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Fan TJ, Xie C, Li L, Jin X, Cui J, Wang JH. HIV-1 Nef activates proviral DNA transcription by recruiting Src kinase to phosphorylate host protein Nef-associated factor 1 to compromise its viral restrictive function. J Virol 2025; 99:e0028025. [PMID: 40272155 DOI: 10.1128/jvi.00280-25] [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: 03/10/2025] [Accepted: 03/25/2025] [Indexed: 04/25/2025] Open
Abstract
HIV-1 accessory protein Nef is a multifunctional pathogenic factor that mediates immune evasion, enhances virion infectivity, antagonizes host restrictive factors, and promotes viral dissemination. However, the modulation of Nef on proviral DNA transcription of latently infected viruses is not well understood. In this study, we found that Nef activated HIV-1 proviral DNA transcription by recruiting Src Family Kinases (SFKs) member Src to stimulate the downstream PI3K/AKT/mTOCR1/CDK9 cellular pathway, and that Naf1 (Nef-associated factor 1), a host protein that is known to suppress HIV-1 transcription, was required for this function of Nef. This seemingly contradictory interplay between Nef and Naf1 was investigated. Naf1 was a repressor of the PI3K/AKT/mTOCR1/CDK9 cellular pathway, but in the presence of Nef, Naf1 was phosphorylated at the Tyrosine-552 by Nef-recruited Src, consequently converting its normal restrictive role to coordinate with Nef to activate proviral DNA transcription. These findings reveal a mechanism by which Nef activates HIV-1 proviral DNA transcription and discover the dual function of Naf1 protein in regulating HIV infection, depending on its phosphorylation status. This study reports a new interaction mode between host factors and viral proteins in regulating HIV-1 replication. IMPORTANCE HIV-1 accessory protein Nef is a multifunctional pathogenic factor; however, the modulation of Nef on proviral DNA transcription of latently infected virus is not well understood. This study demonstrates Nef's role in activating HIV-1 proviral DNA transcription and uncovers the underlying cellular mechanism. Nef recruits Src kinase to phosphorylate Naf1, and the phosphorylation of Naf1 converts its normal restrictive role to coordinate with Nef to activate proviral DNA transcription by stimulating the downstream PI3K/AKT/mTOCR1/CDK9 cellular pathway. These findings also report a new interaction mode between host factors and viral proteins in regulating HIV-1 replication.
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Affiliation(s)
- Tian-Jiao Fan
- Pingyuan Laboratory, State Key Laboratory of Antiviral Drugs, Henan Normal University, Xinxiang, China
- Shanghai Sci-Tech Inno Center for Infection & Immunity, Shanghai, China
| | - Chengzuo Xie
- Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
- Graduate School of Guangzhou Medical University, Guangzhou, China
| | - Lisha Li
- Pingyuan Laboratory, State Key Laboratory of Antiviral Drugs, Henan Normal University, Xinxiang, China
| | - Xia Jin
- Pingyuan Laboratory, State Key Laboratory of Antiviral Drugs, Henan Normal University, Xinxiang, China
| | - Jie Cui
- Shanghai Sci-Tech Inno Center for Infection & Immunity, Shanghai, China
| | - Jian-Hua Wang
- Pingyuan Laboratory, State Key Laboratory of Antiviral Drugs, Henan Normal University, Xinxiang, China
- Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
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Mumby MJ, Prodger JL, Hackman J, Saraf S, Zhu X, Ferreira RC, Tomusange S, Jamiru S, Anok A, Kityamuweesi T, Buule P, Fink C, Edgar CR, Trothen SM, Dekaban GA, Brown EE, Capoferri AA, Baker OR, Klock E, Miller JC, Kirby C, Lynch B, Tobian AAR, Poon AFY, Quinn TC, Galiwango RM, Reynolds SJ, Redd AD, Dikeakos JD. Association between HIV-1 Nef-mediated MHC-I downregulation and the maintenance of the replication-competent latent viral reservoir in individuals with virally suppressed HIV-1 in Uganda: an exploratory cohort study. THE LANCET. MICROBE 2025; 6:101018. [PMID: 40088911 PMCID: PMC12066219 DOI: 10.1016/j.lanmic.2024.101018] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/22/2024] [Revised: 09/24/2024] [Accepted: 10/10/2024] [Indexed: 03/17/2025]
Abstract
BACKGROUND The persistence of a replication-competent latent viral reservoir (RC-LVR) during antiretroviral therapy (ART) is a barrier to the development of a cure for HIV-1, but the role of viral genes in influencing RC-LVR size is unclear. We aimed to assess whether the magnitude by which the HIV-1 accessory protein Nef evades the adaptive immune response by downregulating MHC-I or CD4, or both, from the surface of infected cells is associated with the rate at which the RC-LVR in people with HIV-1 changes during long-term ART (>1 year). METHODS We conducted an exploratory cohort study in which nef genes were sequenced from outgrowth viruses derived from the quantitative viral outgrowth assay (QVOA) for a group of people with ART-suppressed HIV-1 in Uganda between 2015 and 2020. Study participants were selected from the Rakai Health Sciences Program (RHSP) LVR cohort, a cohort of 90 adults (aged ≥18 years) who were HIV-1 positive, receiving ART, and had maintained viral suppression for at least 1 year at the time of study enrolment. For this study, participants were required to have available p24+ QVOA wells that contained a single viral outgrowth isolate, as assessed by next-generation sequencing. In cases where further sequencing identified wells containing multiple viral clones, all sequenced nef variants were included for functional analysis. The unique isolated nef variants were used to generate pseudoviruses, which were employed to measure cell surface CD4 and MHC-I downregulation in infected CD4+ Sup-T1 cells via flow cytometry. The size and rate of change of the RC-LVR in participants was estimated using previous QVOA results and a Bayesian model. We then assessed whether a correlation existed between the extent to which the Nef proteins downregulated cell surface MHC-I and CD4 and the calculated RC-LVR rate of change during the study period. FINDINGS 14 (15%) of 90 participants from the RHSP cohort met the inclusion criteria and were enrolled in this study. 49 nef sequences were isolated from these participants. We observed variability in participant-derived Nef-mediated cell surface MHC-I downregulation (median 114·88% [IQR 104·93-121·51] of the downregulation capacity of NL4-3 Nef) and CD4 downregulation (94·50% [84·05-100·16] of NL4-3 Nef). The estimated rate of change of the RC-LVR was positive for four participants. For one donor, the rate of change was significantly positive (7·4 × 10-4 logit infectious units per million [IUPM] per day [95% credibility interval 3·2 × 10-4 to 1·2 × 10-3]) over the course of the study period (2015-20). The estimated rate of change of the RC-LVR for the remaining ten participants was negative, and significantly negative in four donors (-1·1 × 10-3 logit IUPM per day [95% credibility interval -1·8 × 10-3 to -3·7 × 10-4]; -1·4 × 10-3 [-2·0 × 10-3 to -8·5 × 10-4]; -7·0 × 10-4 [-1·3 × 10-3 to -1·6 × 10-4]; and -2·0 × 10-3 [-2·9 × 10-3 to -1·1 × 10-3]). A significant relationship between Nef-mediated MHC-I downregulation and the RC-LVR rate of change during the 5-year study period (r=0·6088 [95% CI 0·2366 to 0·9810]; p=0·023) was found, in which less efficient MHC-I downregulation correlated with faster RC-LVR decay during long-term ART. By contrast, Nef-mediated CD4 downregulation was not associated with RC-LVR rate of change during the 5-year study period (-0·1604 [-0·7311 to 0·4102]; p=0·58). INTERPRETATION Nef-mediated MHC-I downregulation might contribute to HIV-1 persistence during long-term ART. Strategies to inhibit Nef-mediated MHC-I downregulation could represent a viable therapeutic avenue to reduce the size of the latent reservoir in vivo, improving treatment outcomes in people with HIV-1. FUNDING Canadian Institutes of Health Research, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, and the REACH Martin Delaney Collaboratory.
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Affiliation(s)
- Mitchell J Mumby
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada
| | - Jessica L Prodger
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada
| | - Jada Hackman
- Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Baltimore, Maryland, USA
| | - Sharada Saraf
- Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Baltimore, Maryland, USA
| | - Xianming Zhu
- Department of Pathology, Johns Hopkins University, Baltimore, MA, USA
| | - Roux-Cil Ferreira
- Department of Pathology and Laboratory Medicine, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada
| | | | | | - Aggrey Anok
- Rakai Health Sciences Program, Kalisizo, Uganda
| | | | - Paul Buule
- Rakai Health Sciences Program, Kalisizo, Uganda
| | - Corby Fink
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada
| | - Cassandra R Edgar
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada
| | - Steven M Trothen
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada
| | - Gregory A Dekaban
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada
| | - Erin E Brown
- Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Baltimore, Maryland, USA
| | - Adam A Capoferri
- Department of Medicine, Johns Hopkins University, Baltimore, MA, USA
| | - Owen R Baker
- Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Baltimore, Maryland, USA
| | - Ethan Klock
- Department of Medicine, Johns Hopkins University, Baltimore, MA, USA
| | - Jernelle C Miller
- Department of Medicine, Johns Hopkins University, Baltimore, MA, USA
| | - Charles Kirby
- Department of Pathology, Johns Hopkins University, Baltimore, MA, USA
| | - Briana Lynch
- Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Baltimore, Maryland, USA
| | - Aaron A R Tobian
- Department of Pathology, Johns Hopkins University, Baltimore, MA, USA
| | - Art F Y Poon
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada; Department of Pathology and Laboratory Medicine, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada
| | - Thomas C Quinn
- Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Baltimore, Maryland, USA; Department of Medicine, Johns Hopkins University, Baltimore, MA, USA
| | | | - Steven J Reynolds
- Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Baltimore, Maryland, USA; Department of Medicine, Johns Hopkins University, Baltimore, MA, USA; Rakai Health Sciences Program, Kalisizo, Uganda
| | - Andrew D Redd
- Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Baltimore, Maryland, USA; Department of Medicine, Johns Hopkins University, Baltimore, MA, USA; Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa
| | - Jimmy D Dikeakos
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada.
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Kuse N, Noyori O, Takahashi N, Zhang Y, Suzu S, Takiguchi M. Recognition of HIV-1-infected fibrocytes lacking Nef-mediated HLA-B downregulation by HIV-1-specific T cells. J Virol 2024; 98:e0079124. [PMID: 38940584 PMCID: PMC11264601 DOI: 10.1128/jvi.00791-24] [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: 05/06/2024] [Accepted: 06/01/2024] [Indexed: 06/29/2024] Open
Abstract
Fibrocytes were reported to be host cells for HIV-1, but the immunological recognition of HIV-1-infected fibrocytes has not been studied. Here, we investigated the recognition of HIV-1-infected fibrocytes by HIV-1-specific CD8+ T cells. CD8+ T cells specific for five HIV-1 epitopes (HLA-A*24:02-restricted, HLA-B*52:01-restricted, and HLA-C*12:02-restricted epitopes) produced IFN-γ and expressed CD107a after coculture with HIV-1-infected fibrocytes. HIV-1-infected fibrocytes were effectively killed by HIV-1-specific CD8+ T cells. Although it is well known that HIV-1 Nef-mediated downregulation of HLA-A and HLA-B critically affects the T cell recognition of HIV-1-infected CD4+ T cells and HIV-1-infected macrophages, Nef downregulated HLA-A, but not HLA-B, in HIV-1-infected fibrocytes. These findings suggested that HIV-1-specific CD8+ T cells could recognize HIV-1-infected fibrocytes more strongly than HIV-1-infected CD4+ T cells or HIV-1-infected macrophages. HIV-1-infected fibrocytes were also recognized by HIV-1-specific HLA-DR-restricted T cells, indicating that HIV-1-infected fibrocytes can present HIV-1 epitopes to helper T cells. Collectively, these findings suggest that fibrocytes have an important role as antigen-presenting cells during HIV-1 infection. The present study demonstrates effective recognition of HIV-1-infected fibrocytes by HIV-1-specific T cells and suggests possible roles of fibrocytes in the induction and maintenance of HIV-1-specific T cells. IMPORTANCE Fibrocytes were identified as unique hematopoietic cells with the features of both macrophages and fibroblasts and were demonstrated to be host cells for HIV-1. However, T cell recognition of HIV-1-infected fibrocytes has not been studied. We investigated the recognition of HIV-1-infected fibrocytes by HIV-1-specific T cells. HIV-1-infected fibrocytes were effectively recognized and killed by CD8+ T cells specific for HIV-1 epitopes presented by HLA-A, HLA-B, or HLA-C and were recognized by HIV-1-specific HLA-DR-restricted CD4+ T cells. HIV-1 Nef-mediated downregulation of HLA-A and HLA-B was found in HIV-1-infected CD4+ T cells, whereas Nef did not downregulate HLA-B in HIV-1-infected fibrocytes. These results suggest that HIV-1-specific CD8+ T cells recognize HIV-1-infected fibrocytes more strongly than HIV-1-infected CD4+ T cells. The present study suggests the importance of fibrocytes in the induction and maintenance of HIV-1-specific T cells.
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Affiliation(s)
- Nozomi Kuse
- Division of International Collaboration Research, Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto, Japan
- AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan
| | - Osamu Noyori
- Division of Infection and Hematopoiesis, Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto, Japan
| | - Naofumi Takahashi
- Division of Infection and Hematopoiesis, Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto, Japan
| | - Yu Zhang
- Division of International Collaboration Research, Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto, Japan
| | - Shinya Suzu
- Division of Infection and Hematopoiesis, Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto, Japan
| | - Masafumi Takiguchi
- Division of International Collaboration Research, Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto, Japan
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4
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Ramirez PW, Vollbrecht T, Acosta FM, Suarez M, Angerstein AO, Wallace J, O' Connell RM, Guatelli J. Nef enhances HIV-1 replication and infectivity independently of SERINC5 in CEM T cells. Virology 2023; 578:154-162. [PMID: 36577173 PMCID: PMC10484624 DOI: 10.1016/j.virol.2022.12.008] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2022] [Revised: 12/14/2022] [Accepted: 12/15/2022] [Indexed: 12/25/2022]
Abstract
A primary function of HIV-1 Nef is the enhancement of viral infectivity and replication. Whether counteraction of the antiretroviral proteins SERINC3 and SERINC5 is the cause of this positive influence on viral growth-rate and infectivity remains unclear. Here, we utilized CRISPR/Cas9 to knockout SERINC3 and SERINC5 in a leukemic CD4-positive T cell line (CEM) that displays nef-related infectivity and growth-rate phenotypes. Viral replication was attenuated in CEM cells infected with HIV-1 lacking Nef (HIV-1ΔNef). This attenuated growth-rate phenotype was observed regardless of whether the coding regions of the serinc3 or serinc5 genes were intact. Moreover, knockout of serinc5 alone or of both serinc5 and serinc3 together failed to restore the infectivity of HIV1ΔNef virions produced from infected CEM cells. Our results corroborate a similar study using another T-lymphoid cell line (MOLT-3) and indicate that the antagonism of SERINC3 and SERINC5 does not fully explain the virology of HIV-1 lacking Nef.
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Affiliation(s)
- Peter W Ramirez
- Department of Biological Sciences, California State University Long Beach, Long Beach, CA, USA.
| | - Thomas Vollbrecht
- Department of Medicine, University of California San Diego, La Jolla, CA, USA; VA San Diego Healthcare System, San Diego, CA, USA
| | - Francisco M Acosta
- Department of Biological Sciences, California State University Long Beach, Long Beach, CA, USA
| | | | - Aaron O Angerstein
- Department of Medicine, University of California San Diego, La Jolla, CA, USA; VA San Diego Healthcare System, San Diego, CA, USA
| | - Jared Wallace
- Division of Microbiology and Immunology, Department of Pathology, The University of Utah, Salt Lake City, UT, USA
| | - Ryan M O' Connell
- Division of Microbiology and Immunology, Department of Pathology, The University of Utah, Salt Lake City, UT, USA
| | - John Guatelli
- Department of Medicine, University of California San Diego, La Jolla, CA, USA; VA San Diego Healthcare System, San Diego, CA, USA
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Emert-Sedlak LA, Shi H, Tice CM, Chen L, Alvarado JJ, Shu ST, Du S, Thomas CE, Wrobel JE, Reitz AB, Smithgall TE. Antiretroviral Drug Discovery Targeting the HIV-1 Nef Virulence Factor. Viruses 2022; 14:v14092025. [PMID: 36146831 PMCID: PMC9503669 DOI: 10.3390/v14092025] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2022] [Revised: 09/06/2022] [Accepted: 09/08/2022] [Indexed: 11/16/2022] Open
Abstract
While antiretroviral drugs have transformed the lives of HIV-infected individuals, chronic treatment is required to prevent rebound from viral reservoir cells. People living with HIV also are at higher risk for cardiovascular and neurocognitive complications, as well as cancer. Finding a cure for HIV-1 infection is therefore an essential goal of current AIDS research. This review is focused on the discovery of pharmacological inhibitors of the HIV-1 Nef accessory protein. Nef is well known to enhance HIV-1 infectivity and replication, and to promote immune escape of HIV-infected cells by preventing cell surface MHC-I display of HIV-1 antigens. Recent progress shows that Nef inhibitors not only suppress HIV-1 replication, but also restore sufficient MHC-I to the surface of infected cells to trigger a cytotoxic T lymphocyte response. Combining Nef inhibitors with latency reversal agents and therapeutic vaccines may provide a path to clearance of viral reservoirs.
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Affiliation(s)
- Lori A. Emert-Sedlak
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Haibin Shi
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Colin M. Tice
- Fox Chase Chemical Diversity Center, Inc., Pennsylvania Biotechnology Center, Doylestown, PA 18902, USA
| | - Li Chen
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - John J. Alvarado
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Sherry T. Shu
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Shoucheng Du
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Catherine E. Thomas
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
| | - Jay E. Wrobel
- Fox Chase Chemical Diversity Center, Inc., Pennsylvania Biotechnology Center, Doylestown, PA 18902, USA
| | - Allen B. Reitz
- Fox Chase Chemical Diversity Center, Inc., Pennsylvania Biotechnology Center, Doylestown, PA 18902, USA
| | - Thomas E. Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
- Correspondence:
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6
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Duette G, Hiener B, Morgan H, Mazur FG, Mathivanan V, Horsburgh BA, Fisher K, Tong O, Lee E, Ahn H, Shaik A, Fromentin R, Hoh R, Bacchus-Souffan C, Nasr N, Cunningham AL, Hunt PW, Chomont N, Turville SG, Deeks SG, Kelleher AD, Schlub TE, Palmer S. The HIV-1 proviral landscape reveals that Nef contributes to HIV-1 persistence in effector memory CD4+ T cells. J Clin Invest 2022; 132:154422. [PMID: 35133986 PMCID: PMC8970682 DOI: 10.1172/jci154422] [Citation(s) in RCA: 53] [Impact Index Per Article: 17.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2021] [Accepted: 02/02/2022] [Indexed: 11/17/2022] Open
Abstract
Despite long-term antiretroviral therapy (ART), HIV-1 persists within a reservoir of CD4+ T cells that contribute to viral rebound if treatment is interrupted. Identifying the cellular populations that contribute to the HIV-1 reservoir and understanding the mechanisms of viral persistence are necessary to achieve an effective cure. In this regard, through Full-Length Individual Proviral Sequencing, we observed that the HIV-1 proviral landscape was different and changed with time on ART across naive and memory CD4+ T cell subsets isolated from 24 participants. We found that the proportion of genetically intact HIV-1 proviruses was higher and persisted over time in effector memory CD4+ T cells when compared with naive, central, and transitional memory CD4+ T cells. Interestingly, we found that escape mutations remained stable over time within effector memory T cells during therapy. Finally, we provided evidence that Nef plays a role in the persistence of genetically intact HIV-1. These findings posit effector memory T cells as a key component of the HIV-1 reservoir and suggest Nef as an attractive therapeutic target.
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Affiliation(s)
- Gabriel Duette
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.,Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia
| | - Bonnie Hiener
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.,Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia
| | - Hannah Morgan
- Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia
| | - Fernando G. Mazur
- Post-graduation Program of Evolutionary Genetics and Molecular Biology, Federal University of São Carlos, São Carlos, Brazil
| | - Vennila Mathivanan
- The Kirby Institute, University of New South Wales, Sydney, New South Wales, Australia
| | - Bethany A. Horsburgh
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia
| | - Katie Fisher
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.,Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia
| | - Orion Tong
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia
| | - Eunok Lee
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.,Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia
| | - Haelee Ahn
- Division of Experimental Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Ansari Shaik
- The Kirby Institute, University of New South Wales, Sydney, New South Wales, Australia
| | - Rémi Fromentin
- Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montreal, Quebec, Canada
| | - Rebecca Hoh
- Department of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Charline Bacchus-Souffan
- Division of Experimental Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Najla Nasr
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.,Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia
| | - Anthony L. Cunningham
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.,Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia
| | - Peter W. Hunt
- Division of Experimental Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Nicolas Chomont
- Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montreal, Quebec, Canada.,Department of Microbiology, Infectiology and Immunology, Université de Montréal, Montreal, Quebec, Canada
| | - Stuart G. Turville
- The Kirby Institute, University of New South Wales, Sydney, New South Wales, Australia
| | - Steven G. Deeks
- Department of Medicine, University of California, San Francisco, San Francisco, California, USA
| | - Anthony D. Kelleher
- The Kirby Institute, University of New South Wales, Sydney, New South Wales, Australia
| | - Timothy E. Schlub
- Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia
| | - Sarah Palmer
- Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.,Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia
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7
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Prévost J, Richard J, Gasser R, Medjahed H, Kirchhoff F, Hahn BH, Kappes JC, Ochsenbauer C, Duerr R, Finzi A. Detection of the HIV-1 Accessory Proteins Nef and Vpu by Flow Cytometry Represents a New Tool to Study Their Functional Interplay within a Single Infected CD4 + T Cell. J Virol 2022; 96:e0192921. [PMID: 35080425 PMCID: PMC8941894 DOI: 10.1128/jvi.01929-21] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2021] [Accepted: 01/16/2022] [Indexed: 11/20/2022] Open
Abstract
The HIV-1 Nef and Vpu accessory proteins are known to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) responses by limiting exposure of CD4-induced (CD4i) envelope (Env) epitopes at the cell surface. Although both proteins target the host receptor CD4 for degradation, the extent of their functional redundancy is unknown. Here, we developed an intracellular staining technique that permits the intracellular detection of both Nef and Vpu in primary CD4+ T cells by flow cytometry. Using this method, we show that the combined expression of Nef and Vpu predicts the susceptibility of HIV-1-infected primary CD4+ T cells to ADCC by HIV+ plasma. We also show that Vpu cannot compensate for the absence of Nef, thus providing an explanation for why some infectious molecular clones that carry a LucR reporter gene upstream of Nef render infected cells more susceptible to ADCC responses. Our method thus represents a new tool to dissect the biological activity of Nef and Vpu in the context of other host and viral proteins within single infected CD4+ T cells. IMPORTANCE HIV-1 Nef and Vpu exert several biological functions that are important for viral immune evasion, release, and replication. Here, we developed a new method allowing simultaneous detection of these accessory proteins in their native form together with some of their cellular substrates. This allowed us to show that Vpu cannot compensate for the lack of a functional Nef, which has implications for studies that use Nef-defective viruses to study ADCC responses.
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Affiliation(s)
- Jérémie Prévost
- Centre de Recherche du CHUM, Montreal, Quebec, Canada
- Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montreal, Quebec, Canada
| | - Jonathan Richard
- Centre de Recherche du CHUM, Montreal, Quebec, Canada
- Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montreal, Quebec, Canada
| | - Romain Gasser
- Centre de Recherche du CHUM, Montreal, Quebec, Canada
- Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montreal, Quebec, Canada
| | | | - Frank Kirchhoff
- Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany
| | - Beatrice H. Hahn
- Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
- Department Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - John C. Kappes
- Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Christina Ochsenbauer
- Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Ralf Duerr
- Department of Microbiology, New York University School of Medicine, New York, New York, USA
| | - Andrés Finzi
- Centre de Recherche du CHUM, Montreal, Quebec, Canada
- Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, Montreal, Quebec, Canada
- Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada
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8
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Adzhubei AA, Kulkarni A, Tolstova AP, Anashkina AA, Sviridov D, Makarov AA, Bukrinsky MI. Direct interaction between ABCA1 and HIV-1 Nef: Molecular modeling and virtual screening for inhibitors. Comput Struct Biotechnol J 2021; 19:3876-3884. [PMID: 34584633 PMCID: PMC8440812 DOI: 10.1016/j.csbj.2021.06.050] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2021] [Revised: 06/23/2021] [Accepted: 06/30/2021] [Indexed: 12/18/2022] Open
Abstract
HIV-1 infection impairs cellular cholesterol efflux by downmodulating the cholesterol transporter ABCA1, leading to metabolic co-morbidities like cardio-vascular disease. The main mechanism of this effect is impairment by the HIV-1 protein Nef of the ABCA1 interaction with the endoplasmic reticulum chaperone calnexin, which leads to a block in ABCA1 maturation followed by its degradation. However, ABCA1 is also downmodulated by Nef delivered with the extracellular vesicles, suggesting involvement of a direct Nef:ABCA1 interaction at the plasma membrane. Here, we present an optimized model of the Nef:ABCA1 interaction, which identifies interaction sites and provides an opportunity to perform a virtual screening for potential inhibitors. Interestingly, the predicted sites on Nef involved in the ABCA1 interaction overlap with those involved in the interaction with calnexin. The compounds previously shown to block Nef:calnexin interaction were among the top ranking ligands in docking simulations with ABCA1-interacting sites on Nef, suggesting the possibility that both interactions can be inhibited by the same chemical compounds. This study identifies a series of compounds for potential development as inhibitors of Nef-mediated co-morbidities of HIV infection.
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Affiliation(s)
- Alexei A. Adzhubei
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | - Amol Kulkarni
- Howard University College of Pharmacy, Washington, District of Columbia, USA
| | - Anna P. Tolstova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | | | - Dmitri Sviridov
- Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia
- Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
| | - Alexander A. Makarov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | - Michael I. Bukrinsky
- The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, USA
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9
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Staudt RP, Alvarado JJ, Emert-Sedlak LA, Shi H, Shu ST, Wales TE, Engen JR, Smithgall TE. Structure, function, and inhibitor targeting of HIV-1 Nef-effector kinase complexes. J Biol Chem 2020; 295:15158-15171. [PMID: 32862141 DOI: 10.1074/jbc.rev120.012317] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2020] [Revised: 08/28/2020] [Indexed: 11/06/2022] Open
Abstract
Antiretroviral therapy has revolutionized the treatment of AIDS, turning a deadly disease into a manageable chronic condition. Life-long treatment is required because existing drugs do not eradicate HIV-infected cells. The emergence of drug-resistant viral strains and uncertain vaccine prospects highlight the pressing need for new therapeutic approaches with the potential to clear the virus. The HIV-1 accessory protein Nef is essential for viral pathogenesis, making it a promising target for antiretroviral drug discovery. Nef enhances viral replication and promotes immune escape of HIV-infected cells but lacks intrinsic enzymatic activity. Instead, Nef works through diverse interactions with host cell proteins primarily related to kinase signaling pathways and endosomal trafficking. This review emphasizes the structure, function, and biological relevance of Nef interactions with host cell protein-tyrosine kinases in the broader context of Nef functions related to enhancement of the viral life cycle and immune escape. Drug discovery targeting Nef-mediated kinase activation has allowed identification of promising inhibitors of multiple Nef functions. Pharmacological inhibitors of Nef-induced MHC-I down-regulation restore the adaptive immune response to HIV-infected cells in vitro and have the potential to enhance immune recognition of latent viral reservoirs as part of a strategy for HIV clearance.
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Affiliation(s)
- Ryan P Staudt
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - John J Alvarado
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Lori A Emert-Sedlak
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Haibin Shi
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Sherry T Shu
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - Thomas E Wales
- Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts, USA
| | - John R Engen
- Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts, USA
| | - Thomas E Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
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10
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Burster T, Knippschild U, Molnár F, Zhanapiya A. Cathepsin G and its Dichotomous Role in Modulating Levels of MHC Class I Molecules. Arch Immunol Ther Exp (Warsz) 2020; 68:25. [PMID: 32815043 DOI: 10.1007/s00005-020-00585-3] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2019] [Accepted: 06/11/2020] [Indexed: 12/21/2022]
Abstract
Cathepsin G (CatG) is involved in controlling numerous processes of the innate and adaptive immune system. These features include the proteolytic activity of CatG and play a pivotal role in alteration of chemokines as well as cytokines, clearance of exogenous and internalized pathogens, platelet activation, apoptosis, and antigen processing. This is in contrast to the capability of CatG acting in a proteolytic-independent manner due to the net charge of arginine residues in the CatG sequence which interferes with bacteria. CatG is a double-edged sword; CatG is also responsible in pathophysiological conditions, such as autoimmunity, chronic pulmonary diseases, HIV infection, tumor progression and metastasis, photo-aged human skin, Papillon-Lefèvre syndrome, and chronic inflammatory pain. Here, we summarize the latest findings for functional responsibilities of CatG in immunity, including bivalent regulation of major histocompatibility complex class I molecules, which underscore an additional novel role of CatG within the immune system.
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Affiliation(s)
- Timo Burster
- Department of Biology, School of Sciences and Humanities, Nazarbayev University, Kabanbay Batyr Ave. 53, Nur-Sultan, 010000, Kazakhstan.
| | - Uwe Knippschild
- Department of General and Visceral Surgery, Surgery Center, Ulm University Hospital, 89081, Ulm, Germany
| | - Ferdinand Molnár
- Department of Biology, School of Sciences and Humanities, Nazarbayev University, Kabanbay Batyr Ave. 53, Nur-Sultan, 010000, Kazakhstan
| | - Anuar Zhanapiya
- Department of Biology, School of Sciences and Humanities, Nazarbayev University, Kabanbay Batyr Ave. 53, Nur-Sultan, 010000, Kazakhstan
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11
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Liu B, Zhang X, Zhang W, Wu L, Jing S, Liu W, Xia B, Zou F, Lu L, Ma X, He D, Hu Q, Zhang Y, Deng K, Cai W, Tang X, Peng T, Zhang H, Li L. Lovastatin Inhibits HIV-1-Induced MHC-I Downregulation by Targeting Nef-AP-1 Complex Formation: A New Strategy to Boost Immune Eradication of HIV-1 Infected Cells. Front Immunol 2019; 10:2151. [PMID: 31572371 PMCID: PMC6749138 DOI: 10.3389/fimmu.2019.02151] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2019] [Accepted: 08/27/2019] [Indexed: 01/05/2023] Open
Abstract
Current combined antiretroviral therapy (cART) mainly targets 3 of the 15 HIV proteins leaving many potential viral vulnerabilities unexploited. To purge the HIV-1 latent reservoir, various strategies including “shock and kill” have been developed. A key question is how to restore impaired immune surveillance. HIV-1 protein Nef has long been known to mediate the downregulation of cell-surface MHC-I and assist HIV-1 to evade the immune system. Through high throughput screening of Food and Drug Administration (FDA) approved drugs, we identified lovastatin, a statin drug, to significantly antagonize Nef to downregulate MHC-I, CD4, and SERINC5, and inhibit the intrinsic infectivity of virions. In addition, lovastatin boosted autologous CTLs to eradicate the infected cells and effectively inhibit the subsequent viral rebound in CD4+ T-lymphocytes isolated from HIV-1-infected individuals receiving suppressive cART. Furthermore, we found that lovastatin inhibits Nef-induced MHC-I downregulation by directly binding with Nef and disrupting the Nef–AP-1 complex. These results demonstrate that lovastatin is a promising agent for counteracting Nef-mediated downregulation of MHC-I, CD4, and SERINC5. Lovastatin could potentially be used in the clinic to enhance anti-HIV-1 immune surveillance.
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Affiliation(s)
- Bingfeng Liu
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China.,Department of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Xu Zhang
- Sino-French Hoffmann Institute, Guangzhou Medical University, Guangzhou, China
| | - Wanying Zhang
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Liyang Wu
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Shuliang Jing
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Weiwei Liu
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Baijin Xia
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Fan Zou
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China.,Department of Molecular Therapy, Qianyang Biomedical Research Institute, Guangzhou, China.,Guangzhou Women and Children Hospital, Institute of Pediatrics, Guangzhou Medical University, Guangzhou, China
| | - Lijuan Lu
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Xiancai Ma
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Dalian He
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Qifei Hu
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China.,Department of Molecular Therapy, Qianyang Biomedical Research Institute, Guangzhou, China
| | - Yiwen Zhang
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Kai Deng
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China
| | - Weiping Cai
- Department of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Xiaoping Tang
- Department of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
| | - Tao Peng
- Sino-French Hoffmann Institute, Guangzhou Medical University, Guangzhou, China
| | - Hui Zhang
- Key Laboratory of Tropical Disease Control of Ministry of Education, Guangdong Engineering Research Center for Antimicrobial Agent and Immunotechnology, Zhongshan School of Medicine, Institute of Human Virology, Sun Yat-sen University, Guangzhou, China.,Department of Molecular Therapy, Qianyang Biomedical Research Institute, Guangzhou, China
| | - Linghua Li
- Department of Infectious Diseases, Guangzhou Eighth People's Hospital, Guangzhou Medical University, Guangzhou, China
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12
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Kumari S, Kumar M, Verma R, Ghosh JK, Tripathi RK. HIV-1 Nef-GCC185 interaction regulates assembly of cellular protein complexes at TGN targeting MHC-I downregulation. Life Sci 2019; 229:13-20. [PMID: 30953643 DOI: 10.1016/j.lfs.2019.04.008] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2019] [Revised: 03/27/2019] [Accepted: 04/02/2019] [Indexed: 10/27/2022]
Abstract
AIM HIV-1 Nef downregulates surface MHC-I to protect the infected cells from CTLs-mediated killing. Although MHC-I downregulation has been extensively studied, the Nef-dependent assembly of the multi-protein complex and subsequent pathways activation has not yet been well explored. The present study is aimed for the identification of Nef-mediated sequential recruitment of cellular proteins that constitute the functional multi-protein complex, required for the downregulation of MHC-I. MAIN METHODS Different Cellular protein complexes were identified by co-immunoprecipitation in Nef or NefE4A mutant-expressing Jurkat T, and THP-1 cells followed by exposure to Nef-specific peptides 24 h post infection. The MHC-I downregulation was analyzed by confocal microscopy and flow cytometry. KEY FINDINGS We found the association of Nef with PACS-2, GCC185, PI3K, AP-1, SFK, and MHC-I proteins that probably constitute a functional multi-protein complex. Furthermore, the immunoprecipitations with PACS-2 and GCC185 in the presence or absence of Nef, Nef E4A mutant and Nef with CP-inhibitor divide the functional complex of Nef into Nef-dependent (AP-1 and PI3K) and GCC185-dependent complex (MHC-I and SFK). The molecular mechanisms for activation of cellular pathways have been deciphered on the basis of these interactions that are brought in close proximity through Nef-GCC185 interaction. Knockdown of GCC185 using siRNA in Jurkat T cells showed a direct relationship between the assembly of functional multi-protein complex and MHC-I accumulation at GCC185. SIGNIFICANCE Overall, our study elucidates that GCC185 is a focal point for the assembly of the Nef-mediated multi-protein complex at TGN.
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Affiliation(s)
- Sushila Kumari
- Toxicology and Experimental Medicine Division, CSIR-Central Drug Research Institute, Sector-10, Janakipuram Extension, Sitapur Road, Lucknow 226031, U.P., India
| | - Manjeet Kumar
- Toxicology and Experimental Medicine Division, CSIR-Central Drug Research Institute, Sector-10, Janakipuram Extension, Sitapur Road, Lucknow 226031, U.P., India
| | - Richa Verma
- Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector-10, Janakipuram Extension, Sitapur Road, Lucknow 226031, U.P., India
| | - Jimut Kanti Ghosh
- Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector-10, Janakipuram Extension, Sitapur Road, Lucknow 226031, U.P., India
| | - Raj Kamal Tripathi
- Toxicology and Experimental Medicine Division, CSIR-Central Drug Research Institute, Sector-10, Janakipuram Extension, Sitapur Road, Lucknow 226031, U.P., India.
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13
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Machado Andrade V, Stevenson M. Host and Viral Factors Influencing Interplay between the Macrophage and HIV-1. J Neuroimmune Pharmacol 2018; 14:33-43. [PMID: 29995208 DOI: 10.1007/s11481-018-9795-4] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2018] [Accepted: 06/26/2018] [Indexed: 12/29/2022]
Abstract
HIV-1 persists in cellular reservoirs that cannot be eliminated by antiretroviral therapy (ART). The major reservoir in infected individuals on effective ART is composed of resting memory CD4+ T cells that harbor proviral cDNA, and undergo a state of latency in which viral gene expression is minimal to absent. The CD4+ T cell reservoir has been extensively characterized. However, other HIV-1-permissive cells may contribute to HIV-1 persistence. Lentiviruses have a long recognized association with macrophages. However, the role, if any, played by macrophages in HIV-1 persistence is not well understood. Macrophages are resistant to cell death upon HIV-1 infection, and can survive for long periods of time, making them ideal host cells in which the virus might persist. Studying macrophages is challenging, as these cells reside in nearly all tissues. Moreover, detecting viral DNA or RNA in macrophages does not necessarily indicate that these cells will produce replication-competent viral particles. Currently, the gold standard assay to detect cellular reservoirs is the ex vivo quantitative viral outgrowth assay (QVOA), which requires a patient blood draw. However, macrophages reside deep within tissues that are inaccessible in living subjects, such as the central nervous system (CNS). Therefore, tools other than QVOA must be developed to identify cellular reservoirs that reside in the tissues. In this review, we will focus on the main aspects involved in HIV-1 persistence, including the molecular mechanisms of viral evasion, the main cell types responsible for harboring persistent HIV-1 and the tissue compartments that are likely to be reservoirs for HIV-1.
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Affiliation(s)
- Viviane Machado Andrade
- Molecular Cell and Developmental Biology, Miller School of Medicine, University of Miami, Miami, FL, 33136, USA.
| | - Mario Stevenson
- Division of Infectious Diseases, Department of Medicine, Miller School of Medicine, University of Miami, Miami, FL, 33136, USA
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14
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Wu M, Alvarado JJ, Augelli-Szafran CE, Ptak RG, Smithgall TE. A single β-octyl glucoside molecule induces HIV-1 Nef dimer formation in the absence of partner protein binding. PLoS One 2018; 13:e0192512. [PMID: 29415006 PMCID: PMC5802939 DOI: 10.1371/journal.pone.0192512] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2017] [Accepted: 01/24/2018] [Indexed: 01/05/2023] Open
Abstract
The HIV-1 Nef accessory protein is essential for viral pathogenicity and AIDS progression. Nef forms complexes with multiple host cell factors to facilitate viral replication and promote immune escape of HIV-infected cells. Previous X-ray crystal structures demonstrate that Nef forms homodimers, the orientation of which are influenced by host cell binding partners. In cell-based fluorescence complementation assays, Nef forms homodimers at the plasma membrane. However, recombinant Nef proteins often exist as monomers in solution, suggesting that membrane interaction may also trigger monomer to dimer transitions. In this study, we show that monomeric Nef core proteins can be induced to form dimers in the presence of low concentrations of the non-ionic surfactant, β-octyl glucoside (βOG). X-ray crystallography revealed that a single βOG molecule is present in the Nef dimer, with the 8-carbon acyl chain of the ligand binding to a hydrophobic pocket formed by the dimer interface. This Nef-βOG dimer interface involves helix αB, as observed in previous dimer structures, as well as a helix formed by N-terminal residues 54-66. Nef dimer formation is stabilized in solution by the addition of βOG, providing biochemical validation for the crystal structure. These observations together suggest that the interaction with host cell lipid mediators or other hydrophobic ligands may play a role in Nef dimerization, which has been previously linked to multiple Nef functions including host cell protein kinase activation, CD4 downregulation, and enhancement of HIV-1 replication.
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Affiliation(s)
- Mousheng Wu
- Department of Chemistry, Drug Discovery Division, Southern Research Institute, Birmingham, AL, United States of America
| | - John J. Alvarado
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America
| | - Corinne E. Augelli-Szafran
- Department of Chemistry, Drug Discovery Division, Southern Research Institute, Birmingham, AL, United States of America
| | - Roger G. Ptak
- Department of Infectious Disease Research, Southern Research Institute, Frederick, MD, United States of America
| | - Thomas E. Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America
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15
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Moroco JA, Alvarado JJ, Staudt RP, Shi H, Wales TE, Smithgall TE, Engen JR. Remodeling of HIV-1 Nef Structure by Src-Family Kinase Binding. J Mol Biol 2018; 430:310-321. [PMID: 29258818 PMCID: PMC5801098 DOI: 10.1016/j.jmb.2017.12.008] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2017] [Revised: 12/07/2017] [Accepted: 12/10/2017] [Indexed: 11/25/2022]
Abstract
The HIV-1 accessory protein Nef controls multiple aspects of the viral life cycle and host immune response, making it an attractive therapeutic target. Previous X-ray crystal structures of Nef in complex with key host cell binding partners have shed light on protein-protein interactions critical to Nef function. Crystal structures of Nef in complex with either the SH3 or tandem SH3-SH2 domains of Src-family kinases reveal distinct dimer conformations of Nef. However, the existence of these Nef dimer complexes in solution has not been established. Here we used hydrogen exchange mass spectrometry (HX MS) to compare the solution conformation of Nef alone and in complexes with the SH3 or the SH3-SH2 domains of the Src-family kinase Hck. HX MS revealed that interaction with the Hck SH3 or tandem SH3-SH2 domains induces protection of the Nef αB-helix from deuterium uptake, consistent with a role for αB in dimer formation. HX MS analysis of a Nef mutant (position Asp123, a site buried in the Nef:SH3 dimer but surface exposed in the Nef:SH3-SH2 complex), showed a Hck-induced conformational change in Nef relative to wild-type Nef. These results support a model in which Src-family kinase binding induces conformational changes in Nef to expose residues critical for interaction with the μ1 subunit of adaptor protein 1 and the major histocompatibility complex-1 tail, and subsequent major histocompatibility complex-1 downregulation and immune escape of HIV-infected cells required for functional interactions with downstream binding partners.
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Affiliation(s)
- Jamie A Moroco
- Department of Chemistry and Chemical Biology, Maildrop 412TF, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
| | - John Jeff Alvarado
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 450 Technology Drive, Pittsburgh, PA 15219, USA.
| | - Ryan P Staudt
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 450 Technology Drive, Pittsburgh, PA 15219, USA.
| | - Haibin Shi
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 450 Technology Drive, Pittsburgh, PA 15219, USA.
| | - Thomas E Wales
- Department of Chemistry and Chemical Biology, Maildrop 412TF, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
| | - Thomas E Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 450 Technology Drive, Pittsburgh, PA 15219, USA.
| | - John R Engen
- Department of Chemistry and Chemical Biology, Maildrop 412TF, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
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16
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HIV-I Nef inhibitors: a novel class of HIV-specific immune adjuvants in support of a cure. AIDS Res Ther 2017; 14:53. [PMID: 28893294 PMCID: PMC5594582 DOI: 10.1186/s12981-017-0175-6] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2017] [Accepted: 08/11/2017] [Indexed: 11/22/2022] Open
Abstract
The success of many current vaccines relies on a formulation that incorporates an immune activating adjuvant. This will hold true for the design of a successful therapeutic HIV vaccine targeted at controlling reactivated virus following cessation of combined antiretroviral therapy (cART). The HIV accessory protein Nef functions by interfering with HIV antigen presentation through the major histocompatibility complex I (MHC-I) pathway thereby suppressing CD8+ cytotoxic T cell (CTL)-mediated killing of HIV infected cells. Thus, this important impediment to HIV vaccine success must be circumvented. This review covers our current knowledge of Nef inhibitors that may serve as immune adjuvants that will specifically restore and enhance CTL-mediated killing of reactivated HIV infected cells as part of an overall vaccine strategy to affect a cure for HIV infection.
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17
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Thomas G, Aslan JE, Thomas L, Shinde P, Shinde U, Simmen T. Caught in the act - protein adaptation and the expanding roles of the PACS proteins in tissue homeostasis and disease. J Cell Sci 2017; 130:1865-1876. [PMID: 28476937 PMCID: PMC5482974 DOI: 10.1242/jcs.199463] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Vertebrate proteins that fulfill multiple and seemingly disparate functions are increasingly recognized as vital solutions to maintaining homeostasis in the face of the complex cell and tissue physiology of higher metazoans. However, the molecular adaptations that underpin this increased functionality remain elusive. In this Commentary, we review the PACS proteins - which first appeared in lower metazoans as protein traffic modulators and evolved in vertebrates to integrate cytoplasmic protein traffic and interorganellar communication with nuclear gene expression - as examples of protein adaptation 'caught in the act'. Vertebrate PACS-1 and PACS-2 increased their functional density and roles as metabolic switches by acquiring phosphorylation sites and nuclear trafficking signals within disordered regions of the proteins. These findings illustrate one mechanism by which vertebrates accommodate their complex cell physiology with a limited set of proteins. We will also highlight how pathogenic viruses exploit the PACS sorting pathways as well as recent studies on PACS genes with mutations or altered expression that result in diverse diseases. These discoveries suggest that investigation of the evolving PACS protein family provides a rich opportunity for insight into vertebrate cell and organ homeostasis.
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Affiliation(s)
- Gary Thomas
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15239, USA
- University of Pittsburgh Cancer Institute, Pittsburgh, PA 15239, USA
| | - Joseph E Aslan
- Knight Cardiovascular Institute, Oregon Health & Science University, Portland, OR 97239, USA
| | - Laurel Thomas
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15239, USA
| | - Pushkar Shinde
- Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239, USA
| | - Ujwal Shinde
- Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239, USA
| | - Thomas Simmen
- Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada T6G2H7
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18
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Jacob RA, Johnson AL, Pawlak EN, Dirk BS, Van Nynatten LR, Haeryfar SMM, Dikeakos JD. The interaction between HIV-1 Nef and adaptor protein-2 reduces Nef-mediated CD4 + T cell apoptosis. Virology 2017; 509:1-10. [PMID: 28577469 DOI: 10.1016/j.virol.2017.05.018] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2017] [Revised: 05/24/2017] [Accepted: 05/25/2017] [Indexed: 02/07/2023]
Abstract
Acquired Immune Deficiency Syndrome is characterized by a decline in CD4+ T cells. Here, we elucidated the mechanism underlying apoptosis in Human Immunodeficiency Virus-1 (HIV-1) infection by examining host apoptotic pathways hijacked by the HIV-1 Nef protein in the CD4+ T-cell line Sup-T1. Using a panel of Nef mutants unable to bind specific host proteins we uncovered that Nef generates pro- and anti-apoptotic signals. Apoptosis increased upon mutating the motifs involved in the interaction of Nef:AP-1 (NefM20A or NefEEEE62-65AAAA) or Nef:AP-2 (NefLL164/165AA), implying these interactions limit Nef-mediated apoptosis. In contrast, disrupting the Nef:PAK2 interaction motifs (NefH89A or NefF191A) reduced apoptosis. To validate further, apoptosis was measured after short-hairpin RNA knock-down of AP-1, AP-2 and PAK2. AP-2α depletion enhanced apoptosis, demonstrating that disrupting the Nef:AP-2α interaction limits Nef-mediated apoptosis. Collectively, we describe a mechanism by which HIV-1 regulates cell survival and demonstrate the consequence of interfering with Nef:host protein interactions.
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Affiliation(s)
- Rajesh Abraham Jacob
- Department of Microbiology and Immunology, The University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada
| | - Aaron L Johnson
- Department of Microbiology and Immunology, The University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada
| | - Emily N Pawlak
- Department of Microbiology and Immunology, The University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada
| | - Brennan S Dirk
- Department of Microbiology and Immunology, The University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada
| | - Logan R Van Nynatten
- Department of Microbiology and Immunology, The University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada
| | - S M Mansour Haeryfar
- Department of Microbiology and Immunology, The University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada
| | - Jimmy D Dikeakos
- Department of Microbiology and Immunology, The University of Western Ontario, Schulich School of Medicine and Dentistry, London, Ontario, Canada.
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HIV-1 Nef sequesters MHC-I intracellularly by targeting early stages of endocytosis and recycling. Sci Rep 2016; 6:37021. [PMID: 27841315 PMCID: PMC5107982 DOI: 10.1038/srep37021] [Citation(s) in RCA: 49] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2016] [Accepted: 10/24/2016] [Indexed: 11/25/2022] Open
Abstract
A defining characteristic of HIV-1 infection is the ability of the virus to persist within the host. Specifically, MHC-I downregulation by the HIV-1 accessory protein Nef is of critical importance in preventing infected cells from cytotoxic T-cell mediated killing. Nef downregulates MHC-I by modulating the host membrane trafficking machinery, resulting in the endocytosis and eventual sequestration of MHC-I within the cell. In the current report, we utilized the intracellular protein-protein interaction reporter system, bimolecular fluorescence complementation (BiFC), in combination with super-resolution microscopy, to track the Nef/MHC-I interaction and determine its subcellular localization in cells. We demonstrate that this interaction occurs upon Nef binding the MHC-I cytoplasmic tail early during endocytosis in a Rab5-positive endosome. Disruption of early endosome regulation inhibited Nef-dependent MHC-I downregulation, demonstrating that Nef hijacks the early endosome to sequester MHC-I within the cell. Furthermore, super-resolution imaging identified that the Nef:MHC-I BiFC complex transits through both early and late endosomes before ultimately residing at the trans-Golgi network. Together we demonstrate the importance of the early stages of the endocytic network in the removal of MHC-I from the cell surface and its re-localization within the cell, which allows HIV-1 to optimally evade host immune responses.
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A Highly Conserved Residue in HIV-1 Nef Alpha Helix 2 Modulates Protein Expression. mSphere 2016; 1:mSphere00288-16. [PMID: 27840851 PMCID: PMC5103047 DOI: 10.1128/msphere.00288-16] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2016] [Accepted: 10/21/2016] [Indexed: 01/22/2023] Open
Abstract
The HIV-1 Nef protein has been established as a key pathogenic determinant of HIV/AIDS, but there is little knowledge of how the extensive genetic diversity of HIV-1 affects Nef function. Upon compiling a set of subtype-specific reference strains, we identified a subtype C reference strain, C.BR92025, that contained natural polymorphisms at otherwise highly conserved residues 13, 84, and 92. Interestingly, strain C.BR92025 Nef displayed impaired Nef function and had decreased protein expression. We have demonstrated that strain C.BR92025 Nef has a higher rate of protein turnover than highly expressed Nef proteins and that this higher rate of protein turnover is due to an alanine-to-valine substitution at Nef residue 84. These findings highlight residue A84 as a major determinant of HIV-1 Nef expression. Extensive genetic diversity is a defining characteristic of human immunodeficiency virus type 1 (HIV-1) and poses a significant barrier to the development of an effective vaccine. To better understand the impact of this genetic diversity on the HIV-1 pathogenic factor Nef, we compiled a panel of reference strains from the NIH Los Alamos HIV Database. Initial sequence analysis identified point mutations at Nef residues 13, 84, and 92 in subtype C reference strain C.BR92025 from Brazil. Functional analysis revealed impaired major histocompatibility complex class I and CD4 downregulation of strain C.BR92025 Nef, which corresponded to decreased protein expression. Metabolic labeling demonstrated that strain C.BR92025 Nef has a greater rate of protein turnover than subtype B reference strain B.JRFL that, on the basis of mutational analysis, is related to Nef residue A84. An alanine-to-valine substitution at position 84, located in alpha helix 2 of Nef, was sufficient to alter the rate of turnover of an otherwise highly expressed Nef protein. In conclusion, these findings highlight HIV-1 Nef residue A84 as a major determinant of protein expression that may offer an additional avenue to disrupt or mediate the effects of this key HIV-1 pathogenic factor. IMPORTANCE The HIV-1 Nef protein has been established as a key pathogenic determinant of HIV/AIDS, but there is little knowledge of how the extensive genetic diversity of HIV-1 affects Nef function. Upon compiling a set of subtype-specific reference strains, we identified a subtype C reference strain, C.BR92025, that contained natural polymorphisms at otherwise highly conserved residues 13, 84, and 92. Interestingly, strain C.BR92025 Nef displayed impaired Nef function and had decreased protein expression. We have demonstrated that strain C.BR92025 Nef has a higher rate of protein turnover than highly expressed Nef proteins and that this higher rate of protein turnover is due to an alanine-to-valine substitution at Nef residue 84. These findings highlight residue A84 as a major determinant of HIV-1 Nef expression.
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21
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Pereira EA, daSilva LLP. HIV-1 Nef: Taking Control of Protein Trafficking. Traffic 2016; 17:976-96. [PMID: 27161574 DOI: 10.1111/tra.12412] [Citation(s) in RCA: 96] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2016] [Revised: 05/05/2016] [Accepted: 05/05/2016] [Indexed: 12/25/2022]
Abstract
The Nef protein of the human immunodeficiency virus is a crucial determinant of viral pathogenesis and disease progression. Nef is abundantly expressed early in infection and is thought to optimize the cellular environment for viral replication. Nef controls expression levels of various cell surface molecules that play important roles in immunity and virus life cycle, by directly interfering with the itinerary of these proteins within the endocytic and late secretory pathways. To exert these functions, Nef physically interacts with host proteins that regulate protein trafficking. In recent years, considerable progress was made in identifying host-cell-interacting partners for Nef, and the molecular machinery used by Nef to interfere with protein trafficking has started to be unraveled. Here, we briefly review the knowledge gained and discuss new findings regarding the mechanisms by which Nef modifies the intracellular trafficking pathways to prevent antigen presentation, facilitate viral particle release and enhance the infectivity of HIV-1 virions.
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Affiliation(s)
- Estela A Pereira
- Department of Cell and Molecular Biology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil
| | - Luis L P daSilva
- Department of Cell and Molecular Biology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil
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22
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A Novel Toll-Like Receptor 9 Agonist, MGN1703, Enhances HIV-1 Transcription and NK Cell-Mediated Inhibition of HIV-1-Infected Autologous CD4+ T Cells. J Virol 2016; 90:4441-4453. [PMID: 26889036 DOI: 10.1128/jvi.00222-16] [Citation(s) in RCA: 88] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2016] [Accepted: 02/12/2016] [Indexed: 01/14/2023] Open
Abstract
UNLABELLED Toll-like receptor (TLR) agonists are potent enhancers of innate antiviral immunity and may also reverse HIV-1 latency. Therefore, TLR agonists have a potential role in the context of a "shock-and-kill" approach to eradicate HIV-1. Our extensive preclinical evaluation suggests that a novel TLR9 agonist, MGN1703, may indeed perform both functions in an HIV-1 eradication trial. Peripheral blood mononuclear cells (PBMCs) from aviremic HIV-1-infected donors on antiretroviral therapy (ART) that were incubated with MGN1703 ex vivo exhibited increased secretion of interferon alpha (IFN-α) (P= 0.005) and CXCL10 (P= 0.0005) in culture supernatants. Within the incubated PBMC pool, there were higher proportions of CD69-positive CD56(dim)CD16(+)NK cells (P= 0.001) as well as higher proportions of CD107a-positive (P= 0.002) and IFN-γ-producing (P= 0.038) NK cells. Incubation with MGN1703 also increased the proportions of CD69-expressing CD4(+)and CD8(+)T cells. Furthermore, CD4(+)T cells within the pool of MGN1703-incubated PBMCs showed enhanced levels of unspliced HIV-1 RNA (P= 0.036). Importantly, MGN1703 increased the capacity of NK cells to inhibit virus spread within a culture of autologous CD4(+)T cells assessed by using an HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) (P= 0.03). In conclusion, we show that MGN1703 induced strong antiviral innate immune responses, enhanced HIV-1 transcription, and boosted NK cell-mediated suppression of HIV-1 infection in autologous CD4(+)T cells. These findings support clinical testing of MGN1703 in HIV-1 eradication trials. IMPORTANCE We demonstrate that MGN1703 (a TLR9 agonist currently undergoing phase 3 clinical testing for the treatment of metastatic colorectal cancer) induces potent antiviral responses in immune effector cells from HIV-1-infected individuals on suppressive antiretroviral therapy. The significantly improved safety and tolerability profiles of MGN1703 versus TLR9 agonists of the CpG-oligodeoxynucleotide (CpG-ODN) family are due to its novel "dumbbell-shape" structure made of covalently closed, natural DNA. In our study, we found that incubation of peripheral blood mononuclear cells with MGN1703 results in natural killer cell activation and increased natural killer cell function, which significantly inhibited the spread of HIV in a culture of autologous CD4(+)T cells. Furthermore, we discovered that MGN1703-mediated activation can enhance HIV-1 transcription in CD4(+)T cells, suggesting that this molecule may serve a dual purpose in HIV-1 eradication therapy: enhanced immune function and latency reversal. These findings provide a strong preclinical basis for the inclusion of MGN1703 in an HIV eradication clinical trial.
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23
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PACS-2 mediates the ATM and NF-κB-dependent induction of anti-apoptotic Bcl-xL in response to DNA damage. Cell Death Differ 2016; 23:1448-57. [PMID: 26943323 DOI: 10.1038/cdd.2016.23] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2015] [Revised: 01/28/2016] [Accepted: 02/09/2016] [Indexed: 01/26/2023] Open
Abstract
Nuclear factor kappa B (NF-κB) promotes cell survival in response to genotoxic stress by inducing the expression of anti-apoptotic proteins including Bcl-xL, which protects mitochondria from stress-induced mitochondrial outer membrane permeabilization (MOMP). Here we show that the multifunctional sorting protein Pacs-2 (phosphofurin acidic cluster sorting protein-2) is required for Bcl-xL induction following DNA damage in primary mouse thymocytes. Consequently, in response to DNA damage, Pacs-2(-/-) thymocytes exhibit a blunted induction of Bcl-xL, increased MOMP and accelerated apoptosis. Biochemical studies show that cytoplasmic PACS-2 promotes this DNA damage-induced anti-apoptotic pathway by interacting with ataxia telangiectasia mutated (ATM) to drive NF-κB activation and induction of Bcl-xL. However, Pacs-2 was not required for tumor necrosis factor-α-induced NF-κB activation, suggesting a role for PACS-2 selectively in NF-κB activation in response to DNA damage. These findings identify PACS-2 as an in vivo mediator of the ATM and NF-κB-dependent induction of Bcl-xL that promotes cell survival in response to DNA damage.
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Remodeling of the Host Cell Plasma Membrane by HIV-1 Nef and Vpu: A Strategy to Ensure Viral Fitness and Persistence. Viruses 2016; 8:67. [PMID: 26950141 PMCID: PMC4810257 DOI: 10.3390/v8030067] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2016] [Revised: 02/09/2016] [Accepted: 02/16/2016] [Indexed: 02/07/2023] Open
Abstract
The plasma membrane protects the cell from its surroundings and regulates cellular communication, homing, and metabolism. Not surprisingly, the composition of this membrane is highly controlled through the vesicular trafficking of proteins to and from the cell surface. As intracellular pathogens, most viruses exploit the host plasma membrane to promote viral replication while avoiding immune detection. This is particularly true for the enveloped human immunodeficiency virus (HIV), which assembles and obtains its lipid shell directly at the plasma membrane. HIV-1 encodes two proteins, negative factor (Nef) and viral protein U (Vpu), which function primarily by altering the quantity and localization of cell surface molecules to increase virus fitness despite host antiviral immune responses. These proteins are expressed at different stages in the HIV-1 life cycle and employ a variety of mechanisms to target both unique and redundant surface proteins, including the viral receptor CD4, host restriction factors, immunoreceptors, homing molecules, tetraspanins and membrane transporters. In this review, we discuss recent progress in the study of the Nef and Vpu targeting of host membrane proteins with an emphasis on how remodeling of the cell membrane allows HIV-1 to avoid host antiviral immune responses leading to the establishment of systemic and persistent infection.
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Caetano FA, Dirk BS, Tam JHK, Cavanagh PC, Goiko M, Ferguson SSG, Pasternak SH, Dikeakos JD, de Bruyn JR, Heit B. MIiSR: Molecular Interactions in Super-Resolution Imaging Enables the Analysis of Protein Interactions, Dynamics and Formation of Multi-protein Structures. PLoS Comput Biol 2015; 11:e1004634. [PMID: 26657340 PMCID: PMC4676698 DOI: 10.1371/journal.pcbi.1004634] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2015] [Accepted: 10/27/2015] [Indexed: 11/18/2022] Open
Abstract
Our current understanding of the molecular mechanisms which regulate cellular processes such as vesicular trafficking has been enabled by conventional biochemical and microscopy techniques. However, these methods often obscure the heterogeneity of the cellular environment, thus precluding a quantitative assessment of the molecular interactions regulating these processes. Herein, we present Molecular Interactions in Super Resolution (MIiSR) software which provides quantitative analysis tools for use with super-resolution images. MIiSR combines multiple tools for analyzing intermolecular interactions, molecular clustering and image segmentation. These tools enable quantification, in the native environment of the cell, of molecular interactions and the formation of higher-order molecular complexes. The capabilities and limitations of these analytical tools are demonstrated using both modeled data and examples derived from the vesicular trafficking system, thereby providing an established and validated experimental workflow capable of quantitatively assessing molecular interactions and molecular complex formation within the heterogeneous environment of the cell. In this paper we present the software package Molecular Interactions in Super Resolution (MIiSR), which provides a series of quantitative analytical tools for measuring molecular interactions and the formation of higher-order molecular complexes in super-resolution microscopy images.
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Affiliation(s)
- Fabiana A. Caetano
- The J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute and the Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada
| | - Brennan S. Dirk
- Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada
| | - Joshua H. K. Tam
- The J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute and the Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada
| | - P. Craig Cavanagh
- Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada
| | - Maria Goiko
- Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada
- Department of Physics and Astronomy, The University of Western Ontario, London, Ontario, Canada
| | | | - Stephen H. Pasternak
- The J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute and the Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada
- Department of Clinical Neurological Sciences, Schulich School of Medicine, The University of Western Ontario, London, Ontario, Canada
| | - Jimmy D. Dikeakos
- Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada
| | - John R. de Bruyn
- Department of Physics and Astronomy, The University of Western Ontario, London, Ontario, Canada
| | - Bryan Heit
- Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada
- * E-mail:
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Wales TE, Hochrein JM, Morgan CR, Emert-Sedlak LA, Smithgall TE, Engen JR. Subtle Dynamic Changes Accompany Hck Activation by HIV-1 Nef and are Reversed by an Antiretroviral Kinase Inhibitor. Biochemistry 2015; 54:6382-91. [PMID: 26440750 PMCID: PMC4615603 DOI: 10.1021/acs.biochem.5b00875] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
The HIV-1 virulence factor Nef interacts with the macrophage Src-family kinase Hck, resulting in constitutive kinase activation that contributes to viral replication and immune escape. Previous chemical library screens identified the diphenylfuranopyrimdine kinase inhibitor DFP-4AB, which selectively inhibits Nef-dependent Hck activity in biochemical assays and potently blocks HIV replication in vitro. In the present study, hydrogen exchange mass spectrometry (HX MS) was used to study conformational changes in downregulated Hck that result from Nef binding, as well as the impact of DFP-4AB on these changes. Remarkably, interaction with Nef induced only subtle changes in deuterium uptake by Hck, with the most significant changes in the N-lobe of the kinase domain adjacent to the docking site for Nef on the SH3 domain. No changes in hydrogen exchange were observed in the Hck SH2 domain or C-terminal tail, indicating that this regulatory interaction is unaffected by Nef binding. When HX MS was performed in the presence of DFP-4AB, the effect of Nef on Hck N-lobe dynamics was completely reversed. These results show that constitutive activation of Hck by HIV-1 Nef requires only modest changes to the conformational dynamics of the overall kinase structure. DFP-4AB reverses these effects, consistent with its activity against this Nef-induced signaling event in HIV-infected cells.
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Affiliation(s)
- Thomas E. Wales
- Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115
| | - James M. Hochrein
- Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115
| | - Christopher R. Morgan
- Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115
| | - Lori A. Emert-Sedlak
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219
| | - Thomas E. Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219
| | - John R. Engen
- Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115
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Bose D, Gagnon J, Chebloune Y. Comparative Analysis of Tat-Dependent and Tat-Deficient Natural Lentiviruses. Vet Sci 2015; 2:293-348. [PMID: 29061947 PMCID: PMC5644649 DOI: 10.3390/vetsci2040293] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2015] [Revised: 08/24/2015] [Accepted: 08/24/2015] [Indexed: 01/10/2023] Open
Abstract
The emergence of human immunodeficiency virus (HIV) causing acquired immunodeficiency syndrome (AIDS) in infected humans has resulted in a global pandemic that has killed millions. HIV-1 and HIV-2 belong to the lentivirus genus of the Retroviridae family. This genus also includes viruses that infect other vertebrate animals, among them caprine arthritis-encephalitis virus (CAEV) and Maedi-Visna virus (MVV), the prototypes of a heterogeneous group of viruses known as small ruminant lentiviruses (SRLVs), affecting both goat and sheep worldwide. Despite their long host-SRLV natural history, SRLVs were never found to be responsible for immunodeficiency in contrast to primate lentiviruses. SRLVs only replicate productively in monocytes/macrophages in infected animals but not in CD4+ T cells. The focus of this review is to examine and compare the biological and pathological properties of SRLVs as prototypic Tat-independent lentiviruses with HIV-1 as prototypic Tat-dependent lentiviruses. Results from this analysis will help to improve the understanding of why and how these two prototypic lentiviruses evolved in opposite directions in term of virulence and pathogenicity. Results may also help develop new strategies based on the attenuation of SRLVs to control the highly pathogenic HIV-1 in humans.
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Affiliation(s)
- Deepanwita Bose
- Pathogénèse et Vaccination Lentivirales, PAVAL Lab., Université Joseph Fourier Grenoble 1, Bat. NanoBio2, 570 rue de la Chimie, BP 53, 38041, Grenoble Cedex 9, France.
| | - Jean Gagnon
- Pathogénèse et Vaccination Lentivirales, PAVAL Lab., Université Joseph Fourier Grenoble 1, Bat. NanoBio2, 570 rue de la Chimie, BP 53, 38041, Grenoble Cedex 9, France.
| | - Yahia Chebloune
- Pathogénèse et Vaccination Lentivirales, PAVAL Lab., Université Joseph Fourier Grenoble 1, Bat. NanoBio2, 570 rue de la Chimie, BP 53, 38041, Grenoble Cedex 9, France.
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Association with PAK2 Enables Functional Interactions of Lentiviral Nef Proteins with the Exocyst Complex. mBio 2015; 6:e01309-15. [PMID: 26350970 PMCID: PMC4600113 DOI: 10.1128/mbio.01309-15] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
UNLABELLED Human immunodeficiency virus type 1 (HIV-1) Nef enhances virus replication and contributes to immune evasion in vivo, but the underlying molecular mechanisms remain incompletely defined. Nef interferes with host cell actin dynamics to restrict T lymphocyte responses to chemokine stimulation and T cell receptor engagement. This relies on the assembly of a labile multiprotein complex including the host kinase PAK2 that Nef usurps to phosphorylate and inactivate the actin-severing factor cofilin. Components of the exocyst complex (EXOC), an octameric protein complex involved in vesicular transport and actin remodeling, were recently reported to interact with Nef via the same molecular surface that mediates PAK2 association. Exploring the functional relevance of EXOC in Nef-PAK2 complex assembly/function, we found Nef-EXOC interactions to be specifically mediated by the PAK2 interface of Nef, to occur in infected human T lymphocytes, and to be conserved among lentiviral Nef proteins. In turn, EXOC was dispensable for direct downstream effector functions of Nef-associated PAK2. Surprisingly, PAK2 was essential for Nef-EXOC association, which required a functional Rac1/Cdc42 binding site but not the catalytic activity of PAK2. EXOC was dispensable for Nef functions in vesicular transport but critical for inhibition of actin remodeling and proximal signaling upon T cell receptor engagement. Thus, Nef exploits PAK2 in a stepwise mechanism in which its kinase activity cooperates with an adaptor function for EXOC to inhibit host cell actin dynamics. IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) Nef contributes to AIDS pathogenesis, but the underlying molecular mechanisms remain incompletely understood. An important aspect of Nef function is to facilitate virus replication by disrupting T lymphocyte actin dynamics in response to stimulation via its association with the host cell kinase PAK2. We report here that the molecular surface of Nef for PAK2 association also mediates interaction of Nef with EXOC and establish that PAK2 provides an essential adaptor function for the subsequent formation of Nef-EXOC complexes. PAK2 and EXOC specifically cooperate in the inhibition of actin dynamics and proximal signaling induced by T cell receptor engagement by Nef. These results establish EXOC as a functionally relevant Nef interaction partner, emphasize the suitability of the PAK2 interaction surface for future therapeutic interference with Nef function, and show that such strategies need to target activity-independent PAK2 functions.
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A robust and scalable TCR-based reporter cell assay to measure HIV-1 Nef-mediated T cell immune evasion. J Immunol Methods 2015; 426:104-13. [PMID: 26319395 DOI: 10.1016/j.jim.2015.08.010] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2015] [Revised: 08/20/2015] [Accepted: 08/20/2015] [Indexed: 11/24/2022]
Abstract
HIV-1 evades cytotoxic T cell responses through Nef-mediated downregulation of HLA class I molecules from the infected cell surface. Methods to quantify the impact of Nef on T cell recognition typically employ patient-derived T cell clones; however, these assays are limited by the cost and effort required to isolate and maintain primary cell lines. The variable activity of different T cell clones and the limited number of cells generated by re-stimulation can also hinder assay reproducibility and scalability. Here, we describe a heterologous T cell receptor reporter assay and use it to study immune evasion by Nef. Induction of NFAT-driven luciferase following co-culture with peptide-pulsed or virus-infected target cells serves as a rapid, quantitative and antigen-specific measure of T cell recognition of its cognate peptide/HLA complex. We demonstrate that Nef-mediated downregulation of HLA on target cells correlates inversely with T cell receptor-dependent luminescent signal generated by effector cells. This method provides a robust, flexible and scalable platform that is suitable for studies to measure Nef function in the context of different viral peptide/HLA antigens, to assess the function of patient-derived Nef alleles, or to screen small molecule libraries to identify novel Nef inhibitors.
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30
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Viral bimolecular fluorescence complementation: a novel tool to study intracellular vesicular trafficking pathways. PLoS One 2015; 10:e0125619. [PMID: 25915798 PMCID: PMC4411132 DOI: 10.1371/journal.pone.0125619] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2015] [Accepted: 03/24/2015] [Indexed: 01/23/2023] Open
Abstract
The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Nef interacts with a multitude of cellular proteins, manipulating the host membrane trafficking machinery to evade immune surveillance. Nef interactions have been analyzed using various in vitro assays, co-immunoprecipitation studies, and more recently mass spectrometry. However, these methods do not evaluate Nef interactions in the context of viral infection nor do they define the sub-cellular location of these interactions. In this report, we describe a novel bimolecular fluorescence complementation (BiFC) lentiviral expression tool, termed viral BiFC, to study Nef interactions with host cellular proteins in the context of viral infection. Using the F2A cleavage site from the foot and mouth disease virus we generated a viral BiFC expression vector capable of concurrent expression of Nef and host cellular proteins; PACS-1, MHC-I and SNX18. Our studies confirmed the interaction between Nef and PACS-1, a host membrane trafficking protein involved in Nef-mediated immune evasion, and demonstrated co-localization of this complex with LAMP-1 positive endolysosomal vesicles. Furthermore, we utilized viral BiFC to localize the Nef/MHC-I interaction to an AP-1 positive endosomal compartment. Finally, viral BiFC was observed between Nef and the membrane trafficking regulator SNX18. This novel demonstration of an association between Nef and SNX18 was localized to AP-1 positive vesicles. In summary, viral BiFC is a unique tool designed to analyze the interaction between Nef and host cellular proteins by mapping the sub-cellular locations of their interactions during viral infection.
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Pawlak EN, Dikeakos JD. HIV-1 Nef: a master manipulator of the membrane trafficking machinery mediating immune evasion. Biochim Biophys Acta Gen Subj 2015; 1850:733-41. [PMID: 25585010 DOI: 10.1016/j.bbagen.2015.01.003] [Citation(s) in RCA: 57] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2014] [Revised: 12/09/2014] [Accepted: 01/06/2015] [Indexed: 11/25/2022]
Abstract
BACKGROUND Many viral genomes encode a limited number of proteins, illustrating their innate efficiency in bypassing host immune surveillance. This concept of genomic efficiency is exemplified by the 9 kb RNA genome of human immunodeficiency virus 1 (HIV-1), encoding 15 proteins sub-divided according to function. The enzymatic group includes proteins such as the drug targets reverse transcriptase and protease. In contrast, the accessory proteins lack any known enzymatic or structural function, yet are essential for viral fitness and HIV-1 pathogenesis. Of these, the HIV-1 accessory protein Nef is a master manipulator of host cellular processes, ensuring efficient counterattack against the host immune response, as well as long-term evasion of immune surveillance. In particular, the ability of Nef to downmodulate major histocompatibility complex class I (MHC-I) is a key cellular event that enables HIV-1 to bypass the host's defenses by evading the adaptive immune response. SCOPE OF REVIEW In this article, we briefly review how various pathogenic viruses control cell-surface MHC-I, and then focus on the mechanisms and implications of HIV-1 Nef-mediated MHC-I downregulation via modulation of the host membrane trafficking machinery. CONCLUSION The extensive interaction network formed between Nef and numerous membrane trafficking regulators suggests that Nef's role in evading the immune surveillance system intersects multiple host membrane trafficking pathways. SIGNIFICANCE Nef's ability to evade the immune surveillance system is linked to AIDS pathogenesis. Thus, a complete understanding of the molecular pathways that are subverted by Nef in order to downregulate MHC-I will enhance our understanding of HIV-1's progression to AIDS.
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Affiliation(s)
- Emily N Pawlak
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada, N6A 5C1
| | - Jimmy D Dikeakos
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada, N6A 5C1.
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Daep CA, Muñoz-Jordán JL, Eugenin EA. Flaviviruses, an expanding threat in public health: focus on dengue, West Nile, and Japanese encephalitis virus. J Neurovirol 2014; 20:539-60. [PMID: 25287260 PMCID: PMC4331079 DOI: 10.1007/s13365-014-0285-z] [Citation(s) in RCA: 132] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2014] [Revised: 08/01/2014] [Accepted: 08/26/2014] [Indexed: 10/24/2022]
Abstract
The flaviviruses dengue, West Nile, and Japanese encephalitis represent three major mosquito-borne viruses worldwide. These pathogens impact the lives of millions of individuals and potentially could affect non-endemic areas already colonized by mosquito vectors. Unintentional transport of infected vectors (Aedes and Culex spp.), traveling within endemic areas, rapid adaptation of the insects into new geographic locations, climate change, and lack of medical surveillance have greatly contributed to the increase in flaviviral infections worldwide. The mechanisms by which flaviviruses alter the immune and the central nervous system have only recently been examined despite the alarming number of infections, related deaths, and increasing global distribution. In this review, we will discuss the expansion of the geographic areas affected by flaviviruses, the potential threats to previously unaffected countries, the mechanisms of pathogenesis, and the potential therapeutic interventions to limit the devastating consequences of these viruses.
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Affiliation(s)
- Carlo Amorin Daep
- Public Health Research Institute (PHRI), Rutgers New Jersey Medical School, Rutgers the State University of New Jersey, Newark, NJ, USA
- Department of Microbiology and Molecular Genetics, Rutgers New Jersey Medical School, Rutgers the State University of New Jersey, Newark, NJ, USA
| | - Jorge L. Muñoz-Jordán
- Centers for Disease Control and Prevention Dengue Branch, 1324 Cañada Street, San Juan, PR 00971
| | - Eliseo Alberto Eugenin
- Public Health Research Institute (PHRI), Rutgers New Jersey Medical School, Rutgers the State University of New Jersey, Newark, NJ, USA
- Department of Microbiology and Molecular Genetics, Rutgers New Jersey Medical School, Rutgers the State University of New Jersey, Newark, NJ, USA
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Smithgall TE, Thomas G. Small molecule inhibitors of the HIV-1 virulence factor, Nef. DRUG DISCOVERY TODAY. TECHNOLOGIES 2014; 10:e523-9. [PMID: 24451644 DOI: 10.1016/j.ddtec.2013.07.002] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Although antiretroviral therapy has revolutionized the clinical management of AIDS, life-long treatment is required because these drugs do not eradicate HIV- infected cells. Chronic antiretroviral therapy may not protect AIDS patients from cognitive impairment, raising important quality of life issues. Because of the rise of HIV strains resistant to current drugs and uncertain vaccine prospects, an urgent need exists for the discovery and development of new therapeutic approaches. This review is focused on one such approach, which involves targeting HIV-1 Nef, a viral accessory protein essential for AIDS pathogenesis.
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Alvarado JJ, Tarafdar S, Yeh JI, Smithgall TE. Interaction with the Src homology (SH3-SH2) region of the Src-family kinase Hck structures the HIV-1 Nef dimer for kinase activation and effector recruitment. J Biol Chem 2014; 289:28539-53. [PMID: 25122770 DOI: 10.1074/jbc.m114.600031] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including Hck and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the Hck SH3-SH2 regulatory region to a resolution of 1.86 Å. The complex crystallized as a dimer of complexes, with the conserved Nef PXXPXR motif engaging the Hck SH3 domain. A new intercomplex contact was found between SH3 Glu-93, and Nef Arg-105. Mutagenesis of Hck SH3 Glu-93 interfered with Nef·Hck complex formation and kinase activation in cells. The Hck SH2 domains impinge on the N-terminal region of Nef to stabilize a dimer conformation that exposes Asp-123, a residue critical for Nef function. Our results suggest that in addition to serving as a kinase effector for Nef, Hck binding may reorganize the Nef dimer for functional interaction with other signaling partners.
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Affiliation(s)
- John Jeff Alvarado
- From the Departments of Microbiology and Molecular Genetics and Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 and
| | - Sreya Tarafdar
- From the Departments of Microbiology and Molecular Genetics and Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
| | - Joanne I Yeh
- Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 and
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Hashimoto M, Nasser H, Chihara T, Suzu S. Macropinocytosis and TAK1 mediate anti-inflammatory to pro-inflammatory macrophage differentiation by HIV-1 Nef. Cell Death Dis 2014; 5:e1267. [PMID: 24874739 PMCID: PMC4047869 DOI: 10.1038/cddis.2014.233] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2014] [Revised: 03/23/2014] [Accepted: 03/28/2014] [Indexed: 01/02/2023]
Abstract
Macrophages (MΦ) are functionally classified into two types, anti-inflammatory M2 and pro-inflammatory M1. Importantly, we recently revealed that soluble HIV-1 proteins, particularly the pathogenetic protein Nef, preferentially activate M2-MΦ and drive them towards an M1-like MΦ, which might explain the sustained immune activation seen in HIV-1-infected patients. Here, we show that the preferential effect of Nef on M2-MΦ is mediated by TAK1 (TGF-β-activated kinase 1) and macropinocytosis. As with MAP kinases and NF-κB pathway, Nef markedly activated TAK1 in M-CSF-derived M2-MΦ but not in GM-CSF-derived M1-MΦ. Two Nef mutants, which were unable to activate MAP kinases and NF-κB pathway, failed to activate TAK1. Indeed, the TAK1 inhibitor 5Z-7-oxozeaenol as well as the ectopic expression of a dominant-negative mutant of TAK1 or TRAF2, an upstream molecule of TAK1, inhibited Nef-induced signaling activation and M1-like phenotypic differentiation of M2-MΦ. Meanwhile, the preferential effect of Nef on M2-MΦ correlated with the fact the Nef entered M2-MΦ more efficiently than M1-MΦ. Importantly, the macropinosome formation inhibitor EIPA completely blocked the internalization of Nef into M2-MΦ. Because the macropinocytosis activity of M2-MΦ was higher than that of M1-MΦ, our findings indicate that Nef enters M2-MΦ efficiently by exploiting their higher macropinocytosis activity and drives them towards M1-like MΦ by activating TAK1.
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Affiliation(s)
- M Hashimoto
- Center for AIDS Research, International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto, Japan
| | - H Nasser
- Center for AIDS Research, International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto, Japan
| | - T Chihara
- Center for AIDS Research, International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto, Japan
| | - S Suzu
- Center for AIDS Research, International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto, Japan
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Tarafdar S, Poe JA, Smithgall TE. The accessory factor Nef links HIV-1 to Tec/Btk kinases in an Src homology 3 domain-dependent manner. J Biol Chem 2014; 289:15718-28. [PMID: 24722985 DOI: 10.1074/jbc.m114.572099] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
The HIV-1 Nef virulence factor interacts with multiple host cell-signaling proteins. Nef binds to the Src homology 3 domains of Src family kinases, resulting in kinase activation important for viral infectivity, replication, and MHC-I down-regulation. Itk and other Tec family kinases are also present in HIV target cells, and Itk has been linked to HIV-1 infectivity and replication. However, the molecular mechanism linking Itk to HIV-1 is unknown. In this study, we explored the interaction of Nef with Tec family kinases using a cell-based bimolecular fluorescence complementation assay. In this approach, interaction of Nef with a partner kinase juxtaposes nonfluorescent YFP fragments fused to the C terminus of each protein, resulting in YFP complementation and a bright fluorescent signal. Using bimolecular fluorescence complementation, we observed that Nef interacts with the Tec family members Bmx, Btk, and Itk but not Tec or Txk. Interaction with Nef occurs through the kinase Src homology 3 domains and localizes to the plasma membrane. Allelic variants of Nef from all major HIV-1 subtypes interacted strongly with Itk in this assay, demonstrating the highly conserved nature of this interaction. A selective small molecule inhibitor of Itk kinase activity (BMS-509744) potently blocked wild-type HIV-1 infectivity and replication, but not that of a Nef-defective mutant. Nef induced constitutive Itk activation in transfected cells that was sensitive to inhibitor treatment. Taken together, these results provide the first evidence that Nef interacts with cytoplasmic tyrosine kinases of the Tec family and suggest that Nef provides a mechanistic link between HIV-1 and Itk signaling in the viral life cycle.
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Affiliation(s)
- Sreya Tarafdar
- From the Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 and the Department of Infectious Diseases and Microbiology, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania 15261
| | - Jerrod A Poe
- From the Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 and
| | - Thomas E Smithgall
- From the Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 and
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Trible RP, Narute P, Emert-Sedlak LA, Alvarado JJ, Atkins K, Thomas L, Kodama T, Yanamala N, Korotchenko V, Day BW, Thomas G, Smithgall TE. Discovery of a diaminoquinoxaline benzenesulfonamide antagonist of HIV-1 Nef function using a yeast-based phenotypic screen. Retrovirology 2013; 10:135. [PMID: 24229420 PMCID: PMC3874621 DOI: 10.1186/1742-4690-10-135] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2013] [Accepted: 10/31/2013] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex. RESULTS Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades. CONCLUSIONS Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I.
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Affiliation(s)
- Ronald P Trible
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
| | - Purushottam Narute
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
| | - Lori A Emert-Sedlak
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
| | - John Jeff Alvarado
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
| | - Katelyn Atkins
- School of Medicine, Oregon Health and Science University, 97239, Portland, OR, USA
| | - Laurel Thomas
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
| | - Toshiaki Kodama
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
| | - Naveena Yanamala
- Department of Structural Biology, University of Pittsburgh School of Medicine, 15261, Pittsburgh, PA USA
| | - Vasiliy Korotchenko
- Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, 15261, Pittsburgh, PA USA
| | - Billy W Day
- Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, 15261, Pittsburgh, PA USA
| | - Gary Thomas
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
| | - Thomas E Smithgall
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 15219, Pittsburgh, PA USA
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Witkowski W, Verhasselt B. Contributions of HIV-1 Nef to immune dysregulation in HIV-infected patients: a therapeutic target? Expert Opin Ther Targets 2013; 17:1345-56. [PMID: 23967871 DOI: 10.1517/14728222.2013.830712] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
INTRODUCTION HIV accessory protein Nef is a factor responsible for many of the viral pathogenic effects. Progression to AIDS is dramatically delayed and in some well-documented cases completely abolished on infection with naturally occurring HIV strains lacking intact nef sequences in their genomes. The topic of this review is the contribution of Nef to the immune pathology as a possible target in HIV-infected patients. AREAS COVERED An overview of known Nef functions accounting for its role in pathogenesis is presented, emphasizing interactions with dendritic cells and macrophages, and Nef-induced exosome secretion, all involved in immune dysregulation during the course of HIV infection. Current approaches to Nef inhibition by different classes of compounds are reviewed. EXPERT OPINION Blocking Nef for therapeutic purposes is a challenging endeavor mainly due to intrinsic properties of this HIV accessory protein. Nef has multiple interfaces to interact with host proteins and lacks a catalytic domain. Potential benefits arising from the development of successful inhibitors could however prove beneficial for reducing gradual deterioration of immune system in chronically infected patients in absence of functional cure.
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Affiliation(s)
- Wojciech Witkowski
- Department of Clinical Chemistry, Microbiology and Immunology of Ghent University , Gent , Belgium +32 93323658 ; +32 93323659 ;
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Effector kinase coupling enables high-throughput screens for direct HIV-1 Nef antagonists with antiretroviral activity. ACTA ACUST UNITED AC 2013; 20:82-91. [PMID: 23352142 DOI: 10.1016/j.chembiol.2012.11.005] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2012] [Revised: 11/19/2012] [Accepted: 11/21/2012] [Indexed: 12/12/2022]
Abstract
HIV-1 Nef, a critical AIDS progression factor, represents an important target protein for antiretroviral drug discovery. Because Nef lacks intrinsic enzymatic activity, we developed an assay that couples Nef to the activation of Hck, a Src family member and Nef effector protein. Using this assay, we screened a large, diverse chemical library and identified small molecules that block Nef-dependent Hck activity with low micromolar potency. Of these, a diphenylpyrazolo compound demonstrated submicromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound binds directly to Nef via a pocket formed by the Nef dimerization interface and disrupts Nef dimerization in cells. Coupling of nonenzymatic viral accessory factors to host cell effector proteins amenable to high-throughput screening may represent a general strategy for the discovery of new antimicrobial agents.
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Pagano MA, Tibaldi E, Palù G, Brunati AM. Viral proteins and Src family kinases: Mechanisms of pathogenicity from a “liaison dangereuse”. World J Virol 2013; 2:71-78. [PMID: 24175231 PMCID: PMC3785045 DOI: 10.5501/wjv.v2.i2.71] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2012] [Revised: 01/07/2013] [Accepted: 01/24/2013] [Indexed: 02/05/2023] Open
Abstract
To complete their life cycle and spread, viruses interfere with and gain control of diverse cellular processes, this most often occurring through interaction between viral proteins (VPs) and resident protein partners. Among the latter, Src family kinases (SFKs), a class of non-receptor tyrosine kinases that contributes to the conversion of extracellular signals into intracellular signaling cascades and is involved in virtually all cellular processes, have recently emerged as critical mediators between the cell’s infrastructure and the viral demands. In this scenario, structural or ex novo synthesized VPs are able to bind to the different domains of these enzymes through specific short linear motifs present along their sequences. Proline-rich motifs displaying the conserved minimal consensus PxxP and recognizing the SFK Src homology (SH)3 domain constitute a cardinal signature for the formation of multiprotein complexes and this interaction may promote phosphorylation of VPs by SFKs, thus creating phosphotyrosine motifs that become a docking site for the SH2 domains of SFKs or other SH2 domain-bearing signaling molecules. Importantly, the formation of these assemblies also results in a change in the activity and/or location of SFKs, and these events are critical in perturbing key signaling pathways so that viruses can utilize the cell’s machinery to their own benefit. In the light of these observations, although VPs as such, especially those with enzyme activity, are still regarded as valuable targets for therapeutic strategies, multiprotein complexes composed of viral and host cell proteins are increasingly becoming objects of investigation with a view to deeply characterize the structural aspects that favor their formation and to develop new compounds able to contrast viral diseases in an alternative manner.
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Markle TJ, Philip M, Brockman MA. HIV-1 Nef and T-cell activation: a history of contradictions. Future Virol 2013; 8. [PMID: 24187576 DOI: 10.2217/fvl.13.20] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
HIV-1 Nef is a multifunctional viral protein that contributes to higher plasma viremia and more rapid disease progression. Nef appears to accomplish this, in part, through modulation of T-cell activation; however, the results of these studies over the past 25 years have been inconsistent. Here, the history of contradictory observations related to HIV-1 Nef and its ability to modulate T-cell activation is reviewed, and recent reports that may help to explain Net's apparent ability to both inhibit and activate T cells are highlighted.
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Affiliation(s)
- Tristan J Markle
- Simon Fraser University, 8888 University Drive, Burnaby BC V5A 1S6, Canada
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Jin YJ, Cai CY, Mezei M, Ohlmeyer M, Sanchez R, Burakoff SJ. Identification of a novel binding site between HIV type 1 Nef C-terminal flexible loop and AP2 required for Nef-mediated CD4 downregulation. AIDS Res Hum Retroviruses 2013; 29:725-31. [PMID: 23151229 DOI: 10.1089/aid.2012.0286] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
HIV-1 Nef is an accessory protein necessary for HIV-1 virulence and rapid AIDS development. Nef promotes viral replication and infection by connecting CD4 and several other cell surface receptors to the clathrin adaptor protein AP2, resulting in the internalization and degradation of the receptors interacting with Nef. We investigated how Nef can mediate constitutive receptor endocytosis through the interaction of the dileucine motif in its C-terminal flexible loop (C-loop) with AP2, whereas AP2 binding of the transmembrane receptors usually results in an equilibrated (recycled) endocytosis. Our results indicated that in addition to the dileucine motif, there is a second motif in the Nef C-loop involved in the Nef-AP2 interaction. Nef-mediated CD4 downregulation was impaired when the residue in the hydrophobic region in the Nef C-loop (LL165HPMSLHGM173) was mutated to a basic residue K/R or an acidic residue E/D or to the rigid residue P, or when M168L170, L170H171, or G172M173 was mutated to AA. A pull-down assay indicated that AP2 was not coprecipitated with Nef mutants that did not downregulate CD4. Molecular modeling of the Nef C-terminal flexible loop in complex with AP2 suggests that M168L170 occupies a pocket in the AP2 σ2 subunit. Our data suggest a new model in the Nef-AP2 interaction in which the hydrophobic region in the Nef C-loop with the dileucine (L164L165) motif and M168L170 motif binds to AP2(σ2), while the acidic motif E174 and D175 binds to AP2(α), which explains how Nef through the flexible loop connects CD4 to AP2 for constitutive CD4 downregulation.
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Affiliation(s)
- Yong-Jiu Jin
- Department of Oncological Sciences, Cancer Institute, Mount Sinai School of Medicine, New York, New York
| | - Catherine Yi Cai
- Department of Oncological Sciences, Cancer Institute, Mount Sinai School of Medicine, New York, New York
| | - Mihaly Mezei
- Department of Structural and Chemical Biology, Cancer Institute, Mount Sinai School of Medicine, New York, New York
- Experimental Therapeutics Institute, Cancer Institute, Mount Sinai School of Medicine, New York, New York
| | - Michael Ohlmeyer
- Department of Structural and Chemical Biology, Cancer Institute, Mount Sinai School of Medicine, New York, New York
- Experimental Therapeutics Institute, Cancer Institute, Mount Sinai School of Medicine, New York, New York
| | - Roberto Sanchez
- Department of Structural and Chemical Biology, Cancer Institute, Mount Sinai School of Medicine, New York, New York
- Experimental Therapeutics Institute, Cancer Institute, Mount Sinai School of Medicine, New York, New York
| | - Steven J. Burakoff
- Department of Oncological Sciences, Cancer Institute, Mount Sinai School of Medicine, New York, New York
- Cancer Institute, Mount Sinai School of Medicine, New York, New York
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Saxena SK, Shrivastava G, Tiwari S, Swamy MA, Nair MP. Modulation of HIV pathogenesis and T-cell signaling by HIV-1 Nef. Future Virol 2012; 7:609-620. [PMID: 22844345 DOI: 10.2217/fvl.12.42] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
HIV-1 Nef protein is an approximately 27-kDa myristoylated protein that is a virulence factor essential for efficient viral replication and infection in CD4(+) T cells. The functions of CD4(+) T cells are directly impeded after HIV infection. HIV-1 Nef plays a crucial role in manipulating host cellular machinery and in HIV pathogenesis by reducing the ability of infected lymphocytes to form immunological synapses by promoting virological synapses with APCs, and by affecting T-cell stimulation. This article reviews the current status of the efficient Nef-mediated spread of virus in the unreceptive environment of the immune system by altering CD4(+) T-lymphocyte signaling, intracellular trafficking, cell migration and apoptotic pathways.
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Affiliation(s)
- Shailendra K Saxena
- CSIR-Centre for Cellular & Molecular Biology, Uppal Road, Hyderabad 500007 (AP), India
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Kuo LS, Baugh LL, Denial SJ, Watkins RL, Liu M, Garcia JV, Foster JL. Overlapping effector interfaces define the multiple functions of the HIV-1 Nef polyproline helix. Retrovirology 2012; 9:47. [PMID: 22651890 PMCID: PMC3464899 DOI: 10.1186/1742-4690-9-47] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2012] [Accepted: 05/31/2012] [Indexed: 11/20/2022] Open
Abstract
Background HIV-1 Nef is a multifunctional protein required for full pathogenicity of the virus. As Nef has no known enzymatic activity, it necessarily functions through protein-protein interaction interfaces. A critical Nef protein interaction interface is centered on its polyproline segment (P69VRPQVPLRP78) which contains the helical SH3 domain binding protein motif, PXXPXR. We hypothesized that any Nef-SH3 domain interactions would be lost upon mutation of the prolines or arginine of PXXPXR. Further, mutation of the non-motif “X” residues, (Q73, V74, and L75) would give altered patterns of inhibition for different Nef/SH3 domain protein interactions. Results We found that mutations of either of the prolines or the arginine of PXXPXR are defective for Nef-Hck binding, Nef/activated PAK2 complex formation and enhancement of virion infectivity (EVI). Mutation of the non-motif “X” residues (Q, V and L) gave similar patterns of inhibition for Nef/activated PAK2 complex formation and EVI which were distinct from the pattern for Hck binding. These results implicate an SH3 domain containing protein other than Hck for Nef/activated PAK2 complex formation and EVI. We have also mutated Nef residues at the N-and C-terminal ends of the polyproline segment to explore interactions outside of PXXPXR. We discovered a new locus GFP/F (G67, F68, P69 and F90) that is required for Nef/activated PAK2 complex formation and EVI. MHC Class I (MHCI) downregulation was only partially inhibited by mutating the PXXPXR motif residues, but was fully inhibited by mutating the C-terminal P78. Further, we observed that MHCI downregulation strictly requires G67 and F68. Our mutational analysis confirms the recently reported structure of the complex between Nef, AP-1 μ1 and the cytoplasmic tail of MHCI, but does not support involvement of an SH3 domain protein in MHCI downregulation. Conclusion Nef has evolved to be dependent on interactions with multiple SH3 domain proteins. To the N- and C- terminal sides of the polyproline helix are multifunctional protein interaction sites. The polyproline segment is also adapted to downregulate MHCI with a non-canonical binding surface. Our results demonstrate that Nef polyproline helix is highly adapted to directly interact with multiple host cell proteins.
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Affiliation(s)
- Lillian S Kuo
- Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Y9.206, Dallas, TX 75390, USA
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45
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Chihara T, Hashimoto M, Osman A, Hiyoshi-Yoshidomi Y, Suzu I, Chutiwitoonchai N, Hiyoshi M, Okada S, Suzu S. HIV-1 proteins preferentially activate anti-inflammatory M2-type macrophages. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2012; 188:3620-3627. [PMID: 22407921 DOI: 10.4049/jimmunol.1101593] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
HIV-1 proteins, including Tat, gp120, and Nef, activate macrophages (MΦ), which is consistent with the fact that HIV-1 infection is characterized by sustained immune activation. Meanwhile, MΦ are functionally classified into two types: proinflammatory M1-MΦ and anti-inflammatory M2-MΦ. We show that HIV-1 proteins, particularly Nef, preferentially activate M2-MΦ. Extracellular Tat, gp120, and Nef activated MAPK and NF-κB pathways in human peripheral blood monocyte-derived MΦ. However, the activation was marked in M-CSF-derived M2-MΦ but not GM-CSF-derived M1-MΦ. Nef was the most potent activator, and its signaling activation was comparable to that by TNF-α. Indeed, Nef was internalized more rapidly by M2-MΦ than by M1-MΦ. The myristoylation and proline-rich motif of Nef were responsible for the observed signaling activation. Consistent with the activation of MAPK/NF-κB pathways, Nef stimulated the production of a number of proinflammatory cytokines/chemokines by M2-MΦ. However, Nef reduced the expression of CD163 and phagocytosis, the characteristic markers of M2-MΦ, indicating that Nef drives an M2-like to M1-like phenotypic shift. Because the differentiation of most tissue MΦ depends on M-CSF and its receptor, which is the essential axis for the anti-inflammatory M2-MΦ phenotype, the current study reveals an efficient mechanism by which HIV-1 proteins, such as Nef, induce the proinflammatory MΦ.
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Affiliation(s)
- Takashi Chihara
- Center for AIDS Research, Kumamoto University, Kumamoto 860-0811, Japan
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Dikeakos JD, Thomas L, Kwon G, Elferich J, Shinde U, Thomas G. An interdomain binding site on HIV-1 Nef interacts with PACS-1 and PACS-2 on endosomes to down-regulate MHC-I. Mol Biol Cell 2012; 23:2184-97. [PMID: 22496420 PMCID: PMC3364181 DOI: 10.1091/mbc.e11-11-0928] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
HIV-1 Nef pirates PACS-1 and PACS-2 to downregulate MHC-I, but little is known about the Nef–PACS interaction. The sites on Nef and the PACS proteins required for their interaction are identified, and their importance for Nef trafficking and Nef-induced MHC-I downregulation is discussed. The results provide insight into the molecular basis of Nef action. The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef directs virus escape from immune surveillance by subverting host cell intracellular signaling and membrane traffic to down-regulate cell-surface major histocompatibility complex class I (MHC-I). The interaction of Nef with the sorting proteins PACS-1 and PACS-2 mediates key signaling and trafficking steps required for Nef-mediated MHC-I down-regulation. Little is known, however, about the molecular basis underlying the Nef–PACS interaction. Here we identify the sites on Nef and the PACS proteins required for their interaction and describe the consequences of disrupting this interaction for Nef action. A previously unidentified cargo subsite on PACS-1 and PACS-2 interacted with a bipartite site on Nef formed by the EEEE65 acidic cluster on the N-terminal domain and W113 in the core domain. Mutation of these sites prevented the interaction between Nef and the PACS proteins on Rab5 (PACS-2 and PACS-1)- or Rab7 (PACS-1)-positive endosomes as determined by bimolecular fluorescence complementation and caused a Nef mutant defective in PACS binding to localize to distorted endosomal compartments. Consequently, disruption of the Nef–PACS interaction repressed Nef-induced MHC-I down-regulation in peripheral blood mononuclear cells. Our results provide insight into the molecular basis of Nef action and suggest new strategies to combat HIV-1.
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Affiliation(s)
- Jimmy D Dikeakos
- Vollum Institute, Oregon Health and Science University, Portland, OR 97239, USA
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47
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Compeer EB, Flinsenberg TWH, van der Grein SG, Boes M. Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation. Front Immunol 2012; 3:37. [PMID: 22566920 PMCID: PMC3342355 DOI: 10.3389/fimmu.2012.00037] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2011] [Accepted: 02/16/2012] [Indexed: 12/29/2022] Open
Abstract
Cross-presentation of endocytosed antigen as peptide/class I major histocompatibility complex complexes plays a central role in the elicitation of CD8+ T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells capable of antigen cross-presentation, identification of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC), there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlights DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, maturation-induced endosomal sorting of membrane proteins, dynamic remodeling of endosomal structures and cell surface-directed endosomal trafficking. We will conclude with the description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.
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Affiliation(s)
- Ewoud Bernardus Compeer
- Department of Pediatric Immunology, University Medical Center Utrecht/Wilhelmina Children's Hospital Utrecht, Netherlands
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48
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Single-domain antibody-SH3 fusions for efficient neutralization of HIV-1 Nef functions. J Virol 2012; 86:4856-67. [PMID: 22345475 DOI: 10.1128/jvi.06329-11] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
HIV-1 Nef is essential for AIDS pathogenesis, but this viral protein is not targeted by antiviral strategies. The functions of Nef are largely related to perturbations of intracellular trafficking and signaling pathways through leucine-based and polyproline motifs that are required for interactions with clathrin-associated adaptor protein complexes and SH3 domain-containing proteins, such as the phagocyte-specific kinase Hck. We previously described a single-domain antibody (sdAb) targeting Nef and inhibiting many, but not all, of its biological activities. We now report a further development of this anti-Nef strategy through the demonstration of the remarkable inhibitory activity of artificial Nef ligands, called Neffins, comprised of the anti-Nef sdAb fused to modified SH3 domains. The Neffins inhibited all key activities of Nef, including Nef-mediated CD4 and major histocompatibility complex class I (MHC-I) cell surface downregulation and enhancement of virus infectivity. When expressed in T lymphocytes, Neffins specifically inhibited the Nef-induced mislocalization of the Lck kinase, which contributes to the alteration of the formation of the immunological synapse. In macrophages, Neffins inhibited the Nef-induced formation of multinucleated giant cells and podosome rosettes, and it counteracted the inhibitory activity of Nef on phagocytosis. Since we show here that these effects of Nef on macrophage and T cell functions were both dependent on the leucine-based and polyproline motifs, we confirmed that Neffins disrupted interactions of Nef with both AP complexes and Hck. These results demonstrate that it is possible to inhibit all functions of Nef, both in T lymphocytes and macrophages, with a single ligand that represents an efficient tool to develop new antiviral strategies targeting Nef.
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The human immunodeficiency virus type 1 Nef and Vpu proteins downregulate the natural killer cell-activating ligand PVR. J Virol 2012; 86:4496-504. [PMID: 22301152 DOI: 10.1128/jvi.05788-11] [Citation(s) in RCA: 103] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
The human immunodeficiency virus type 1 (HIV-1) evades the immune responses of natural killer (NK) cells through mechanisms that have been partially deciphered. Here we show that in HIV-1-infected T lymphocytes, the early viral Nef protein downmodulates PVR (CD155, Necl-5), a ligand for the activating receptor DNAM-1 (CD226) expressed by all NK cells, CD8(+) T cells, and other cell types. This novel Nef activity is conserved by Nef proteins of laboratory HIV-1 strains (NL4-3, SF2) and of a patient-derived virus, but it is not maintained by HIV-2. Nef uses the same motifs to downregulate PVR and HLA-I molecules, likely by the same mechanisms. Indeed, as previously demonstrated for HLA-I, Nef reduces the total amounts of cell-associated PVR. Optimal downregulation of cell surface PVR by Nef also requires the presence of the late viral factor Vpu. In line with PVR reduction, the NK cell-mediated lysis of T cells infected by a wild-type but not Nef-deficient virus is virtually abrogated upon blocking of both DNAM-1 and another activating receptor, NKG2D, previously shown to mediate killing of HIV-infected cells. Together, these data demonstrate that the PVR downmodulation by Nef and Vpu is a strategy evolved by HIV-1 to prevent NK cell-mediated lysis of infected cells. The PVR downregulation reported here has the potential to affect the immune responses of other DNAM-1-positive cells besides NK cells and to alter multiple PVR-mediated cellular processes, such as adhesion and migration, and may thus greatly influence HIV-1 pathogenesis.
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Chutiwitoonchai N, Hiyoshi M, Mwimanzi P, Ueno T, Adachi A, Ode H, Sato H, Fackler OT, Okada S, Suzu S. The identification of a small molecule compound that reduces HIV-1 Nef-mediated viral infectivity enhancement. PLoS One 2011; 6:e27696. [PMID: 22110726 PMCID: PMC3217016 DOI: 10.1371/journal.pone.0027696] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2011] [Accepted: 10/22/2011] [Indexed: 12/14/2022] Open
Abstract
Nef is a multifunctional HIV-1 protein that accelerates progression to AIDS, and enhances the infectivity of progeny viruses through a mechanism that is not yet understood. Here, we show that the small molecule compound 2c reduces Nef-mediated viral infectivity enhancement. When added to viral producer cells, 2c did not affect the efficiency of viral production itself. However, the infectivity of the viruses produced in the presence of 2c was significantly lower than that of control viruses. Importantly, an inhibitory effect was observed with Nef(+) wild-type viruses, but not with viruses produced in the absence of Nef or in the presence of proline-rich PxxP motif-disrupted Nef, both of which displayed significantly reduced intrinsic infectivity. Meanwhile, the overexpression of the SH3 domain of the tyrosine kinase Hck, which binds to a PxxP motif in Nef, also reduced viral infectivity. Importantly, 2c inhibited Hck SH3-Nef binding, which was more marked when Nef was pre-incubated with 2c prior to its incubation with Hck, indicating that both Hck SH3 and 2c directly bind to Nef and that their binding sites overlap. These results imply that both 2c and the Hck SH3 domain inhibit the interaction of Nef with an unidentified host protein and thereby reduce Nef-mediated infectivity enhancement. The first inhibitory compound 2c is therefore a valuable chemical probe for revealing the underlying molecular mechanism by which Nef enhances the infectivity of HIV-1.
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Affiliation(s)
| | | | - Philip Mwimanzi
- Center for AIDS Research, Kumamoto University, Kumamoto, Japan
| | - Takamasa Ueno
- Center for AIDS Research, Kumamoto University, Kumamoto, Japan
| | - Akio Adachi
- Department of Microbiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
| | - Hirotaka Ode
- Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan
| | - Hironori Sato
- Pathogen Genomics Center, National Institute of Infectious Diseases, Tokyo, Japan
| | - Oliver T. Fackler
- Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany
| | - Seiji Okada
- Center for AIDS Research, Kumamoto University, Kumamoto, Japan
| | - Shinya Suzu
- Center for AIDS Research, Kumamoto University, Kumamoto, Japan
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