexed: 02/28/2024] Open
Abstract
Human cytomegalovirus (HCMV) is a prevalent pathogen that establishes life-long latent infection in hematopoietic cells. While this infection is usually asymptomatic, immune dysregulation leads to viral reactivation, which can cause significant morbidity and mortality. However, the mechanisms underpinning reactivation remain incompletely understood. The HCMV major immediate early promoter (MIEP)/enhancer is a key factor in this process, as its transactivation from a repressed to active state helps drive viral gene transcription necessary for reactivation from latency. Numerous host transcription factors bind the MIE locus and recruit repressive chromatin modifiers, thus impeding virus reactivation. One such factor is CCCTC-binding protein (CTCF), a highly conserved host zinc finger protein that mediates chromatin conformation and nuclear architecture. However, the mechanisms by which CTCF contributes to HCMV latency were previously unexplored. Here, we confirm that CTCF binds two convergent sites within the MIE locus during latency in primary CD14+ monocytes, and following cellular differentiation, CTCF association is lost as the virus reactivates. While mutation of the MIE enhancer CTCF binding site does not impact viral lytic growth in fibroblasts, this mutant virus fails to maintain latency in myeloid cells. Furthermore, we show the two convergent CTCF binding sites allow looping to occur across the MIEP, supporting transcriptional repression during latency. Indeed, looping between the two sites diminishes during virus reactivation, concurrent with activation of MIE transcription. Taken together, our data reveal that three-dimensional chromatin looping aids in the regulation of HCMV latency and provides insight into promoter/enhancer regulation that may prove broadly applicable across biological systems.
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Affiliation(s)
- Ian J. Groves
- Infection Biology Program, Sheikha Fatima bint Mubarak Global Center for Pathogen and Human Health Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH44195
- Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland Clinic, Cleveland, OH44195
- Case Comprehensive Cancer Center, Cleveland, OH44106
| | - Stephen M. Matthews
- Infection Biology Program, Sheikha Fatima bint Mubarak Global Center for Pathogen and Human Health Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH44195
- Case Comprehensive Cancer Center, Cleveland, OH44106
| | - Christine M. O’Connor
- Infection Biology Program, Sheikha Fatima bint Mubarak Global Center for Pathogen and Human Health Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH44195
- Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland Clinic, Cleveland, OH44195
- Case Comprehensive Cancer Center, Cleveland, OH44106
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10
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Dochnal SA, Whitford AL, Francois AK, Krakowiak PA, Cuddy S, Cliffe AR. c-Jun signaling during initial HSV-1 infection modulates latency to enhance later reactivation in addition to directly promoting the progression to full reactivation. J Virol 2024; 98:e0176423. [PMID: 38193709 PMCID: PMC10878265 DOI: 10.1128/jvi.01764-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2023] [Accepted: 12/12/2023] [Indexed: 01/10/2024] Open
Abstract
Herpes simplex virus-1 (HSV-1) establishes a latent infection in peripheral neurons and periodically reactivates to permit transmission, which can result in clinical manifestations. Viral transactivators required for lytic infection are largely absent during latent infection, and therefore, HSV-1 relies on the co-option of neuronal host signaling pathways to initiate its gene expression. The activation of the neuronal c-Jun N-terminal kinase (JNK) cell stress pathway is central to initiating biphasic reactivation in response to multiple stimuli. However, how host factors work with JNK to stimulate the initial wave of gene expression (known as Phase I) or the progression to full Phase II reactivation remains unclear. Here, we found that c-Jun, the primary target downstream of neuronal JNK cell stress signaling, functions during reactivation but not during the JNK-mediated initiation of Phase I gene expression. Instead, c-Jun was required to transition from Phase I to full HSV-1 reactivation and was detected in viral replication compartments of reactivating neurons. Interestingly, we also identified a role for both c-Jun and enhanced neuronal stress during initial neuronal infection in promoting a more reactivation-competent form of HSV-1 latency. Therefore, c-Jun functions at multiple stages during the HSV latent infection of neurons to promote reactivation but not during the initial JNK-dependent Phase I. Importantly, by demonstrating that initial infection conditions can contribute to later reactivation abilities, this study highlights the potential for latently infected neurons to maintain a molecular scar of previous exposure to neuronal stressors.IMPORTANCEThe molecular mechanisms that regulate the reactivation of herpes simplex virus-1 (HSV-1) from latent infection are unknown. The host transcription and pioneer factor c-Jun is the main target of the JNK cell stress pathway that is known to be important in exit of HSV from latency. Surprisingly, we found that c-Jun does not act with JNK during exit from latency but instead promotes the transition to full reactivation. Moreover, c-Jun and enhanced neuronal stress during initial neuronal infection promoted a more reactivation-competent form of HSV-1 latency. c-Jun, therefore, functions at multiple stages during HSV-1 latent infection of neurons to promote reactivation. Importantly, this study contributes to a growing body of evidence that de novo HSV-1 infection conditions can modulate latent infection and impact future reactivation events, raising important questions on the clinical impact of stress during initial HSV-1 acquisition on future reactivation events and consequences.
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Affiliation(s)
- Sara A. Dochnal
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA
| | - Abigail L. Whitford
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA
| | - Alison K. Francois
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA
| | - Patryk A. Krakowiak
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA
| | - Sean Cuddy
- Neuroscience Graduate Program, University of Virginia, Charlottesville, Virginia, USA
| | - Anna R. Cliffe
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA
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11
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Holtappels R, Büttner JK, Freitag K, Reddehase MJ, Lemmermann NA. Modulation of cytomegalovirus immune evasion identifies direct antigen presentation as the predominant mode of CD8 T-cell priming during immune reconstitution after hematopoietic cell transplantation. Front Immunol 2024; 15:1355153. [PMID: 38426094 PMCID: PMC10902149 DOI: 10.3389/fimmu.2024.1355153] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2023] [Accepted: 02/01/2024] [Indexed: 03/02/2024] Open
Abstract
Cytomegalovirus (CMV) infection is the most critical infectious complication in recipients of hematopoietic cell transplantation (HCT) in the period between a therapeutic hematoablative treatment and the hematopoietic reconstitution of the immune system. Clinical investigation as well as the mouse model of experimental HCT have consistently shown that timely reconstitution of antiviral CD8 T cells is critical for preventing CMV disease in HCT recipients. Reconstitution of cells of the T-cell lineage generates naïve CD8 T cells with random specificities among which CMV-specific cells need to be primed by presentation of viral antigen for antigen-specific clonal expansion and generation of protective antiviral effector CD8 T cells. For CD8 T-cell priming two pathways are discussed: "direct antigen presentation" by infected professional antigen-presenting cells (pAPCs) and "antigen cross-presentation" by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Current view in CMV immunology favors the cross-priming hypothesis with the argument that viral immune evasion proteins, known to interfere with the MHC class-I pathway of direct antigen presentation by infected cells, would inhibit the CD8 T-cell response. While the mode of antigen presentation in the mouse model of CMV infection has been studied in the immunocompetent host under genetic or experimental conditions excluding either pathway of antigen presentation, we are not aware of any study addressing the medically relevant question of how newly generated naïve CD8 T cells become primed in the phase of lympho-hematopoietic reconstitution after HCT. Here we used the well-established mouse model of experimental HCT and infection with murine CMV (mCMV) and pursued the recently described approach of up- or down-modulating direct antigen presentation by using recombinant viruses lacking or overexpressing the central immune evasion protein m152 of mCMV, respectively. Our data reveal that the magnitude of the CD8 T-cell response directly reflects the level of direct antigen presentation.
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Affiliation(s)
- Rafaela Holtappels
- Institute for Virology and Research Center for Immunotherapy (FZI) at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Julia K. Büttner
- Institute for Virology and Research Center for Immunotherapy (FZI) at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Kirsten Freitag
- Institute for Virology and Research Center for Immunotherapy (FZI) at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Matthias J. Reddehase
- Institute for Virology and Research Center for Immunotherapy (FZI) at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
| | - Niels A. Lemmermann
- Institute for Virology and Research Center for Immunotherapy (FZI) at the University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany
- Institute of Virology, Medical Faculty, University of Bonn, Bonn, Germany
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12
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Lawrence SM, Goshia T, Sinha M, Fraley SI, Williams M. Decoding human cytomegalovirus for the development of innovative diagnostics to detect congenital infection. Pediatr Res 2024; 95:532-542. [PMID: 38146009 PMCID: PMC10837078 DOI: 10.1038/s41390-023-02957-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/02/2023] [Revised: 11/14/2023] [Accepted: 11/27/2023] [Indexed: 12/27/2023]
Abstract
Cytomegalovirus is the most common cause of congenital infectious disease and the leading nongenetic etiology of sensorineural hearing loss. Although most infected neonates are asymptomatic at birth, congenital cytomegalovirus infection is responsible for nearly 400 infant deaths annually in the United States and may lead to significant long-term neurodevelopmental impairments in survivors. The resulting financial and social burdens of congenital cytomegalovirus infection have led many medical centers to initiate targeted testing after birth, with a growing advocacy to advance universal newborn screening. While no cures or vaccines are currently available to eliminate or prevent cytomegalovirus infection, much has been learned over the last five years regarding disease pathophysiology and viral replication cycles that may enable the development of innovative diagnostics and therapeutics. This Review will detail our current understanding of congenital cytomegalovirus infection, while focusing our discussion on routine and emerging diagnostics for viral detection, quantification, and long-term prognostication. IMPACT: This review highlights our current understanding of the fetal transmission of human cytomegalovirus. It details clinical signs and physical findings of congenital cytomegalovirus infection. This submission discusses currently available cytomegalovirus diagnostics and introduces emerging platforms that promise improved sensitivity, specificity, limit of detection, viral quantification, detection of genomic antiviral resistance, and infection staging (primary, latency, reactivation, reinfection).
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Affiliation(s)
- Shelley M Lawrence
- University of Utah, College of Medicine, Department of Pediatrics, Division of Neonatology, Salt Lake City, UT, USA.
| | - Tyler Goshia
- Department of Bioengineering, University of California, San Diego, San Diego, CA, USA
| | | | - Stephanie I Fraley
- Department of Bioengineering, University of California, San Diego, San Diego, CA, USA
| | - Marvin Williams
- University of Oklahoma, College of Medicine, Department of Obstetrics and Gynecology, Division of Fetal-Maternal Medicine, Oklahoma City, OK, USA
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13
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Francois AK, Rohani A, Loftus M, Dochnal S, Hrit J, McFarlane S, Whitford A, Lewis A, Krakowiak P, Boutell C, Rothbart SB, Kashatus D, Cliffe AR. Single-genome analysis reveals heterogeneous association of the Herpes Simplex Virus genome with H3K27me2 and the reader PHF20L1 following infection of human fibroblasts. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.12.03.569766. [PMID: 38076966 PMCID: PMC10705572 DOI: 10.1101/2023.12.03.569766] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/19/2023]
Abstract
The fate of herpesvirus genomes following entry into different cell types is thought to regulate the outcome of infection. For the Herpes simplex virus 1 (HSV-1), latent infection of neurons is characterized by association with repressive heterochromatin marked with Polycomb silencing-associated lysine 27 methylation on histone H3 (H3K27me). However, whether H3K27 methylation plays a role in repressing lytic gene expression in non-neuronal cells is unclear. To address this gap in knowledge, and with consideration that the fate of the viral genome and outcome of HSV-1 infection could be heterogeneous, we developed an assay to quantify the abundance of histone modifications within single viral genome foci of infected fibroblasts. Using this approach, combined with bulk epigenetic techniques, we were unable to detect any role for H3K27me3 during HSV-1 lytic infection of fibroblasts. In contrast, we could detect the lesser studied H3K27me2 on a subpopulation of viral genomes, which was consistent with a role for H3K27 demethylases in promoting lytic gene expression. This was consistent with a role for H3K27 demethylases in promoting lytic gene expression. In addition, viral genomes co-localized with the H3K27me2 reader protein PHF20L1, and this association was enhanced by inhibition of the H3K27 demethylases UTX and JMJD3. Notably, targeting of H3K27me2 to viral genomes was enhanced following infection with a transcriptionally defective virus in the absence of Promyelocytic leukemia nuclear bodies. Collectively, these studies implicate a role for H3K27me2 in fibroblast-associated HSV genome silencing in a manner dependent on genome sub-nuclear localization and transcriptional activity.
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Affiliation(s)
- Alison K Francois
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, 22908
| | - Ali Rohani
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, 22908
| | - Matt Loftus
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, 22908
| | - Sara Dochnal
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, 22908
| | - Joel Hrit
- Department of Epigenetics, Van Andel Institute, Grand Rapids, MI, 49503
| | - Steven McFarlane
- MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow, Scotland
| | - Abigail Whitford
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, 22908
| | - Anna Lewis
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, 22908
| | - Patryk Krakowiak
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, 22908
| | - Chris Boutell
- MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow, Scotland
| | - Scott B. Rothbart
- Department of Epigenetics, Van Andel Institute, Grand Rapids, MI, 49503
| | - David Kashatus
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, 22908
| | - Anna R Cliffe
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, 22908
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14
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Zehner M, Alt M, Ashurov A, Goldsmith JA, Spies R, Weiler N, Lerma J, Gieselmann L, Stöhr D, Gruell H, Schultz EP, Kreer C, Schlachter L, Janicki H, Laib Sampaio K, Stegmann C, Nemetchek MD, Dähling S, Ullrich L, Dittmer U, Witzke O, Koch M, Ryckman BJ, Lotfi R, McLellan JS, Krawczyk A, Sinzger C, Klein F. Single-cell analysis of memory B cells from top neutralizers reveals multiple sites of vulnerability within HCMV Trimer and Pentamer. Immunity 2023; 56:2602-2620.e10. [PMID: 37967532 DOI: 10.1016/j.immuni.2023.10.009] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2023] [Revised: 07/02/2023] [Accepted: 10/18/2023] [Indexed: 11/17/2023]
Abstract
Human cytomegalovirus (HCMV) can cause severe diseases in fetuses, newborns, and immunocompromised individuals. Currently, no vaccines are approved, and treatment options are limited. Here, we analyzed the human B cell response of four HCMV top neutralizers from a cohort of 9,000 individuals. By single-cell analyses of memory B cells targeting the pentameric and trimeric HCMV surface complexes, we identified vulnerable sites on the shared gH/gL subunits as well as complex-specific subunits UL128/130/131A and gO. Using high-resolution cryogenic electron microscopy, we revealed the structural basis of the neutralization mechanisms of antibodies targeting various binding sites. Moreover, we identified highly potent antibodies that neutralized a broad spectrum of HCMV strains, including primary clinical isolates, that outperform known antibodies used in clinical trials. Our study provides a deep understanding of the mechanisms of HCMV neutralization and identifies promising antibody candidates to prevent and treat HCMV infection.
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Affiliation(s)
- Matthias Zehner
- Laboratory of Experimental Immunology, Institute of Virology, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany.
| | - Mira Alt
- Department of Infectious Diseases, West German Centre of Infectious Diseases, University Hospital Essen, University Duisburg-Essen, 45147 Essen, Germany
| | - Artem Ashurov
- Laboratory of Experimental Immunology, Institute of Virology, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany
| | - Jory A Goldsmith
- Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA
| | - Rebecca Spies
- Institute for Virology, Ulm University Medical Center, 89081 Ulm, Germany
| | - Nina Weiler
- Institute for Virology, Ulm University Medical Center, 89081 Ulm, Germany
| | - Justin Lerma
- Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA
| | - Lutz Gieselmann
- Laboratory of Experimental Immunology, Institute of Virology, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany; German Center for Infection Research, Partner Site Bonn-Cologne, 50931 Cologne, Germany
| | - Dagmar Stöhr
- Institute for Virology, Ulm University Medical Center, 89081 Ulm, Germany
| | - Henning Gruell
- Laboratory of Experimental Immunology, Institute of Virology, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany
| | - Eric P Schultz
- Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA; Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA
| | - Christoph Kreer
- Laboratory of Experimental Immunology, Institute of Virology, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany
| | - Linda Schlachter
- Laboratory of Experimental Immunology, Institute of Virology, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany
| | - Hanna Janicki
- Laboratory of Experimental Immunology, Institute of Virology, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany
| | | | - Cora Stegmann
- Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA; Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA
| | - Michelle D Nemetchek
- Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA; Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA
| | - Sabrina Dähling
- Laboratory of Experimental Immunology, Institute of Virology, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany
| | - Leon Ullrich
- Laboratory of Experimental Immunology, Institute of Virology, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany
| | - Ulf Dittmer
- Institute for Virology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany
| | - Oliver Witzke
- Department of Infectious Diseases, West German Centre of Infectious Diseases, University Hospital Essen, University Duisburg-Essen, 45147 Essen, Germany
| | - Manuel Koch
- Institute for Dental Research and Oral Musculoskeletal Biology, Center for Biochemistry, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany
| | - Brent J Ryckman
- Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA; Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, USA
| | - Ramin Lotfi
- Institute for Transfusion Medicine, Ulm University Medical Center, 89081 Ulm, Germany
| | - Jason S McLellan
- Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA
| | - Adalbert Krawczyk
- Department of Infectious Diseases, West German Centre of Infectious Diseases, University Hospital Essen, University Duisburg-Essen, 45147 Essen, Germany; Institute for Virology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany
| | - Christian Sinzger
- Institute for Virology, Ulm University Medical Center, 89081 Ulm, Germany
| | - Florian Klein
- Laboratory of Experimental Immunology, Institute of Virology, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany; German Center for Infection Research, Partner Site Bonn-Cologne, 50931 Cologne, Germany; Center for Molecular Medicine Cologne (CMMC), University Hospital of Cologne, 50931 Cologne, Germany.
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15
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Dochnal SA, Whitford AL, Francois AK, Krakowiak PA, Cuddy S, Cliffe AR. c-Jun Signaling During Initial HSV-1 Infection Modulates Latency to Enhance Later Reactivation in addition to Directly Promoting the Progression to Full Reactivation. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.11.10.566462. [PMID: 37986840 PMCID: PMC10659354 DOI: 10.1101/2023.11.10.566462] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/22/2023]
Abstract
Herpes simplex virus-1 (HSV-1) establishes a latent infection in peripheral neurons and can periodically reactivate to permit transmission and clinical manifestations. Viral transactivators required for lytic infection are largely absent during latent infection and therefore HSV-1 relies on the co-option of neuronal host signaling pathways to initiate its gene expression. Activation of the neuronal c-Jun N-terminal kinase (JNK) cell stress pathway is central to initiating biphasic reactivation in response to multiple stimuli. However, how host factors work with JNK to stimulate the initial wave of gene expression (known as Phase I) or the progression to full, Phase II reactivation remains unclear. Here, we found that c-Jun, the primary target downstream of neuronal JNK cell stress signaling, functions during reactivation but not during the JNK-mediated initiation of Phase I gene expression. Instead, c-Jun was required for the transition from Phase I to full HSV-1 reactivation and was detected in viral replication compartments of reactivating neurons. Interestingly, we also identified a role for both c-Jun and enhanced neuronal stress during initial neuronal infection in promoting a more reactivation-competent form of HSV-1 latency. Therefore, c-Jun functions at multiple stages during HSV latent infection of neurons to promote reactivation. Importantly, by demonstrating that initial infection conditions can contribute to later reactivation abilities, this study highlights the potential for latently infected neurons to maintain a molecular scar of previous exposure to neuronal stressors.
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Affiliation(s)
- Sara A. Dochnal
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, 22908
| | - Abigail L. Whitford
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, 22908
| | - Alison K. Francois
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, 22908
| | - Patryk A. Krakowiak
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, 22908
| | - Sean Cuddy
- Neuroscience Graduate Program, University of Virginia, Charlottesville, VA, 22908
| | - Anna R. Cliffe
- Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, 22908
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16
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Weiler N, Sampaio KL, Scherer M, Sinzger C. Generation of UL128-shRNA transduced fibroblasts for the release of cell-free virus from clinical human cytomegalovirus isolates. Biotechniques 2023; 75:183-194. [PMID: 37846844 DOI: 10.2144/btn-2023-0046] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2023] Open
Abstract
Working with recent isolates of human cytomegalovirus (HCMV) is complicated by their strictly cell-associated growth with lack of infectivity in the supernatant. Adaptation to cell-free growth is associated with disruption of the viral UL128 gene locus. The authors transduced fibroblasts with a lentiviral vector encoding UL128-specific-shRNA to allow the release of cell-free infectivity without genetic alteration. Transduced cells were cocultured with fibroblasts containing cell-associated isolates, and knockdown of the UL128 protein was validated by immunoblotting. Cell-free infectivity increased 1000-fold in isolate cocultures with UL128-shRNA compared with controls, and virions could be purified by density gradients. Transduced fibroblasts also allowed direct isolation of HCMV from a clinical specimen and cell-free transfer to other cell types. In conclusion, UL128-shRNA-transduced fibroblasts allow applications previously unsuitable for recent isolates.
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Affiliation(s)
- Nina Weiler
- Institute of Virology, Ulm University Medical Center, Ulm, Germany
| | | | - Myriam Scherer
- Institute of Virology, Ulm University Medical Center, Ulm, Germany
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17
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Chu C, Yu S, Min F, Sun L, Liu M, Meng Q. Establishment and application of a point-of-care testing and diagnosis method for early immediate expression gene IE1 of cytomegalovirus in maternal urine based on isothermal amplification. Virus Res 2023; 337:199229. [PMID: 37769815 PMCID: PMC10579523 DOI: 10.1016/j.virusres.2023.199229] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2023] [Revised: 09/17/2023] [Accepted: 09/25/2023] [Indexed: 10/03/2023]
Abstract
BACKGROUND Human Cytomegalovirus virus (HCMV) is a worldwide virus that causes no serious symptoms in most adults. However, HCMV infection during pregnancy, it may lead to a series of serious complications, such as hearing loss, mental retardation, visual impairment, microcephaly and developmental retardation. AIM The aim of this study was to develop a simple, low dependence on equipment and accurate method for HCMV detection based on the recombinase polymerase amplification (RPA) and lateral flow chromatography strip (LFS) reading. METHODS In order to meet the feasibility of HCMV early screening, three pairs of RPA primers were designed based on the UL123 gene encoding IE1, which was expressed immediately in the early stage of HCMV. In order to improve the specificity of the reaction and satisfy the visual detection, a specific probe was designed to insert THF site between upstream and downstream primers, fluorescein isothiocyanate (FITC) and C3spacer were used to modify the 5' end and the 3' end respectively, and Biotin was used to modify the 5' end of the reverse primer. HCMV standard strain AD169 was enriched by ARPE-19 cells culture, and its genome was extracted. The primers and probes were screened by RPA-LFS test, and the optimal reaction temperature and time were determined The specificity was verified in different viruses, bacteria and parasites. The standard curve was drawn based on the constructed recombinant plasmid of pMD18T-HCMV-UL123 and used for HCMV genomic DNA quantification and determination of the detection sensitivity. Urine samples from artificial HCMV contamination or clinical collection were prepared to evaluate the consistency with the results of real-time quantitative PCR. RESULTS The results showed that the primers and probes for HCMV RPA-LFS detection based on UL123 gene were successfully screened, the amplification of HCMV genomic DNA with as low as 30 copies could be completed at 37 °C within 15 min, it did not react with Human herpesvirus 1, Streptococcus pyogenes, Candida albicans, Listeria monocytogenes, Y. enterocolitica, Klebsiella Pneumoniae, Enterobacter cloacae, Citrobacter freundii, Vibrio alginnolyfificus, Vibrio parahaemolyticus, S. typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa and Trichomonas vaginalis. The positive rate of PCR was 96.67 % in 30 simulated urine samples and 100 % in 127 clinical urine samples with the same UL123 gene detection. CONCLUSIONS To sum up, we developed a diagnostic method for HCMV based on UL123 gene combined with RPA and LFS, which is low dependent on equipment, fast, sensitive and specific, provide reference for point-of-care testing HCMV in grass-roots laboratories and remote areas.
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Affiliation(s)
- Chu Chu
- Obstetrical Department, Lianyungang Maternal and Child Health Care Hospital, Lianyungang, Jiangsu 222006, China
| | - Shijiao Yu
- Obstetrical Department, Lianyungang Maternal and Child Health Care Hospital, Lianyungang, Jiangsu 222006, China
| | - Fanli Min
- Obstetrical Department, Lianyungang Maternal and Child Health Care Hospital, Lianyungang, Jiangsu 222006, China
| | - Lizhou Sun
- Obstetrical Department, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 222006, China
| | - Meilin Liu
- Obstetrical Department, Lianyungang Maternal and Child Health Care Hospital, Lianyungang, Jiangsu 222006, China.
| | - Qian Meng
- Obstetrical Department, Lianyungang Maternal and Child Health Care Hospital, Lianyungang, Jiangsu 222006, China.
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18
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Nappi F, Alzamil A, Avtaar Singh SS, Spadaccio C, Bonnet N. Current Knowledge on the Interaction of Human Cytomegalovirus Infection, Encoded miRNAs, and Acute Aortic Syndrome. Viruses 2023; 15:2027. [PMID: 37896804 PMCID: PMC10611417 DOI: 10.3390/v15102027] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2023] [Revised: 09/21/2023] [Accepted: 09/28/2023] [Indexed: 10/29/2023] Open
Abstract
Aortic dissection is a clinicopathological entity caused by rupture of the intima, leading to a high mortality if not treated. Over time, diagnostic and investigative methods, antihypertensive therapy, and early referrals have resulted in improved outcomes according to registry data. Some data have also emerged from recent studies suggesting a link between Human Cytomegalovirus (HCMV) infection and aortic dissection. Furthermore, the use of microRNAs has also become increasingly widespread in the literature. These have been noted to play a role in aortic dissections with elevated levels noted in studies as early as 2017. This review aims to provide a broad and holistic overview of the role of miRNAs, while studying the role of HCMV infection in the context of aortic dissections. The roles of long non-coding RNAs, circular RNAs, and microRNAs are explored to identify changes in expression during aortic dissections. The use of such biomarkers may one day be translated into clinical practice to allow early detection and prognostication of outcomes and drive preventative and therapeutic options in the future.
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Affiliation(s)
- Francesco Nappi
- Department of Cardiac Surgery, Centre Cardiologique du Nord, 93200 Saint-Denis, France; (A.A.); (N.B.)
| | - Almothana Alzamil
- Department of Cardiac Surgery, Centre Cardiologique du Nord, 93200 Saint-Denis, France; (A.A.); (N.B.)
| | | | - Cristiano Spadaccio
- Department of Cardiothoracic Surgery, Mayo Clinic, Rochester, Rochester, MN 55905, USA;
| | - Nicolas Bonnet
- Department of Cardiac Surgery, Centre Cardiologique du Nord, 93200 Saint-Denis, France; (A.A.); (N.B.)
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19
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Abstract
Human cytomegalovirus (HCMV) is a betaherpesvirus that establishes lifelong infection in its host and can cause severe comorbidities in individuals with suppressed or compromised immune systems. The lifecycle of HCMV consists of lytic and latent phases, largely dependent upon the cell type infected and whether transcription from the major immediate early locus can ensue. Control of this locus, which acts as a critical "switch" region from where the lytic gene expression cascade originates, as well as regulation of the additional ~235 kilobases of virus genome, occurs through chromatinization with cellular histone proteins after infection. Upon infection of a host cell, an initial intrinsic antiviral response represses gene expression from the incoming genome, which is relieved in permissive cells by viral and host factors in concert. Latency is established in a subset of hematopoietic cells, during which viral transcription is largely repressed while the genome is maintained. As these latently infected cells differentiate, the cellular milieu and epigenetic modifications change, giving rise to the initial stages of virus reactivation from latency. Thus, throughout the cycle of infection, chromatinization, chromatin modifiers, and the recruitment of specific transcription factors influence the expression of genes from the HCMV genome. In this review, we discuss epigenetic regulation of the HCMV genome during the different phases of infection, with an emphasis on recent reports that add to our current perspective.
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Affiliation(s)
- Stephen M. Matthews
- Infection Biology, Global Center for Pathogen and Human Health Research, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA
| | - Ian J. Groves
- Infection Biology, Global Center for Pathogen and Human Health Research, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA
- Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland Clinic, Cleveland, Ohio, USA
| | - Christine M. O'Connor
- Infection Biology, Global Center for Pathogen and Human Health Research, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA
- Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland Clinic, Cleveland, Ohio, USA
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20
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Zeng J, Cao D, Yang S, Jaijyan DK, Liu X, Wu S, Cruz-Cosme R, Tang Q, Zhu H. Insights into the Transcriptome of Human Cytomegalovirus: A Comprehensive Review. Viruses 2023; 15:1703. [PMID: 37632045 PMCID: PMC10458407 DOI: 10.3390/v15081703] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2023] [Revised: 08/03/2023] [Accepted: 08/04/2023] [Indexed: 08/27/2023] Open
Abstract
Human cytomegalovirus (HCMV) is a widespread pathogen that poses significant risks to immunocompromised individuals. Its genome spans over 230 kbp and potentially encodes over 200 open-reading frames. The HCMV transcriptome consists of various types of RNAs, including messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs), with emerging insights into their biological functions. HCMV mRNAs are involved in crucial viral processes, such as viral replication, transcription, and translation regulation, as well as immune modulation and other effects on host cells. Additionally, four lncRNAs (RNA1.2, RNA2.7, RNA4.9, and RNA5.0) have been identified in HCMV, which play important roles in lytic replication like bypassing acute antiviral responses, promoting cell movement and viral spread, and maintaining HCMV latency. CircRNAs have gained attention for their important and diverse biological functions, including association with different diseases, acting as microRNA sponges, regulating parental gene expression, and serving as translation templates. Remarkably, HCMV encodes miRNAs which play critical roles in silencing human genes and other functions. This review gives an overview of human cytomegalovirus and current research on the HCMV transcriptome during lytic and latent infection.
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Affiliation(s)
- Janine Zeng
- Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, 225 Warren Street, Newark, NJ 070101, USA
| | - Di Cao
- Department of Pain Medicine, Huazhong University of Science and Technology Union Shenzhen Hospital, Shenzhen 518052, China
| | - Shaomin Yang
- Department of Pain Medicine, Huazhong University of Science and Technology Union Shenzhen Hospital, Shenzhen 518052, China
| | - Dabbu Kumar Jaijyan
- Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, 225 Warren Street, Newark, NJ 070101, USA
| | - Xiaolian Liu
- Institute of Pathogenic Organisms, Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China
| | - Songbin Wu
- Department of Pain Medicine, Huazhong University of Science and Technology Union Shenzhen Hospital, Shenzhen 518052, China
| | - Ruth Cruz-Cosme
- Department of Microbiology, Howard University College of Medicine, 520 W Street NW, Washington, DC 20059, USA
| | - Qiyi Tang
- Department of Microbiology, Howard University College of Medicine, 520 W Street NW, Washington, DC 20059, USA
| | - Hua Zhu
- Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, 225 Warren Street, Newark, NJ 070101, USA
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21
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Crawford LB. Hematopoietic stem cells and betaherpesvirus latency. Front Cell Infect Microbiol 2023; 13:1189805. [PMID: 37346032 PMCID: PMC10279960 DOI: 10.3389/fcimb.2023.1189805] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2023] [Accepted: 05/11/2023] [Indexed: 06/23/2023] Open
Abstract
The human betaherpesviruses including human cytomegalovirus (HCMV), human herpesvirus (HHV)-6a and HHV-6b, and HHV-7 infect and establish latency in CD34+ hematopoietic stem and progenitor cells (HPCs). The diverse repertoire of HPCs in humans and the complex interactions between these viruses and host HPCs regulate the viral lifecycle, including latency. Precise manipulation of host and viral factors contribute to preferential maintenance of the viral genome, increased host cell survival, and specific manipulation of the cellular environment including suppression of neighboring cells and immune control. The dynamic control of these processes by the virus regulate inter- and intra-host signals critical to the establishment of chronic infection. Regulation occurs through direct viral protein interactions and cellular signaling, miRNA regulation, and viral mimics of cellular receptors and ligands, all leading to control of cell proliferation, survival, and differentiation. Hematopoietic stem cells have unique biological properties and the tandem control of virus and host make this a unique environment for chronic herpesvirus infection in the bone marrow. This review highlights the elegant complexities of the betaherpesvirus latency and HPC virus-host interactions.
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Affiliation(s)
- Lindsey B Crawford
- Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, United States
- Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE, United States
- Nebraska Center for Integrated Biomolecular Communication, University of Nebraska-Lincoln, Lincoln, NE, United States
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22
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Forte E, Li M, Ayaloglu Butun F, Hu Q, Borst EM, Schipma MJ, Piunti A, Shilatifard A, Terhune SS, Abecassis M, Meier JL, Hummel M. Critical Role for the Human Cytomegalovirus Major Immediate Early Proteins in Recruitment of RNA Polymerase II and H3K27Ac To an Enhancer-Like Element in Ori Lyt. Microbiol Spectr 2023; 11:e0314422. [PMID: 36645269 PMCID: PMC9927211 DOI: 10.1128/spectrum.03144-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2022] [Accepted: 12/16/2022] [Indexed: 01/17/2023] Open
Abstract
Human cytomegalovirus (HCMV) is an opportunistic pathogen that infects most of the population. The complex 236 kbp genome encodes more than 170 open reading frames, whose expression is temporally regulated by both viral transcriptional regulators and cellular factors that control chromatin and transcription. Here, we have used state of the art genomic technologies to investigate the viral transcriptome in conjunction with 2 key transcriptional regulators: Pol II and H3K27Ac. Although it is well known that the major immediate early (IE) proteins activate early gene expression through both direct and indirect interactions, and that histone modifications play an important role in regulating viral gene expression, the role of the IE proteins in modulating viral chromatin is not fully understood. To address this question, we have used a virus engineered for conditional expression of the IE proteins combined with RNA and Chromatin immunoprecipitation (ChIP) analyses to assess the role of these proteins in modulating both viral chromatin and gene expression. Our results show that (i) there is an enhancer-like element in OriLyt that is extraordinarily enriched in H3K27Ac; (ii) in addition to activation of viral gene expression, the IE proteins play a critical role in recruitment of Pol II and H3K27Ac to this element. IMPORTANCE HCMV is an important human pathogen associated with complications in transplant patients and birth defects. The complex program of viral gene expression is regulated by both viral proteins and host factors. Here, we have investigated the role of the immediate early proteins in regulating the viral epigenome. Our results show that the viral immediate early proteins bring about an enormous enrichment of H3K27Ac marks at the OriLyt RNA4.9 promoter, concomitant with an increase in RNA4.9 expression. This epigenetic characteristic adds importantly to the view that OriLyt has structural and functional characteristics of a strong enhancer that, we now discover, is regulated by IE proteins.
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Affiliation(s)
- Eleonora Forte
- Comprehensive Transplant Center, Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
- Proteomics Center of Excellence, Northwestern University, Evanston, Illinois, USA
| | - Ming Li
- Departments of Internal Medicine and Epidemiology, University of Iowa and Iowa City Veterans Affairs Health Care System, Iowa City, Iowa, USA
| | - Fatma Ayaloglu Butun
- Comprehensive Transplant Center, Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
- Proteomics Center of Excellence, Northwestern University, Evanston, Illinois, USA
| | - Qiaolin Hu
- Departments of Internal Medicine and Epidemiology, University of Iowa and Iowa City Veterans Affairs Health Care System, Iowa City, Iowa, USA
| | - Eva Maria Borst
- Department of Virology, Hannover Medical School, Hannover, Germany
| | - Matthew J. Schipma
- NUSeq Core, Quantitative Data Science Core, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
| | - Andrea Piunti
- Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
| | - Ali Shilatifard
- Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
| | - Scott S. Terhune
- Department of Microbiology and Immunology and Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin, USA
| | - Michael Abecassis
- Comprehensive Transplant Center, Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
| | - Jeffery L. Meier
- Departments of Internal Medicine and Epidemiology, University of Iowa and Iowa City Veterans Affairs Health Care System, Iowa City, Iowa, USA
| | - Mary Hummel
- Comprehensive Transplant Center, Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
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23
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Cheng Y, Du Y, Wang Q, Lv Q, Xue Y, Zhou W, Zhang C, Chen X, Wang D. Human cytomegalovirus-encoded microRNAs expression profile in plasma of patients with aortic dissection. J Cardiothorac Surg 2023; 18:39. [PMID: 36653806 PMCID: PMC9848029 DOI: 10.1186/s13019-023-02122-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2022] [Accepted: 01/02/2023] [Indexed: 01/20/2023] Open
Abstract
BACKGROUND Aortic dissection (AD) is a rare disease with high mortality for which no effective diagnostic biomarkers are available. Human cytomegalovirus (HCMV) infection is an important cause of the occurrence and progression of many diseases, but the relationship between HCMV infection and AD is not clear. METHODS In this study, we first used quantitative reverse transcription polymerase chain reaction (qRT-PCR) to determine the expression profile of 25 HCMV-encoded microRNAs (HCMV miRNAs) in the plasma within a training set consisting of 20 AD patients and 20 healthy controls. Then, abnormal expressed HCMV miRNAs were verified in a validation set of 12 AD patients and 12 healthy controls. In addition, HCMV infection was detected in the third cohort consisting of 20 AD patients and 20 healthy controls. RESULTS The 95% quantile of the expression levels of HCMV miRNAs in the training set was used as the threshold for distinction between AD patients and healthy controls. The proportion of individuals with high level of five types of HCMV miRNAs was significantly different between AD patients and healthy controls. In the validation set, only the proportion of individuals with high levels of hcmv-miR-UL112-5p and hcmv-miR-UL22A-5p, two of the five HCMV miRNAs obtained in the preliminary screening, showed significant difference between AD patients and healthy controls. In the third cohort, there was no significant difference in HCMV DNA levels and anti-HCMV IgG concentrations between AD patients and healthy controls. CONCLUSIONS The HCMV miRNAs levels in plasma differed in AD patients and healthy controls. This finding may contribute to a further understanding of the relationship between HCMV infection and AD and are worthy of future research on the diagnosis and etiology of AD.
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Affiliation(s)
- Yongqing Cheng
- grid.41156.370000 0001 2314 964XDepartment of Cardio-Thoracic Surgery, Drum Tower Hospital Affiliated to Nanjing University Medical School, Nanjing, 210008 Jiangsu China
| | - Yufan Du
- grid.41156.370000 0001 2314 964XPresent Address: State Key Laboratory of Pharmaceutical Biotechnology, Collaborative Innovation Center of Chemistry for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute for Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, 210033 Jiangsu China
| | - Qi Wang
- grid.41156.370000 0001 2314 964XPresent Address: State Key Laboratory of Pharmaceutical Biotechnology, Collaborative Innovation Center of Chemistry for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute for Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, 210033 Jiangsu China
| | - Qinghe Lv
- grid.41156.370000 0001 2314 964XPresent Address: State Key Laboratory of Pharmaceutical Biotechnology, Collaborative Innovation Center of Chemistry for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute for Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, 210033 Jiangsu China
| | - Yunxin Xue
- grid.41156.370000 0001 2314 964XDepartment of Cardio-Thoracic Surgery, Drum Tower Hospital Affiliated to Nanjing University Medical School, Nanjing, 210008 Jiangsu China
| | - Weihong Zhou
- grid.41156.370000 0001 2314 964XDepartment of Health Management Centre, Drum Tower Hospital Affiliated to Nanjing University Medical School, Nanjing, 210008 Jiangsu China
| | - Chenyu Zhang
- grid.41156.370000 0001 2314 964XPresent Address: State Key Laboratory of Pharmaceutical Biotechnology, Collaborative Innovation Center of Chemistry for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute for Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, 210033 Jiangsu China
| | - Xi Chen
- grid.41156.370000 0001 2314 964XPresent Address: State Key Laboratory of Pharmaceutical Biotechnology, Collaborative Innovation Center of Chemistry for Life Sciences, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute for Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, 210033 Jiangsu China
| | - Dongjin Wang
- grid.41156.370000 0001 2314 964XDepartment of Cardio-Thoracic Surgery, Drum Tower Hospital Affiliated to Nanjing University Medical School, Nanjing, 210008 Jiangsu China
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24
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Vezzani G, Pimazzoni S, Ferranti R, Calò S, Monda G, Amendola D, Frigimelica E, Maione D, Cortese M, Merola M. Human immunoglobulins are transported to HCMV viral envelope by viral Fc gamma receptors-dependent and independent mechanisms. Front Microbiol 2023; 13:1106401. [PMID: 36726564 PMCID: PMC9885202 DOI: 10.3389/fmicb.2022.1106401] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2022] [Accepted: 12/20/2022] [Indexed: 01/18/2023] Open
Abstract
Human cytomegaloviruses (HCMVs) employ many different mechanisms to escape and subvert the host immune system, including expression of the viral IgG Fcγ receptors (vFcγRs) RL11 (gp34), RL12 (gp95), RL13 (gpRL13), and UL119 (gp68) gene products. The role of vFcγRs in HCMV pathogenesis has been reported to operate in infected cells by interfering with IgG-mediated effector functions. We found that gp34 and gp68 are envelope proteins that bind and internalize human IgGs on the surface of infected cells. Internalized IgGs are then transported on the envelope of viral particles in a vFcR-dependent mechanism. This mechanism is also responsible for the incorporation on the virions of the anti-gH neutralizing antibody MSL-109. Intriguingly, we show that gp68 is responsible for MSL-109 incorporation, but it is dispensable for other anti-HCMV antibodies that do not need this function to be transported on mature virions. HCMV-infected cells grown in presence of anti-HCMV monoclonal antibodies generate a viral progeny still infective and possible to be neutralized. This is the first example of a virus carrying neutralizing IgGs on its surface and their possible role is discussed.
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Affiliation(s)
| | | | | | | | | | | | | | | | - Mirko Cortese
- GSK, Siena, Italy,Department of Environmental Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, Caserta, Italy,Mirko Cortese, ✉
| | - Marcello Merola
- GSK, Siena, Italy,Department of Biology, University of Naples Federico II, Naples, Italy,*Correspondence: Marcello Merola, ✉
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25
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Mosher BS, Kowalik TF, Yurochko AD. Overview of how HCMV manipulation of host cell intracellular trafficking networks can promote productive infection. FRONTIERS IN VIROLOGY 2022. [DOI: 10.3389/fviro.2022.1026452] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in the immunocompromised and developing fetuses. Infection has also been linked to chronic inflammatory diseases, cardiovascular disease, and the development of certain cancers. The wide range of pathologies associated with HCMV infection is attributable to the broad cellular tropism of the virus where infection affects every organ system. Like other viruses, HCMV must tailor host cells to support productive infection. In particular, HCMV dedicates many resources and various strategies to manipulate host intracellular trafficking networks to facilitate various aspects of infection across all infected cell types. The dysregulation of host intracellular trafficking networks allows the virus to translocate to the host cell nucleus for genome replication, facilitate nuclear import/export of viral proteins and immature virions, subvert the host immune response, form new organelles for progeny virion assembly, maturation and egress, and promote cellular migration and viral spread. However, due to their complex nature, many aspects of these processes are not well-studied. New research and omics-based technologies have recently begun to elucidate the extent to which HCMV dysregulates host cell trafficking machinery. Here we review the variety of strategies HCMV utilizes to dysregulate intracellular trafficking networks to promote productive infection.
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26
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Jentzer A, Fauteux-Daniel S, Verhoeven P, Cantais A, Novoa MY, Jospin F, Chanut B, Rochereau N, Bourlet T, Roblin X, Pozzetto B, Pillet S. Impact of Dextran-Sodium-Sulfate-Induced Enteritis on Murine Cytomegalovirus Reactivation. Viruses 2022; 14:2595. [PMID: 36560599 PMCID: PMC9781000 DOI: 10.3390/v14122595] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2022] [Revised: 11/11/2022] [Accepted: 11/18/2022] [Indexed: 11/24/2022] Open
Abstract
(1) Background: Ulcerative colitis (UC) is an inflammatory bowel disease that causes inflammation of the intestines, which participates in human cytomegalovirus (HCMV) reactivation from its latent reservoir. CMV-associated colitis plays a pejorative role in the clinical course of UC. We took advantage of a model of chemically induced enteritis to study the viral reactivation of murine CMV (MCMV) in the context of gut inflammation. (2) Methods: Seven-week-old BALB/c mice were infected by 3 × 103 plaque-forming units (PFU) of MCMV; 2.5% (w/v) DSS was administered in the drinking water from day (D) 30 to D37 post-infection to induce enteritis. (3) Results: MCMV DNA levels in the circulation decreased from D21 after infection until resolution of the acute infection. DSS administration resulted in weight loss, high disease activity index, elevated Nancy index shortening of the colon length and increase in fecal lipocalin. However, chemically induced enteritis had no impact on MCMV reactivation as determined by qPCR and immunohistochemistry of intestinal tissues. (4) Conclusions: Despite the persistence of MCMV in the digestive tissues after the acute phase of infection, the gut inflammation induced by DSS did not induce MCMV reactivation in intestinal tissues, thus failing to recapitulate inflammation-driven HCMV reactivation in human UC.
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Affiliation(s)
- Alexandre Jentzer
- CIRI, Centre International de Recherche en Infectiologie, GIMAP Team, Univ Lyon, Univ St-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1, 42023 Saint-Etienne, France
| | - Sébastien Fauteux-Daniel
- French Blood Establishment Auvergne-Rhône-Alpes, Scientific Department, 42270 Saint-Etienne, France
| | - Paul Verhoeven
- CIRI, Centre International de Recherche en Infectiologie, GIMAP Team, Univ Lyon, Univ St-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1, 42023 Saint-Etienne, France
- Department of Infectious Agents and Hygiene, University-Hospital of Saint-Etienne, 42055 Saint-Etienne, France
| | - Aymeric Cantais
- CIRI, Centre International de Recherche en Infectiologie, GIMAP Team, Univ Lyon, Univ St-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1, 42023 Saint-Etienne, France
| | - Melyssa Yaugel Novoa
- CIRI, Centre International de Recherche en Infectiologie, GIMAP Team, Univ Lyon, Univ St-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1, 42023 Saint-Etienne, France
| | - Fabienne Jospin
- CIRI, Centre International de Recherche en Infectiologie, GIMAP Team, Univ Lyon, Univ St-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1, 42023 Saint-Etienne, France
| | - Blandine Chanut
- CIRI, Centre International de Recherche en Infectiologie, GIMAP Team, Univ Lyon, Univ St-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1, 42023 Saint-Etienne, France
| | - Nicolas Rochereau
- CIRI, Centre International de Recherche en Infectiologie, GIMAP Team, Univ Lyon, Univ St-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1, 42023 Saint-Etienne, France
| | - Thomas Bourlet
- CIRI, Centre International de Recherche en Infectiologie, GIMAP Team, Univ Lyon, Univ St-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1, 42023 Saint-Etienne, France
- Department of Infectious Agents and Hygiene, University-Hospital of Saint-Etienne, 42055 Saint-Etienne, France
| | - Xavier Roblin
- CIRI, Centre International de Recherche en Infectiologie, GIMAP Team, Univ Lyon, Univ St-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1, 42023 Saint-Etienne, France
- Department of Gastroenterology, University-Hospital of Saint-Etienne, 42055 Saint-Etienne, France
| | - Bruno Pozzetto
- CIRI, Centre International de Recherche en Infectiologie, GIMAP Team, Univ Lyon, Univ St-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1, 42023 Saint-Etienne, France
- Department of Infectious Agents and Hygiene, University-Hospital of Saint-Etienne, 42055 Saint-Etienne, France
| | - Sylvie Pillet
- CIRI, Centre International de Recherche en Infectiologie, GIMAP Team, Univ Lyon, Univ St-Etienne, INSERM U1111, CNRS UMR5308, ENS de Lyon, UCBL1, 42023 Saint-Etienne, France
- Department of Infectious Agents and Hygiene, University-Hospital of Saint-Etienne, 42055 Saint-Etienne, France
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27
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Complete Genome and Molecular Characterization of a New Cyprinid Herpesvirus 2 (CyHV-2) SH-01 Strain Isolated from Cultured Crucian Carp. Viruses 2022; 14:v14092068. [PMID: 36146873 PMCID: PMC9503944 DOI: 10.3390/v14092068] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2022] [Revised: 09/09/2022] [Accepted: 09/13/2022] [Indexed: 11/17/2022] Open
Abstract
Cyprinid herpesvirus 2 (CyHV-2) is a causative factor of herpesviral hematopoietic necrosis (HVHN) in farmed crucian carp (Carassius carassius) and goldfish (Carassius auratus). In this study, we analyzed the genomic characteristics of a new strain, CyHV-2 SH-01, isolated during outbreaks in crucian carp at a local fish farm near Shanghai, China. CyHV-2 SH-01 exhibited a high sensitivity to goldfish and crucian carp in our previous research. The complete genome of SH-01 is 290,428 bp with 154 potential open reading frames (ORFs) and terminal repeat (TR) regions at both ends. Compared to the sequenced genomes of other CyHVs, Carassius auratus herpesvirus (CaHV) and Anguillid herpesvirus 1 (AngHV-1), several variations were found in SH-01, including nucleotide mutations, deletions, and insertions, as well as gene duplications, rearrangements, and horizontal transfers. Overall, the genome of SH-01 shares 99.60% of its identity with that of ST-J1. Genomic collinearity analysis showed that SH-01 has a high degree of collinearity with another three CyHV-2 isolates, and it is generally closely related to CaHV, CyHV-1, and CyHV-3, although it contains many differences in locally collinear blocks (LCBs). The lowest degree of collinearity was found with AngHV-1, despite some homologous LCBs, indicating that they are evolutionarily the most distantly related. The results provide new clues to better understand the CyHV-2 genome through sequencing and sequence mining.
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Külekci B, Schwarz S, Brait N, Perkmann-Nagele N, Jaksch P, Hoetzenecker K, Puchhammer-Stöckl E, Goerzer I. Human cytomegalovirus strain diversity and dynamics reveal the donor lung as a major contributor after transplantation. Virus Evol 2022; 8:veac076. [PMID: 36128049 PMCID: PMC9477073 DOI: 10.1093/ve/veac076] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2022] [Revised: 07/05/2022] [Accepted: 08/23/2022] [Indexed: 11/17/2022] Open
Abstract
Mixed human cytomegalovirus (HCMV) strain infections are frequent in lung transplant recipients (LTRs). To date, the influence of the donor (D) and recipient (R) HCMV serostatus on intra-host HCMV strain composition and viral population dynamics after transplantation is only poorly understood. Here, we investigated ten pre-transplant lungs from HCMV-seropositive donors and 163 sequential HCMV-DNA-positive plasma and bronchoalveolar lavage samples from fifty LTRs with multiviremic episodes post-transplantation. The study cohort included D+R+ (38 per cent), D+R- (36 per cent), and D-R+ (26 per cent) patients. All samples were subjected to quantitative genotyping by short amplicon deep sequencing, and twenty-four of them were additionally PacBio long-read sequenced for genotype linkages. We find that D+R+ patients show a significantly elevated intra-host strain diversity compared to D+R- and D-R+ patients (P = 0.0089). Both D+ patient groups display significantly higher viral population dynamics than D- patients (P = 0.0061). Five out of ten pre-transplant donor lungs were HCMV DNA positive, whereof three multiple HCMV strains were detected, indicating that multi-strain transmission via lung transplantation is likely. Using long reads, we show that intra-host haplotypes can share distinctly linked genotypes, which limits overall intra-host diversity in mixed infections. Together, our findings demonstrate donor-derived strains as the main source of increased HCMV strain diversity and dynamics post-transplantation. These results foster strategies to mitigate the potential transmission of the donor strain reservoir to the allograft, such as ex vivo delivery of HCMV-selective immunotoxins prior to transplantation to reduce latent HCMV.
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Affiliation(s)
- Büsra Külekci
- Center for Virology, Medical University of Vienna, Kinderspitalgasse 15, Vienna 1090, Austria
| | - Stefan Schwarz
- Department of Thoracic Surgery, Medical University of Vienna, Währinger Gürtel 18-20, Vienna 1090, Austria
| | - Nadja Brait
- Cluster of Microbial Ecology, Groningen Institute for Evolutionary Life Sciences, University of Groningen, Nijenborgh 7, Groningen 9747 AG, The Netherlands
| | - Nicole Perkmann-Nagele
- Division of Clinical Virology, Medical University of Vienna, Währinger Gürtel 18-20, Vienna 1090, Austria
| | - Peter Jaksch
- Department of Thoracic Surgery, Medical University of Vienna, Währinger Gürtel 18-20, Vienna 1090, Austria
| | - Konrad Hoetzenecker
- Department of Thoracic Surgery, Medical University of Vienna, Währinger Gürtel 18-20, Vienna 1090, Austria
| | | | - Irene Goerzer
- Center for Virology, Medical University of Vienna, Kinderspitalgasse 15, Vienna 1090, Austria
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29
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Effect of Cytomegalovirus on the Immune System: Implications for Aging and Mental Health. Curr Top Behav Neurosci 2022; 61:181-214. [PMID: 35871707 DOI: 10.1007/7854_2022_376] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/16/2022]
Abstract
Human cytomegalovirus (HCMV) is a major modulator of the immune system leading to long-term changes in T-lymphocytes, macrophages, and natural killer (NK) cells among others. Perhaps because of this immunomodulatory capacity, HCMV infection has been linked with a host of deleterious effects including accelerated immune aging (premature mortality, increased expression of immunosenescence-linked markers, telomere shortening, speeding-up of epigenetic "clocks"), decreased vaccine immunogenicity, and greater vulnerability to infectious diseases (e.g., tuberculosis) or infectious disease-associated pathology (e.g., HIV). Perhaps not surprisingly given the long co-evolution between HCMV and humans, the virus has also been associated with beneficial effects, such as increased vaccine responsiveness, heterologous protection against infections, and protection against relapse in the context of leukemia. Here, we provide an overview of this literature. Ultimately, we focus on one other deleterious effect of HCMV, namely the emerging literature suggesting that HCMV plays a pathophysiological role in psychiatric illness, particularly depression and schizophrenia. We discuss this literature through the lens of psychological stress and inflammation, two well-established risk factors for psychiatric illness that are also known to predispose to reactivation of HCMV.
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30
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Combined knockdown of RL13 and UL128 for release of cell-free infectivity from recent HCMV isolates. J Virol Methods 2022; 305:114537. [PMID: 35526667 DOI: 10.1016/j.jviromet.2022.114537] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2022] [Accepted: 04/28/2022] [Indexed: 11/23/2022]
Abstract
Due to strictly cell-associated growth, experiments requiring cell-free virus are not applicable to recent clinical HCMV isolates to date. On the other hand, adaptation to cell-free growth is associated with undesirable changes in the viral gene regions RL13 and UL128. We had previously found that siRNA-mediated reduction of UL128 expression allowed transient release of cell-free virus by clinical isolates, and now hypothesized that virus yield could be further increased by additional knockdown of RL13. Despite the extensive polymorphism of RL13, effective RL13-specific siRNAs could be designed for three recent isolates and the Merlin strain. Knockdown efficiency was demonstrated at the protein level with a Merlin variant expressing V5-tagged pRL13. Knockdown of RL13 alone did not result in measurable release of cell-free virus, but combined knockdown of RL13 and UL128 increased infectivity in cell-free supernatants by a factor of 10-2000 compared to knockdown of UL128 alone. These supernatants could be used in dose-response assays to compare the effect of a neutralizing antibody on the various HCMV isolates. In summary, combined knockdown of RL13 and UL128 by specific siRNAs allows reliable release of cell-free infectivity from otherwise strictly cell-associated HCMV isolates without the need to modify the viral genome.
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31
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Kumar M, Saadaoui M, Al Khodor S. Infections and Pregnancy: Effects on Maternal and Child Health. Front Cell Infect Microbiol 2022; 12:873253. [PMID: 35755838 PMCID: PMC9217740 DOI: 10.3389/fcimb.2022.873253] [Citation(s) in RCA: 47] [Impact Index Per Article: 15.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2022] [Accepted: 05/04/2022] [Indexed: 12/22/2022] Open
Abstract
Pregnancy causes physiological and immunological adaptations that allow the mother and fetus to communicate with precision in order to promote a healthy pregnancy. At the same time, these adaptations may make pregnant women more susceptible to infections, resulting in a variety of pregnancy complications; those pathogens may also be vertically transmitted to the fetus, resulting in adverse pregnancy outcomes. Even though the placenta has developed a robust microbial defense to restrict vertical microbial transmission, certain microbial pathogens have evolved mechanisms to avoid the placental barrier and cause congenital diseases. Recent mechanistic studies have begun to uncover the striking role of the maternal microbiota in pregnancy outcomes. In this review, we discuss how microbial pathogens overcome the placental barrier to cause congenital diseases. A better understanding of the placental control of fetal infection should provide new insights into future translational research.
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Affiliation(s)
- Manoj Kumar
- Research Department, Sidra Medicine, Doha, Qatar
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32
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O’Brien BS, Mokry RL, Schumacher ML, Pulakanti K, Rao S, Terhune SS, Ebert AD. Downregulation of neurodevelopmental gene expression in iPSC-derived cerebral organoids upon infection by human cytomegalovirus. iScience 2022; 25:104098. [PMID: 35391828 PMCID: PMC8980761 DOI: 10.1016/j.isci.2022.104098] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2021] [Revised: 01/18/2022] [Accepted: 03/15/2022] [Indexed: 11/25/2022] Open
Abstract
Human cytomegalovirus (HCMV) is a betaherpesvirus that can cause severe birth defects including vision and hearing loss, microcephaly, and seizures. Currently, no approved treatment options exist for in utero infections. Here, we aimed to determine the impact of HCMV infection on the transcriptome of developing neurons in an organoid model system. Cell populations isolated from organoids based on a marker for infection and transcriptomes were defined. We uncovered downregulation in key cortical, neurodevelopmental, and functional gene pathways which occurred regardless of the degree of infection. To test the contributions of specific HCMV immediate early proteins known to disrupt neural differentiation, we infected NPCs using a recombinant virus harboring a destabilization domain. Despite suppressing their expression, HCMV-mediated transcriptional downregulation still occurred. Together, our studies have revealed that HCMV infection causes a profound downregulation of neurodevelopmental genes and suggest a role for other viral factors in this process.
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Affiliation(s)
- Benjamin S. O’Brien
- Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA
| | - Rebekah L. Mokry
- Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI 53226, USA
| | - Megan L. Schumacher
- Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI 53226, USA
| | | | - Sridhar Rao
- Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA
- Blood Research Institute, Versiti, Milwaukee, WI 53226, USA
| | - Scott S. Terhune
- Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, WI 53226, USA
| | - Allison D. Ebert
- Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA
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Hong YM, Min SY, Kim D, Kim S, Seo D, Lee KH, Han SH. Human MicroRNAs Attenuate the Expression of Immediate Early Proteins and HCMV Replication during Lytic and Latent Infection in Connection with Enhancement of Phosphorylated RelA/p65 (Serine 536) That Binds to MIEP. Int J Mol Sci 2022; 23:ijms23052769. [PMID: 35269913 PMCID: PMC8911160 DOI: 10.3390/ijms23052769] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2022] [Revised: 02/24/2022] [Accepted: 02/28/2022] [Indexed: 02/05/2023] Open
Abstract
Attenuating the expression of immediate early (IE) proteins is essential for controlling the lytic replication of human cytomegalovirus (HCMV). The human microRNAs (hsa-miRs), miR-200b-3p and miR-200c-3p, have been identified to bind the 3′-untranslated region (3′-UTR) of the mRNA encoding IE proteins. However, whether hsa-miRs can reduce IE72 expression and HCMV viral load or exhibit a crosstalk with the host cellular signaling machinery, most importantly the NF-κB cascade, has not been evaluated. In this study, argonaute-crosslinking and immunoprecipitation-seq revealed that miR-200b-3p and miR-200c-3p bind the 3′-UTR of UL123, which is a gene that encodes IE72. The binding of these miRNAs to the 3′-UTR of UL123 was verified in transfected cells stably expressing GFP. We used miR-200b-3p/miR-200c-3p mimics to counteract the downregulation of these miRNA after acute HCMV infection. This resulted in reduced IE72/IE86 expression and HCMV VL during lytic infection. We determined that IE72/IE86 alone can inhibit the phosphorylation of RelA/p65 at the Ser536 residue and that p-Ser536 RelA/p65 binds to the major IE promoter/enhancer (MIEP). The upregulation of miR-200b-3p and miR-200c-3p resulted in the phosphorylation of RelA/p65 at Ser536 through the downregulation of IE, and the binding of the resultant p-Ser536 RelA/p65 to MIEP resulted in a decreased production of pro-inflammatory cytokines. Overall, miR-200b-3p and miR-200c-3p—together with p-Ser536 RelA/p65—can prevent lytic HCMV replication during acute and latent infection
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Affiliation(s)
- Yeon-Mi Hong
- Division of Infectious Disease, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 06273, Korea; (Y.-M.H.); (S.Y.M.); (D.K.); (S.K.); (K.H.L.)
| | - Seo Yeon Min
- Division of Infectious Disease, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 06273, Korea; (Y.-M.H.); (S.Y.M.); (D.K.); (S.K.); (K.H.L.)
| | - Dayeong Kim
- Division of Infectious Disease, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 06273, Korea; (Y.-M.H.); (S.Y.M.); (D.K.); (S.K.); (K.H.L.)
| | - Subin Kim
- Division of Infectious Disease, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 06273, Korea; (Y.-M.H.); (S.Y.M.); (D.K.); (S.K.); (K.H.L.)
| | - Daekwan Seo
- Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 06273, Korea;
| | - Kyoung Hwa Lee
- Division of Infectious Disease, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 06273, Korea; (Y.-M.H.); (S.Y.M.); (D.K.); (S.K.); (K.H.L.)
| | - Sang Hoon Han
- Division of Infectious Disease, Department of Internal Medicine, Yonsei University College of Medicine, Seoul 06273, Korea; (Y.-M.H.); (S.Y.M.); (D.K.); (S.K.); (K.H.L.)
- Correspondence: ; Tel.: +82-2-2019-3319; Fax: +82-2-3463-3882
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34
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Ribeiro RVP, Ku T, Wang A, Pires L, Ferreira VH, Michaelsen V, Ali A, Galasso M, Moshkelgosha S, Gazzalle A, Jeppesen MG, Rosenkilde MM, Liu M, Singer LG, Kumar D, Keshavjee S, Sinclair J, Kledal TN, Humar A, Cypel M. Ex vivo treatment of cytomegalovirus in human donor lungs using a novel chemokine-based immunotoxin. J Heart Lung Transplant 2022; 41:287-297. [PMID: 34802874 DOI: 10.1016/j.healun.2021.10.010] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2021] [Revised: 10/12/2021] [Accepted: 10/15/2021] [Indexed: 01/07/2023] Open
Abstract
BACKGROUND Transmission of latent human cytomegalovirus (HCMV) via organ transplantation with post-transplant viral reactivation is extremely prevalent and results in substantial adverse impact on outcomes. Therapies targeting the latent reservoir within the allograft to mitigate viral transmission would represent a major advance. Here, we delivered an immunotoxin (F49A-FTP) that targets and kills latent HCMV aiming at reducing the HCMV reservoir from donor lungs using ex-vivo lung perfusion (EVLP). METHODS HCMV seropositive human lungs were placed on EVLP alone or EVLP + 1mg/L of F49A-FTP for 6 hours (n = 6, each). CD14+ monocytes isolated from biopsies pre and post EVLP underwent HCMV reactivation assay designed to evaluate viral reactivation capacity. Off-target effects of F49A-FTP were studied evaluating cell death markers of CD34+ and CD14+ cells using flow cytometry. Lung function on EVLP and inflammatory cytokine production were evaluated as safety endpoints. RESULTS We demonstrate that lungs treated ex-vivo with F49A-FTP had a significant reduction in HCMV reactivation compared to controls, suggesting successful targeting of latent virus (76% median reduction in F49A-FTP vs 15% increase in controls, p = 0.0087). Furthermore, there was comparable cell death rates of the targeted cells between both groups, suggesting no off-target effects. Ex-vivo lung function was stable over 6 hours and no differences in key inflammatory cytokines were observed demonstrating safety of this novel treatment. CONCLUSIONS Ex-vivo F49A-FTP treatment of human lungs targets and kills latent HCMV, markedly attenuating HCMV reactivation. This approach demonstrates the first experiments targeting latent HCMV in a donor organ with promising results towards clinical translation.
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Affiliation(s)
- Rafaela V P Ribeiro
- Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada
| | - Terrance Ku
- Ajmera Transplant Centre, University Health Network, Toronto, Ontario, Canada
| | - Aizhou Wang
- Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada
| | - Layla Pires
- Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada
| | - Victor H Ferreira
- Ajmera Transplant Centre, University Health Network, Toronto, Ontario, Canada
| | - Vinicius Michaelsen
- Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada
| | - Aadil Ali
- Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada
| | - Marcos Galasso
- Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada
| | - Sajad Moshkelgosha
- Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada
| | - Anajara Gazzalle
- Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada
| | | | - Mette M Rosenkilde
- Synklino ApS, Ole Måløes vej X, Copenhagen, Denmark; Laboratory of Molecular Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Mingyao Liu
- Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada
| | - Lianne G Singer
- Ajmera Transplant Centre, University Health Network, Toronto, Ontario, Canada
| | - Deepali Kumar
- Ajmera Transplant Centre, University Health Network, Toronto, Ontario, Canada
| | - Shaf Keshavjee
- Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada; Ajmera Transplant Centre, University Health Network, Toronto, Ontario, Canada
| | - John Sinclair
- Department of Medicine, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK
| | | | - Atul Humar
- Ajmera Transplant Centre, University Health Network, Toronto, Ontario, Canada
| | - Marcelo Cypel
- Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada; Ajmera Transplant Centre, University Health Network, Toronto, Ontario, Canada.
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35
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Abstract
Human cytomegalovirus (HCMV) is a highly prevalent beta-herpesvirus and a significant cause of morbidity and mortality following hematopoietic and solid organ transplant, as well as the leading viral cause of congenital abnormalities. A key feature of the pathogenesis of HCMV is the ability of the virus to establish a latent infection in hematopoietic progenitor and myeloid lineage cells. The study of HCMV latency has been hampered by difficulties in obtaining and culturing primary cells, as well as an inability to quantitatively measure reactivating virus, but recent advances in both in vitro and in vivo models of HCMV latency and reactivation have led to a greater understanding of the interplay between host and virus. Key differences in established model systems have also led to controversy surrounding the role of viral gene products in latency establishment, maintenance, and reactivation. This review will discuss the details and challenges of various models including hematopoietic progenitor cells, monocytes, cell lines, and humanized mice. We highlight the utility and functional differences between these models and the necessary experimental design required to define latency and reactivation, which will help to generate a more complete picture of HCMV infection of myeloid-lineage cells.
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36
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Ahn R, Schaenman J, Qian Z, Pickering H, Groysberg V, Rossetti M, Llamas M, Hoffmann A, Gjertson D, Deng M, Bunnapradist S, Reed EF. Acute and Chronic Changes in Gene Expression After CMV DNAemia in Kidney Transplant Recipients. Front Immunol 2021; 12:750659. [PMID: 34867983 PMCID: PMC8634678 DOI: 10.3389/fimmu.2021.750659] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2021] [Accepted: 10/20/2021] [Indexed: 12/13/2022] Open
Abstract
Cytomegalovirus (CMV) viremia continues to cause significant morbidity and mortality in kidney transplant patients with clinical complications including organ rejection and death. Whole blood gene expression dynamics in CMV viremic patients from onset of DNAemia through convalescence has not been well studied to date in humans. To evaluate how CMV infection impacts whole blood leukocyte gene expression over time, we evaluated a matched cohort of 62 kidney transplant recipients with and without CMV DNAemia using blood samples collected at multiple time points during the 12-month period after transplant. While transcriptomic differences were minimal at baseline between DNAemic and non-DNAemic patients, hundreds of genes were differentially expressed at the long-term timepoint, including genes enriching for pathways important for macrophages, interferon, and IL-8 signaling. Amongst patients with CMV DNAemia, the greatest amount of transcriptomic change occurred between baseline and 1-week post-DNAemia, with increase in pathways for interferon signaling and cytotoxic T cell function. Time-course gene set analysis of these differentially expressed genes revealed that most of the enriched pathways had a significant time-trend. While many pathways that were significantly down- or upregulated at 1 week returned to baseline-like levels, we noted that several pathways important in adaptive and innate cell function remained upregulated at the long-term timepoint after resolution of CMV DNAemia. Differential expression analysis and time-course gene set analysis revealed the dynamics of genes and pathways involved in the immune response to CMV DNAemia in kidney transplant patients. Understanding transcriptional changes caused by CMV DNAemia may identify the mechanism behind patient vulnerability to CMV reactivation and increased risk of rejection in transplant recipients and suggest protective strategies to counter the negative immunologic impact of CMV. These findings provide a framework to identify immune correlates for risk assessment and guiding need for extending antiviral prophylaxis.
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Affiliation(s)
- Richard Ahn
- Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, CA, United States.,Institute for Quantitative and Computational Biosciences, University of California Los Angeles, Los Angeles, CA, United States
| | - Joanna Schaenman
- Department of Medicine, University of California Los Angeles, Los Angeles, CA, United States
| | - Zachary Qian
- Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, CA, United States.,Institute for Quantitative and Computational Biosciences, University of California Los Angeles, Los Angeles, CA, United States
| | - Harry Pickering
- Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, CA, United States
| | - Victoria Groysberg
- Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, CA, United States
| | - Maura Rossetti
- Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, CA, United States
| | - Megan Llamas
- Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, CA, United States
| | - Alexander Hoffmann
- Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, CA, United States.,Institute for Quantitative and Computational Biosciences, University of California Los Angeles, Los Angeles, CA, United States
| | - David Gjertson
- Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, CA, United States.,Department of Biostatistics, University of California Los Angeles, Los Angeles, CA, United States
| | - Mario Deng
- Department of Medicine, University of California Los Angeles, Los Angeles, CA, United States
| | - Suphamai Bunnapradist
- Department of Medicine, University of California Los Angeles, Los Angeles, CA, United States
| | - Elaine F Reed
- Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, CA, United States
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Hale AE, Moorman NJ. The Ends Dictate the Means: Promoter Switching in Herpesvirus Gene Expression. Annu Rev Virol 2021; 8:201-218. [PMID: 34129370 DOI: 10.1146/annurev-virology-091919-072841] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Herpesvirus gene expression is dynamic and complex, with distinct complements of viral genes expressed at specific times in different infection contexts. These complex patterns of viral gene expression arise in part from the integration of multiple cellular and viral signals that affect the transcription of viral genes. The use of alternative promoters provides an increased level of control, allowing different promoters to direct the transcription of the same gene in response to distinct temporal and contextual cues. While once considered rare, herpesvirus alternative promoter usage was recently found to be far more pervasive and impactful than previously thought. Here we review several examples of promoter switching in herpesviruses and discuss the functional consequences on the transcriptional and post-transcriptional regulation of viral gene expression.
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Affiliation(s)
- Andrew E Hale
- Department of Microbiology and Immunology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA;
| | - Nathaniel J Moorman
- Department of Microbiology and Immunology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA;
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38
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Abstract
Despite the prevalence and medical significance of human cytomegalovirus (HCMV) infections, a systematic analysis of the targets of T cell recognition in humans that spans the entire genome and includes recently described potential novel ORFs is not available. Here, we screened a library of epitopes predicted to bind HLA class II that spans over 350 different HCMV ORFs and includes ∼150 previously described and ∼200 recently described potential novel ORFs using an ex vivo IFNγ fluorospot assay. We identified 235 unique HCMV specific epitopes derived from 100 ORFs, some previously described as immunodominant and others that were not previously described to be immunogenic. Of those, 41 belong to the set of recently reported novel ORFs, thus providing evidence that at least some of these are actually expressed in vivo in humans. These data reveal that the breadth of the human T cell response to HCMV is much greater than previously thought. The ORFs and epitopes identified will help elucidate how T cell immunity relates to HCMV pathogenesis and instruct ongoing HCMV vaccine research. Importance To understand the crucial role of adaptive immunity in controlling cytomegalovirus infection and disease, we systematically analyzed the CMV 'ORFeome' to identify new CMV epitopes targeted primarily by CD4 T cells in humans. Our study identified >200 new T cell epitopes derived from both canonical and novel ORFs, highlighting the substantial breadth of anti-CMV T cell response and providing new targets for vaccine design.
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Evasion of the Host Immune Response by Betaherpesviruses. Int J Mol Sci 2021; 22:ijms22147503. [PMID: 34299120 PMCID: PMC8306455 DOI: 10.3390/ijms22147503] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2021] [Revised: 07/11/2021] [Accepted: 07/12/2021] [Indexed: 02/07/2023] Open
Abstract
The human immune system boasts a diverse array of strategies for recognizing and eradicating invading pathogens. Human betaherpesviruses, a highly prevalent subfamily of viruses, include human cytomegalovirus (HCMV), human herpesvirus (HHV) 6A, HHV-6B, and HHV-7. These viruses have evolved numerous mechanisms for evading the host response. In this review, we will highlight the complex interplay between betaherpesviruses and the human immune response, focusing on protein function. We will explore methods by which the immune system first responds to betaherpesvirus infection as well as mechanisms by which viruses subvert normal cellular functions to evade the immune system and facilitate viral latency, persistence, and reactivation. Lastly, we will briefly discuss recent advances in vaccine technology targeting betaherpesviruses. This review aims to further elucidate the dynamic interactions between betaherpesviruses and the human immune system.
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40
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Poole EL, Nevels MM. Editorial: Cytomegalovirus Pathogenesis and Host Interactions. Front Cell Infect Microbiol 2021; 11:711551. [PMID: 34307201 PMCID: PMC8293988 DOI: 10.3389/fcimb.2021.711551] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2021] [Accepted: 06/24/2021] [Indexed: 12/02/2022] Open
Affiliation(s)
- Emma L. Poole
- Division of Infectious Diseases, Department of Medicine, University of Cambridge, Cambridge, United Kingdom
| | - Michael M. Nevels
- Biomedical Sciences Research Complex, School of Biology, University of St Andrews, St Andrews, United Kingdom
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41
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Krstanović F, Britt WJ, Jonjić S, Brizić I. Cytomegalovirus Infection and Inflammation in Developing Brain. Viruses 2021; 13:1078. [PMID: 34200083 PMCID: PMC8227981 DOI: 10.3390/v13061078] [Citation(s) in RCA: 44] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2021] [Revised: 06/01/2021] [Accepted: 06/03/2021] [Indexed: 02/06/2023] Open
Abstract
Human cytomegalovirus (HCMV) is a highly prevalent herpesvirus that can cause severe disease in immunocompromised individuals and immunologically immature fetuses and newborns. Most infected newborns are able to resolve the infection without developing sequelae. However, in severe cases, congenital HCMV infection can result in life-threatening pathologies and permanent damage of organ systems that possess a low regenerative capacity. Despite the severity of the problem, HCMV infection of the central nervous system (CNS) remains inadequately characterized to date. Cytomegaloviruses (CMVs) show strict species specificity, limiting the use of HCMV in experimental animals. Infection following intraperitoneal administration of mouse cytomegalovirus (MCMV) into newborn mice efficiently recapitulates many aspects of congenital HCMV infection in CNS. Upon entering the CNS, CMV targets all resident brain cells, consequently leading to the development of widespread histopathology and inflammation. Effector functions from both resident cells and infiltrating immune cells efficiently resolve acute MCMV infection in the CNS. However, host-mediated inflammatory factors can also mediate the development of immunopathologies during CMV infection of the brain. Here, we provide an overview of the cytomegalovirus infection in the brain, local immune response to infection, and mechanisms leading to CNS sequelae.
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Affiliation(s)
- Fran Krstanović
- Center for Proteomics and Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, 51000 Rijeka, Croatia; (F.K.); (S.J.)
| | - William J. Britt
- Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL 35294, USA;
| | - Stipan Jonjić
- Center for Proteomics and Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, 51000 Rijeka, Croatia; (F.K.); (S.J.)
| | - Ilija Brizić
- Center for Proteomics and Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, 51000 Rijeka, Croatia; (F.K.); (S.J.)
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Lee BJ, Min CK, Hancock M, Streblow DN, Caposio P, Goodrum FD, Yurochko AD. Human Cytomegalovirus Host Interactions: EGFR and Host Cell Signaling Is a Point of Convergence Between Viral Infection and Functional Changes in Infected Cells. Front Microbiol 2021; 12:660901. [PMID: 34025614 PMCID: PMC8138183 DOI: 10.3389/fmicb.2021.660901] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2021] [Accepted: 04/07/2021] [Indexed: 12/22/2022] Open
Abstract
Viruses have evolved diverse strategies to manipulate cellular signaling pathways in order to promote infection and/or persistence. Human cytomegalovirus (HCMV) possesses a number of unique properties that allow the virus to alter cellular events required for infection of a diverse array of host cell types and long-term persistence. Of specific importance is infection of bone marrow derived and myeloid lineage cells, such as peripheral blood monocytes and CD34+ hematopoietic progenitor cells (HPCs) because of their essential role in dissemination of the virus and for the establishment of latency. Viral induced signaling through the Epidermal Growth Factor Receptor (EGFR) and other receptors such as integrins are key control points for viral-induced cellular changes and productive and latent infection in host organ systems. This review will explore the current understanding of HCMV strategies utilized to hijack cellular signaling pathways, such as EGFR, to promote the wide-spread dissemination and the classic life-long herpesvirus persistence.
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Affiliation(s)
- Byeong-Jae Lee
- Department of Microbiology & Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA, United States.,Center for Applied Immunology and Pathological Processes, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA, United States.,Center of Excellence for Emerging Viral Threats, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA, United States
| | - Chan-Ki Min
- Department of Microbiology & Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA, United States.,Center for Applied Immunology and Pathological Processes, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA, United States.,Center of Excellence for Emerging Viral Threats, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA, United States
| | - Meaghan Hancock
- Vaccine and Gene Therapy Institute, Oregon Health & Science University, Beaverton, OR, United States
| | - Daniel N Streblow
- Vaccine and Gene Therapy Institute, Oregon Health & Science University, Beaverton, OR, United States
| | - Patrizia Caposio
- Vaccine and Gene Therapy Institute, Oregon Health & Science University, Beaverton, OR, United States
| | | | - Andrew D Yurochko
- Department of Microbiology & Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA, United States.,Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA, United States.,Center for Cardiovascular Diseases and Sciences, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA, United States.,Center of Excellence in Arthritis and Rheumatology, Louisiana State University Health Sciences Center Shreveport, Shreveport, LA, United States
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43
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Perera MR, Wills MR, Sinclair JH. HCMV Antivirals and Strategies to Target the Latent Reservoir. Viruses 2021; 13:817. [PMID: 34062863 PMCID: PMC8147263 DOI: 10.3390/v13050817] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2021] [Revised: 04/26/2021] [Accepted: 04/28/2021] [Indexed: 12/11/2022] Open
Abstract
Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus. In healthy people, primary infection is generally asymptomatic, and the virus can go on to establish lifelong latency in cells of the myeloid lineage. However, HCMV often causes severe disease in the immunosuppressed: transplant recipients and people living with AIDS, and also in the immunonaive foetus. At present, there are several antiviral drugs licensed to control HCMV disease. However, these are all faced with problems of poor bioavailability, toxicity and rapidly emerging viral resistance. Furthermore, none of them are capable of fully clearing the virus from the host, as they do not target latent infection. Consequently, reactivation from latency is a significant source of disease, and there remains an unmet need for treatments that also target latent infection. This review briefly summarises the most common HCMV antivirals used in clinic at present and discusses current research into targeting the latent HCMV reservoir.
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Affiliation(s)
| | | | - John H. Sinclair
- Department of Medicine, Cambridge Institute of Therapeutic Immunology and Infectious Disease, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 0QQ, UK; (M.R.P.); (M.R.W.)
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Ramendra R, Isnard S, Lin J, Fombuena B, Ouyang J, Mehraj V, Zhang Y, Finkelman M, Costiniuk C, Lebouché B, Chartrand-Lefebvre C, Durand M, Tremblay C, Ancuta P, Boivin G, Routy JP. Cytomegalovirus Seropositivity Is Associated With Increased Microbial Translocation in People Living With Human Immunodeficiency Virus and Uninfected Controls. Clin Infect Dis 2021; 71:1438-1446. [PMID: 31608409 PMCID: PMC7486843 DOI: 10.1093/cid/ciz1001] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2019] [Accepted: 10/07/2019] [Indexed: 12/11/2022] Open
Abstract
Background Cytomegalovirus (CMV) seropositivity and anti-CMV immunoglobulin G (IgG) levels are associated with adverse health outcomes in elderly populations. Among people living with human immunodeficiency virus (PLWH), CMV seropositivity has been associated with persistent CD8 T-cell elevation and increased risk of developing non-AIDS comorbidities despite long-term antiretroviral therapy (ART). Herein, we investigated whether CMV seropositivity and elevation of anti-CMV IgG levels were associated with increased epithelial gut damage, microbial translocation, and systemic inflammation. Methods A total of 150 PLWH (79 ART-naive and 71 ART-treated) were compared to 26 without human immunodeficiency virus (HIV) infection (uninfected controls). Plasma markers of HIV disease progression, epithelial gut damage, microbial translocation, nonspecific B-cell activation, anti-CMV and anti–Epstein-Barr virus (EBV) IgG levels, and proinflammatory cytokines were measured. Results CMV seropositivity and elevated anti-CMV IgG levels were associated with markers of epithelial gut damage, microbial translocation, and inflammation in PLWH and participants without HIV infection. In contrast, total nonspecific IgG, immunoglobulin M, immunoglobulin A, and anti-EBV IgG levels were not associated with these markers. CMV seropositivity was associated with markers of epithelial gut damage, microbial translocation, and inflammation independent of sociodemographic and behavioral characteristics of the study population. Conclusions CMV-seropositive people with and without HIV had increased epithelial gut damage, microbial translocation, and inflammation. Furthermore, anti-CMV IgG levels were independently associated with increased epithelial gut damage and microbial translocation. CMV coinfection may partially explain persistent gut damage, microbial translocation, and inflammation in ART-treated PLWH.
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Affiliation(s)
- Rayoun Ramendra
- Chronic Viral Illness Service, McGill University Health Centre, Montreal, Quebec, Canada.,Infectious Diseases and Immunity in Global Health Program, Research Institute, McGill University Health Centre, Montreal, Quebec, Canada.,Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada
| | - Stéphane Isnard
- Chronic Viral Illness Service, McGill University Health Centre, Montreal, Quebec, Canada.,Infectious Diseases and Immunity in Global Health Program, Research Institute, McGill University Health Centre, Montreal, Quebec, Canada
| | - John Lin
- Chronic Viral Illness Service, McGill University Health Centre, Montreal, Quebec, Canada.,Infectious Diseases and Immunity in Global Health Program, Research Institute, McGill University Health Centre, Montreal, Quebec, Canada
| | - Brandon Fombuena
- Chronic Viral Illness Service, McGill University Health Centre, Montreal, Quebec, Canada.,Infectious Diseases and Immunity in Global Health Program, Research Institute, McGill University Health Centre, Montreal, Quebec, Canada.,Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada
| | - Jing Ouyang
- Chronic Viral Illness Service, McGill University Health Centre, Montreal, Quebec, Canada.,Infectious Diseases and Immunity in Global Health Program, Research Institute, McGill University Health Centre, Montreal, Quebec, Canada.,Chongqing Public Health Medical Center, Chongqing, China
| | - Vikram Mehraj
- Chronic Viral Illness Service, McGill University Health Centre, Montreal, Quebec, Canada.,Infectious Diseases and Immunity in Global Health Program, Research Institute, McGill University Health Centre, Montreal, Quebec, Canada.,Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montréal, Quebec, Canada
| | - Yonglong Zhang
- Associates of Cape Cod Inc, Falmouth, Massachusetts, USA
| | | | - Cecilia Costiniuk
- Chronic Viral Illness Service, McGill University Health Centre, Montreal, Quebec, Canada.,Infectious Diseases and Immunity in Global Health Program, Research Institute, McGill University Health Centre, Montreal, Quebec, Canada
| | - Bertrand Lebouché
- Chronic Viral Illness Service, McGill University Health Centre, Montreal, Quebec, Canada.,Infectious Diseases and Immunity in Global Health Program, Research Institute, McGill University Health Centre, Montreal, Quebec, Canada.,Department of Family Medicine, McGill University, Montreal, Quebec, Canada
| | - Carl Chartrand-Lefebvre
- Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montréal, Quebec, Canada
| | - Madeleine Durand
- Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montréal, Quebec, Canada
| | - Cécile Tremblay
- Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.,Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montréal, Quebec, Canada
| | - Petronela Ancuta
- Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.,Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montréal, Quebec, Canada
| | - Guy Boivin
- Department of Microbiology-Immunology and Infectious Diseases, Laval University, Quebec City, Quebec, Canada
| | - Jean-Pierre Routy
- Chronic Viral Illness Service, McGill University Health Centre, Montreal, Quebec, Canada.,Infectious Diseases and Immunity in Global Health Program, Research Institute, McGill University Health Centre, Montreal, Quebec, Canada.,Division of Hematology, McGill University Health Centre, Montreal, Quebec, Canada
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Vezzani G, Amendola D, Yu D, Chandramouli S, Frigimelica E, Maione D, Merola M. The Human Cytomegalovirus UL116 Glycoprotein Is a Chaperone to Control gH-Based Complexes Levels on Virions. Front Microbiol 2021; 12:630121. [PMID: 33889136 PMCID: PMC8056026 DOI: 10.3389/fmicb.2021.630121] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2020] [Accepted: 02/22/2021] [Indexed: 01/14/2023] Open
Abstract
Human cytomegalovirus (HCMV) relies in large part upon the viral membrane fusion glycoprotein B and two alternative gH/gL complexes, gH/gL/gO (Trimer) and gH/gL/UL128/UL130/UL131A (Pentamer) to enter into cells. The relative amounts of Trimer and Pentamer vary among HCMV strains and contribute to differences in cell tropism. Although the viral ER resident protein UL148 has been shown to interact with gH to facilitate gO incorporation, the mechanisms that favor the assembly and maturation of one complex over another remain poorly understood. HCMV virions also contain an alternative non-disulfide bound heterodimer comprised of gH and UL116 whose function remains unknown. Here, we show that disruption of HCMV gene UL116 causes infectivity defects of ∼10-fold relative to wild-type virus and leads to reduced expression of both gH/gL complexes in virions. Furthermore, gH that is not covalently bound to other viral glycoproteins, which are readily detected in wild-type HCMV virions, become undetectable in the absence of UL116 suggesting that the gH/UL116 complex is abundant in virions. We find evidence that UL116 and UL148 interact during infection indicating that the two proteins might cooperate to regulate the abundance of HCMV gH complexes. Altogether, these results are consistent with a role of UL116 as a chaperone for gH during the assembly and maturation of gH complexes in infected cells.
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Affiliation(s)
- Giacomo Vezzani
- GSK, Siena, Italy.,Department of Pharmacy and Biotechnology (FABIT), University of Bologna, Bologna, Italy
| | | | - Dong Yu
- GSK, Rockville, MD, United States
| | | | | | | | - Marcello Merola
- GSK, Siena, Italy.,Department of Biology, University of Naples Federico II, Naples, Italy
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Diggins NL, Crawford LB, Hancock MH, Mitchell J, Nelson JA. Human Cytomegalovirus miR-US25-1 Targets the GTPase RhoA To Inhibit CD34 + Hematopoietic Progenitor Cell Proliferation To Maintain the Latent Viral Genome. mBio 2021; 12:e00621-21. [PMID: 33824207 PMCID: PMC8092260 DOI: 10.1128/mbio.00621-21] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2021] [Accepted: 03/09/2021] [Indexed: 12/25/2022] Open
Abstract
Human cytomegalovirus (HCMV) microRNAs play essential roles in latency and reactivation in CD34+ hematopoietic progenitor cells (HPCs) via regulation of viral and cellular gene expression. In the present study, we show that HCMV miR-US25-1 targets RhoA, a small GTPase required for CD34+ HPC self-renewal, proliferation, and hematopoiesis. Expression of miR-US25-1 impairs signaling through the nonmuscle myosin II light chain, which leads to a block in cytokinesis and an inhibition of proliferation. Moreover, infection with an HCMV mutant lacking miR-US25-1 resulted in increased proliferation of CD34+ HPCs and a decrease in the proportion of genome-containing cells at the end of latency culture. These observations provide a mechanism by which HCMV limits proliferation to maintain latent viral genomes in CD34+ HPCs.IMPORTANCE Each herpesvirus family establishes latency in a unique cell type. Since herpesvirus genomes are maintained as episomes, the virus needs to devise mechanisms to retain the latent genome during cell division. Alphaherpesviruses overcome this obstacle by infecting nondividing neurons, while gammaherpesviruses tether their genome to the host chromosome in dividing B cells. The betaherpesvirus human cytomegalovirus (HCMV) establishes latency in CD34+ hematopoietic progenitor cells (HPCs), but the mechanism used to maintain the viral genome is unknown. In this report, we demonstrate that HCMV miR-US25-1 downregulates expression of RhoA, a key cell cycle regulator, which results in inhibition of CD34+ HPC proliferation by blocking mitosis. Mutation of miR-US25-1 during viral infection results in enhanced cellular proliferation and a decreased frequency of genome-containing CD34+ HPCs. These results reveal a novel mechanism through which HCMV is able to regulate cell division to prevent viral genome loss during proliferation.
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Affiliation(s)
- Nicole L Diggins
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon, USA
| | - Lindsey B Crawford
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon, USA
| | - Meaghan H Hancock
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon, USA
| | - Jennifer Mitchell
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon, USA
| | - Jay A Nelson
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon, USA
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Abstract
Herpesviruses such as herpes simplex virus (HSV) type 1 and 2, varicella-zoster virus (VZV), and cytomegalovirus (CMV) maintain lifelong latency in the host after primary infection and can reactivate periodically either as asymptomatic viral shedding or as clinical disease. Immunosuppression, including biologic therapy, may increase frequency and severity of herpesvirus reactivation and infection. Licensed biologics are reviewed regarding their risks of potentiating HSV, VZV, and CMV reactivation and infection. Approaches to prophylaxis against HSV, VZV, and CMV infection or reactivation are discussed.
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Affiliation(s)
- Dora Y Ho
- Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Lane Building L-135, Stanford, CA 94305-5107, USA.
| | - Kyle Enriquez
- Stanford University, 450 Serra Mall, Stanford, CA 94305, USA
| | - Ashrit Multani
- Division of Infectious Diseases, Department of Medicine, David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue CHS 37-121, Los Angeles, CA 90095-1688, USA
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Chen YC. CRISPR based genome editing and removal of human viruses. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2021; 179:93-116. [PMID: 33785179 DOI: 10.1016/bs.pmbts.2020.12.014] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins 9 (Cas9), a gene-editing technology, has been extensively applied as a tool for genetic engineering in basic research. Efficient genome engineering has been performed in viruses, human cells, bacteria, fungi, plants and animals, etc. Currently, it has been employed to edit human viruses for studying viral molecular biology, pathogenesis and oncogenesis, and facilitate the development of antiviral agents and vaccine. The virus is ubiquitous worldwide and elicits global health problems, many human diseases are associated with virus infections. Although traditional drugs can be used to treat or prevent productive viral infections, their efficacy is limited because of toxicity, side effects and other problems. Additionally, no current drugs are approved to be indicated for latent infections. Therefore, the next highlight is to develop antiviral approaches to against both productive and latent infections. Fortunately, CRISPR has been successfully applied in the removal of human viruses ex vivo and/or in vivo, and has the potential to be used to manufacture antiviral agents for clinical application. CRISPR/Cas9 is promising in applications, even though some technical challenges, safety concerns, ethic concerns need to be improved. In this article, the discovery and application of genome editing and removal of human viruses based on CRISPR are explored. Additionally, we evaluate the prospects and limitations of this novel antiviral strategies.
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Affiliation(s)
- Yuan-Chuan Chen
- Jenteh Junior College of Medicine, Nursing and Management, Miaoli County, Taiwan; Program in Comparative Biochemistry, University of California, Berkeley, CA, United States.
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Krishna BA, Wass AB, Dooley AL, O'Connor CM. CMV-encoded GPCR pUL33 activates CREB and facilitates its recruitment to the MIE locus for efficient viral reactivation. J Cell Sci 2021; 134:jcs254268. [PMID: 33199520 PMCID: PMC7860128 DOI: 10.1242/jcs.254268] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2020] [Accepted: 11/02/2020] [Indexed: 12/12/2022] Open
Abstract
Human cytomegalovirus (HCMV) establishes life-long latent infection in hematopoietic progenitor cells and circulating monocytes in infected individuals. Myeloid differentiation coupled with immune dysregulation leads to viral reactivation, which can cause severe disease and mortality. Reactivation of latent virus requires chromatin reorganization and the removal of transcriptional repressors in exchange for transcriptional activators. While some factors involved in these processes are identified, a complete characterization of the viral and cellular factors involved in their upstream regulation remains elusive. Herein, we show the HCMV-encoded G protein-coupled receptor (GPCR), UL33, is expressed during latency. Although this viral GPCR is not required to maintain latent infection, our data reveal UL33-mediated signaling is important for efficient viral reactivation. Additionally, UL33 signaling induces cellular cyclic AMP response element binding protein (CREB1, referred to here as CREB) phosphorylation, a transcription factor that promotes reactivation when recruited to the major immediate early (MIE) enhancer/promoter. Finally, targeted pharmacological inhibition of CREB activity reverses the reactivation phenotype of the UL33 signaling-deficient mutant. In sum, our data reveal UL33-mediated signaling functions to activate CREB, resulting in successful viral reactivation.
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Affiliation(s)
- Benjamin A Krishna
- Genomic Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
| | - Amanda B Wass
- Genomic Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
| | - Abigail L Dooley
- Genomic Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
| | - Christine M O'Connor
- Genomic Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
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CD34 + Hematopoietic Progenitor Cell Subsets Exhibit Differential Ability To Maintain Human Cytomegalovirus Latency and Persistence. J Virol 2021; 95:JVI.02105-20. [PMID: 33177198 DOI: 10.1128/jvi.02105-20] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2020] [Accepted: 11/02/2020] [Indexed: 11/20/2022] Open
Abstract
In human cytomegalovirus (HCMV)-seropositive patients, CD34+ hematopoietic progenitor cells (HPCs) provide an important source of latent virus that reactivates following cellular differentiation into tissue macrophages. Multiple groups have used primary CD34+ HPCs to investigate mechanisms of viral latency. However, analyses of mechanisms of HCMV latency have been hampered by the genetic variability of CD34+ HPCs from different donors, availability of cells, and low frequency of reactivation. In addition, multiple progenitor cell types express surface CD34, and the frequencies of these populations differ depending on the tissue source of the cells and culture conditions in vitro In this study, we generated CD34+ progenitor cells from two different embryonic stem cell (ESC) lines, WA01 and WA09, to circumvent limitations associated with primary CD34+ HPCs. HCMV infection of CD34+ HPCs derived from either WA01 or WA09 ESCs supported HCMV latency and induced myelosuppression similar to infection of primary CD34+ HPCs. Analysis of HCMV-infected primary or ESC-derived CD34+ HPC subpopulations indicated that HCMV was able to establish latency and reactivate in CD38+ CD90+ and CD38+/low CD90- HPCs but persistently infected CD38- CD90+ cells to produce infectious virus. These results indicate that ESC-derived CD34+ HPCs can be used as a model for HCMV latency and that the virus either latently or persistently infects specific subpopulations of CD34+ cells.IMPORTANCE Human cytomegalovirus infection is associated with severe disease in transplant patients and understanding how latency and reactivation occur in stem cell populations is essential to understand disease. CD34+ hematopoietic progenitor cells (HPCs) are a critical viral reservoir; however, these cells are a heterogeneous pool with donor-to-donor variation in functional, genetic, and phenotypic characteristics. We generated a novel system using embryonic stem cell lines to model HCMV latency and reactivation in HPCs with a consistent cellular background. Our study defined three key stem cell subsets with differentially regulated latent and replicative states, which provide cellular candidates for isolation and treatment of transplant-mediated disease. This work provides a direction toward developing strategies to control the switch between latency and reactivation.
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