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Deng Y, Navarro-Forero S, Yang Z. Temporal expression classes and functions of vaccinia virus and mpox (monkeypox) virus genes. mBio 2025; 16:e0380924. [PMID: 40111027 PMCID: PMC11980589 DOI: 10.1128/mbio.03809-24] [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] [Indexed: 03/22/2025] Open
Abstract
Poxviruses comprise pathogens that are highly pathogenic to humans and animals, causing diseases such as smallpox and mpox (formerly monkeypox). The family also contains members developed as vaccine vectors and oncolytic agents to fight other diseases. Vaccinia virus is the prototype poxvirus and the vaccine used to eradicate smallpox. Poxvirus genes follow a cascade temporal expression pattern, categorized into early, intermediate, and late stages using distinct transcription factors. This review comprehensively summarized the temporal expression classification of over 200 vaccinia virus genes. The relationships between expression classes and functions, as well as different branches of immune responses, were discussed. Based on the vaccinia virus orthologs, we classified the temporal expression classes of all the mpox virus genes, including a few that were not previously annotated with orthologs in vaccinia viruses. Additionally, we reviewed the functions of all vaccinia virus genes based on the up-to-date published papers. This review provides a readily usable resource for researchers working on poxvirus biology, medical countermeasures, and poxvirus utility development.
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Affiliation(s)
- Yining Deng
- Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, Texas, USA
| | - Santiago Navarro-Forero
- Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, Texas, USA
| | - Zhilong Yang
- Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, Texas, USA
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2
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Molteni C, Forni D, Cagliani R, Bravo IG, Sironi M. Evolution and diversity of nucleotide and dinucleotide composition in poxviruses. J Gen Virol 2023; 104. [PMID: 37792576 DOI: 10.1099/jgv.0.001897] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/06/2023] Open
Abstract
Poxviruses (family Poxviridae) have long dsDNA genomes and infect a wide range of hosts, including insects, birds, reptiles and mammals. These viruses have substantial incidence, prevalence and disease burden in humans and in other animals. Nucleotide and dinucleotide composition, mostly CpG and TpA, have been largely studied in viral genomes because of their evolutionary and functional implications. We analysed here the nucleotide and dinucleotide composition, as well as codon usage bias, of a set of representative poxvirus genomes, with a very diverse host spectrum. After correcting for overall nucleotide composition, entomopoxviruses displayed low overall GC content, no enrichment in TpA and large variation in CpG enrichment, while chordopoxviruses showed large variation in nucleotide composition, no obvious depletion in CpG and a weak trend for TpA depletion in GC-rich genomes. Overall, intergenome variation in dinucleotide composition in poxviruses is largely accounted for by variation in overall genomic GC levels. Nonetheless, using vaccinia virus as a model, we found that genes expressed at the earliest times in infection are more CpG-depleted than genes expressed at later stages. This observation has parallels in betahepesviruses (also large dsDNA viruses) and suggests an antiviral role for the innate immune system (e.g. via the zinc-finger antiviral protein ZAP) in the early phases of poxvirus infection. We also analysed codon usage bias in poxviruses and we observed that it is mostly determined by genomic GC content, and that stratification after host taxonomy does not contribute to explaining codon usage bias diversity. By analysis of within-species diversity, we show that genomic GC content is the result of mutational biases. Poxvirus genomes that encode a DNA ligase are significantly AT-richer than those that do not, suggesting that DNA repair systems shape mutation biases. Our data shed light on the evolution of poxviruses and inform strategies for their genetic manipulation for therapeutic purposes.
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Affiliation(s)
- Cristian Molteni
- Scientific Institute IRCCS E. MEDEA, Bioinformatics, Bosisio Parini, Italy
| | - Diego Forni
- Scientific Institute IRCCS E. MEDEA, Bioinformatics, Bosisio Parini, Italy
| | - Rachele Cagliani
- Scientific Institute IRCCS E. MEDEA, Bioinformatics, Bosisio Parini, Italy
| | - Ignacio G Bravo
- Laboratoire MIVEGEC (Univ Montpellier CNRS, IRD), Centre National de la Recherche Scientifique, Montpellier, France
| | - Manuela Sironi
- Scientific Institute IRCCS E. MEDEA, Bioinformatics, Bosisio Parini, Italy
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3
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Kang Y. Landscape of NcRNAs involved in drug resistance of breast cancer. Clin Transl Oncol 2023; 25:1869-1892. [PMID: 37067729 PMCID: PMC10250522 DOI: 10.1007/s12094-023-03189-3] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2022] [Accepted: 07/02/2022] [Indexed: 04/18/2023]
Abstract
Breast cancer (BC) leads to the most amounts of deaths among women. Chemo-, endocrine-, and targeted therapies are the mainstay drug treatments for BC in the clinic. However, drug resistance is a major obstacle for BC patients, and it leads to poor prognosis. Accumulating evidences suggested that noncoding RNAs (ncRNAs) are intricately linked to a wide range of pathological processes, including drug resistance. Till date, the correlation between drug resistance and ncRNAs is not completely understood in BC. Herein, we comprehensively summarized a dysregulated ncRNAs landscape that promotes or inhibits drug resistance in chemo-, endocrine-, and targeted BC therapies. Our review will pave way for the effective management of drug resistance by targeting oncogenic ncRNAs, which, in turn will promote drug sensitivity of BC in the future.
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Affiliation(s)
- Yujuan Kang
- Department of Breast Surgery, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China.
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Byareddy SN, Sharma K, Sachdev S, Reddy AS, Acharya A, Klaustermeier KM, Lorson CL, Singh K. Potential therapeutic targets for Mpox: the evidence to date. Expert Opin Ther Targets 2023; 27:419-431. [PMID: 37368464 PMCID: PMC10722886 DOI: 10.1080/14728222.2023.2230361] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2023] [Revised: 06/07/2023] [Accepted: 06/23/2023] [Indexed: 06/28/2023]
Abstract
INTRODUCTION The global Mpox (MPX) disease outbreak caused by the Mpox virus (MPXV) in 2022 alarmed the World Health Organization (WHO) and health regulation agencies of individual countries leading to the declaration of MPX as a Public Health Emergency. Owing to the genetic similarities between smallpox-causing poxvirus and MPXV, vaccine JYNNEOS, and anti-smallpox drugs Brincidofovir and Tecovirimat were granted emergency use authorization by the United States Food and Drug Administration. The WHO also included cidofovir, NIOCH-14, and other vaccines as treatment options. AREAS COVERED This article covers the historical development of EUA-granted antivirals, resistance to these antivirals, and the projected impact of signature mutations on the potency of antivirals against currently circulating MPXV. Since a high prevalence of MPXV infections in individuals coinfected with HIV and MPXV, the treatment results among these individuals have been included. EXPERT OPINION All EUA-granted drugs have been approved for smallpox treatment. These antivirals show good potency against Mpox. However, conserved resistance mutation positions in MPXV and related poxviruses, and the signature mutations in the 2022 MPXV can potentially compromise the efficacy of the EUA-granted treatments. Therefore, MPXV-specific medications are required not only for the current but also for possible future outbreaks.
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Affiliation(s)
- Siddappa N Byareddy
- Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | | | - Shrikesh Sachdev
- Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA
| | - Athreya S. Reddy
- Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA
| | - Arpan Acharya
- Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | | | - Christian L Lorson
- Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA
- Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211, USA
| | - Kamal Singh
- Department of Pharmaceutical Chemistry, DPSRU, New Delhi-110017
- Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA
- Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211, USA
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Abstract
Genetic recombination is used as a tool for modifying the composition of poxvirus genomes in both discovery and applied research. This review documents the history behind the development of these tools as well as what has been learned about the processes that catalyze virus recombination and the links between it and DNA replication and repair. The study of poxvirus recombination extends back to the 1930s with the discovery that one virus can reactivate another by a process later shown to generate recombinants. In the years that followed it was shown that recombinants can be produced in virus-by-virus crosses within a genus (e.g., variola-by-rabbitpox) and efforts were made to produce recombination-based genetic maps with modest success. The marker rescue mapping method proved more useful and led to methods for making genetically engineered viruses. Many further insights into the mechanism of recombination have been provided by transfection studies which have shown that this is a high-frequency process associated with hybrid DNA formation and inextricably linked to replication. The links reflect the fact that poxvirus DNA polymerases, specifically the vaccinia virus E9 enzyme, can catalyze strand transfer in in vivo and in vitro reactions dependent on the 3'-to-5' proofreading exonuclease and enhanced by the I3 replicative single-strand DNA binding protein. These reactions have shaped the composition of virus genomes and are modulated by constraints imposed on virus-virus interactions by viral replication in cytoplasmic factories. As recombination reactions are used for replication fork assembly and repair in many biological systems, further study of these reactions may provide new insights into still poorly understood features of poxvirus DNA replication.
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Affiliation(s)
- David Hugh Evans
- Department of Medical Microbiology & Immunology and Li Ka Shing Institute of Virology, The University of Alberta, Edmonton, AB T6G 2J7, Canada
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6
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Abstract
Poxviruses, of which vaccinia virus is the prototype, are a large family of double-stranded DNA viruses that replicate exclusively in the cytoplasm of infected cells. This physical and genetic autonomy from the host cell nucleus necessitates that these viruses encode most, if not all, of the proteins required for replication in the cytoplasm. In this review, we follow the life of the viral genome through space and time to address some of the unique challenges that arise from replicating a 195-kb DNA genome in the cytoplasm. We focus on how the genome is released from the incoming virion and deposited into the cytoplasm; how the endoplasmic reticulum is reorganized to form a replication factory, thereby compartmentalizing and helping to protect the replicating genome from immune sensors; how the cellular milieu is tailored to support high-fidelity replication of the genome; and finally, how newly synthesized genomes are faithfully and specifically encapsidated into new virions. Expected final online publication date for the Annual Review of Virology, Volume 9 is September 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Affiliation(s)
- Matthew D Greseth
- Department of Biochemistry and Molecular Biology, The Medical University of South Carolina, Charleston, South Carolina, USA;
| | - Paula Traktman
- Department of Biochemistry and Molecular Biology, The Medical University of South Carolina, Charleston, South Carolina, USA; .,Department of Microbiology and Immunology, The Medical University of South Carolina, Charleston, South Carolina, USA
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Templeton CW, Traktman P. UV Irradiation of Vaccinia Virus-Infected Cells Impairs Cellular Functions, Introduces Lesions into the Viral Genome, and Uncovers Repair Capabilities for the Viral Replication Machinery. J Virol 2022; 96:e0213721. [PMID: 35404095 PMCID: PMC9093118 DOI: 10.1128/jvi.02137-21] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2021] [Accepted: 03/17/2022] [Indexed: 11/20/2022] Open
Abstract
Vaccinia virus (VV), the prototypic poxvirus, encodes a repertoire of proteins responsible for the metabolism of its large dsDNA genome. Previous work has furthered our understanding of how poxviruses replicate and recombine their genomes, but little is known about whether the poxvirus genome undergoes DNA repair. Our studies here are aimed at understanding how VV responds to exogenous DNA damage introduced by UV irradiation. Irradiation of cells prior to infection decreased protein synthesis and led to an ∼12-fold reduction in viral yield. On top of these cell-specific insults, irradiation of VV infections at 4 h postinfection (hpi) introduced both cyclobutene pyrimidine dimer (CPD) and 6,4-photoproduct (6,4-PP) lesions into the viral genome led to a nearly complete halt to further DNA synthesis and to a further reduction in viral yield (∼35-fold). DNA lesions persisted throughout infection and were indeed present in the genomes encapsidated into nascent virions. Depletion of several cellular proteins that mediate nucleotide excision repair (XP-A, -F, and -G) did not render viral infections hypersensitive to UV. We next investigated whether viral proteins were involved in combatting DNA damage. Infections performed with a virus lacking the A50 DNA ligase were moderately hypersensitive to UV irradiation (∼3-fold). More strikingly, when the DNA polymerase inhibitor cytosine arabinoside (araC) was added to wild-type infections at the time of UV irradiation (4 hpi), an even greater hypersensitivity to UV irradiation was seen (∼11-fold). Virions produced under the latter condition contained elevated levels of CPD adducts, strongly suggesting that the viral polymerase contributes to the repair of UV lesions introduced into the viral genome. IMPORTANCE Poxviruses remain of significant interest because of their continuing clinical relevance, their utility for the development of vaccines and oncolytic therapies, and their illustration of fundamental principles of viral replication and virus/cell interactions. These viruses are unique in that they replicate exclusively in the cytoplasm of infected mammalian cells, providing novel challenges for DNA viruses. How poxviruses replicate, recombine, and possibly repair their genomes is still only partially understood. Using UV irradiation as a form of exogenous DNA damage, we have examined how vaccinia virus metabolizes its genome following insult. We show that even UV irradiation of cells prior to infection diminishes viral yield, while UV irradiation during infection damages the genome, causes a halt in DNA accumulation, and reduces the viral yield more severely. Furthermore, we show that viral proteins, but not the cellular machinery, contribute to a partial repair of the viral genome following UV irradiation.
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Affiliation(s)
- Conor W. Templeton
- Departments of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, USA
| | - Paula Traktman
- Departments of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, USA
- Departments of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina, USA
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8
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Ke F, Zhang QY. ADRV 12L: A Ranaviral Putative Rad2 Family Protein Involved in DNA Recombination and Repair. Viruses 2022; 14:v14050908. [PMID: 35632650 PMCID: PMC9146916 DOI: 10.3390/v14050908] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2022] [Revised: 04/24/2022] [Accepted: 04/26/2022] [Indexed: 02/01/2023] Open
Abstract
The Andrias davidianus ranavirus (ADRV) is a member of the family Iridoviridae and belongs to the nucleocytoplasmic large DNA viruses. Based on genomic analysis, an ADRV-encoding protein, ADRV 12L, and its homologs from other iridoviruses were predicted as Rad2 family proteins based on the conserved amino acids, domains, and secondary structures. Expression analysis showed that the transcription of ADRV 12L started at 4 h post infection, and its expression was not inhibited by a DNA-replication inhibitor. Meanwhile, immunofluorescence localization showed that ADRV 12L mainly localized in viral factories and colocalized with the viral nascent DNA, which hinted at a possible role in DNA replication. Furthermore, a mutant ADRV lacking 12L (ADRV-Δ12L) was constructed. In both luciferase assays based on homologous recombination (HR) and double-strand break repair (DSBR) that followed, ADRV-Δ12L induced less luciferase activity than the wild-type ADRV, indicating that HR and DSBR were impaired in ADRV-Δ12L infected cells. In addition, infection with ADRV-Δ12L resulted in smaller plaque sizes and lower viral titers than that with wild-type ADRV, indicating an important role for 12L in efficient virus infection. Therefore, the results suggest that Rad2 homologs encoded by iridovirus have important roles in HR- and DSBR-process of the viral DNA and, thus, affect virus replication and the production of progeny virions.
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Affiliation(s)
- Fei Ke
- Institute of Hydrobiology, The Innovation Academy of Seed Design, Chinese Academy of Sciences, Wuhan 430072, China;
- College of Modern Agriculture Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
- Correspondence: ; Tel.: +86-027-6878-0002
| | - Qi-Ya Zhang
- Institute of Hydrobiology, The Innovation Academy of Seed Design, Chinese Academy of Sciences, Wuhan 430072, China;
- College of Modern Agriculture Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
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9
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Zheng Q, You YL, Li F, Lai QN, Chen JM. Interaction between 038R and 125R of Cherax quadricarinatus iridovirus (CQIV) and their effects on virus replication. J Invertebr Pathol 2021; 187:107699. [PMID: 34838791 DOI: 10.1016/j.jip.2021.107699] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2021] [Revised: 11/15/2021] [Accepted: 11/22/2021] [Indexed: 11/28/2022]
Abstract
Iridovirids are a group icosahedral viruses containing linear double-stranded DNA, and mainly infect invertebrates and poikilothermic vertebrates. Cherax quadricarinatus iridovirus (CQIV) is a new species of the family Iridoviridae and can cause high mortality in shrimps. In CQIV genome, there are 25 conserved genes and the putative products are involved in several viral processes. In this study, three core protein including CQIV-032R, CQIV-125R and CQIV-160L were identified to interact with CQIV-038R by yeast two-hybrid (Y2H), and the interaction between CQIV-038R and CQIV-125R was further confirmed by co-immunoprecipitation (Co-IP) assays. In the expression system, EGFP-038R and mCherry-125R were colocalized in the cytoplasm when co-expressed in Sf9 cells. Moreover, silencing the expression of 038R, 125R or both of these two proteins respectively in C. quadricarinatus cells by small interfering RNAs showed significantly inhibit CQIV replication. Collectively, we identified the interaction between 038R and 125R, and demonstrated they are essential for CQIV replication.
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Affiliation(s)
- Qin Zheng
- Institute of Oceanography, Minjiang University, Fuzhou 350108, China
| | - Yan-Lin You
- College of Biological Sciences and Engineering, Fuzhou University, Fuzhou 350108, China
| | - Fang Li
- Key Laboratory of Marine Genetic Resources, Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China
| | - Qing-Na Lai
- Institute of Oceanography, Minjiang University, Fuzhou 350108, China
| | - Jian-Ming Chen
- Institute of Oceanography, Minjiang University, Fuzhou 350108, China.
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10
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Vallée G, Norris P, Paszkowski P, Noyce RS, Evans DH. Vaccinia Virus Gene Acquisition through Nonhomologous Recombination. J Virol 2021; 95:e0031821. [PMID: 33910949 PMCID: PMC8223923 DOI: 10.1128/jvi.00318-21] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2021] [Accepted: 04/19/2021] [Indexed: 01/04/2023] Open
Abstract
Many of the genes encoded by poxviruses are orthologs of cellular genes. These virus genes serve different purposes, but perhaps of most interest is the way some have been repurposed to inhibit the antiviral pathways that their cellular homologs still regulate. What is unclear is how these virus genes were acquired, although it is presumed to have been catalyzed by some form(s) of nonhomologous recombination (NHR). We used transfection assays and substrates encoding a fluorescent and drug-selectable marker to examine the NHR frequency in vaccinia virus (VAC)-infected cells. These studies showed that when cells were transfected with linear duplex DNAs bearing VAC N2L gene homology, it yielded a recombinant frequency (RF) of 6.7 × 10-4. In contrast, DNA lacking any VAC homology reduced the yield of recombinants ∼400-fold (RF = 1.6 × 10-6). DNA-RNA hybrids were also substrates, although homologous molecules yielded fewer recombinants (RF = 2.1 × 10-5), and nonhomologous substrates yielded only rare recombinants (RF ≤ 3 × 10-8). NHR was associated with genome rearrangements ranging from simple insertions with flanking sequence duplications to large-scale indels that produced helper-dependent viruses. The insert was often also partially duplicated and would rapidly rearrange through homologous recombination. Most of the virus-insert junctions exhibited little or no preexiting microhomology, although a few encoded VAC topoisomerase recognition sites (C/T·CCTT). These studies show that VAC can catalyze NHR through a process that may reflect a form of aberrant replication fork repair. Although it is less efficient than classical homologous recombination, the rates of NHR may still be high enough to drive virus evolution. IMPORTANCE Large DNA viruses sometimes interfere in antiviral defenses using repurposed and mutant forms of the cellular proteins that mediate these same reactions. Such virus orthologs of cellular genes were presumably captured through nonhomologous recombination, perhaps in the distant past, but nothing is known about the processes that might promote "gene capture" or even how often these events occur over the course of an infectious cycle. This study shows that nonhomologous recombination in vaccinia virus-infected cells is frequent enough to seed a small but still significant portion of novel recombinants into large populations of newly replicated virus particles. This offers a route by which a pool of virus might survey the host genome for sequences that offer a selective growth advantage and potentially drive discontinuous virus evolution (saltation) through the acquisition of adventitious traits.
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Affiliation(s)
- Greg Vallée
- Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
- Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
| | - Peter Norris
- Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
- Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
| | - Patrick Paszkowski
- Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
- Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
| | - Ryan S. Noyce
- Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
- Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
| | - David H. Evans
- Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
- Li Ka Shing Institute of Virology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
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11
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M. Iyer L, Anantharaman V, Krishnan A, Burroughs AM, Aravind L. Jumbo Phages: A Comparative Genomic Overview of Core Functions and Adaptions for Biological Conflicts. Viruses 2021; 13:v13010063. [PMID: 33466489 PMCID: PMC7824862 DOI: 10.3390/v13010063] [Citation(s) in RCA: 61] [Impact Index Per Article: 15.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2020] [Revised: 12/31/2020] [Accepted: 12/31/2020] [Indexed: 02/07/2023] Open
Abstract
Jumbo phages have attracted much attention by virtue of their extraordinary genome size and unusual aspects of biology. By performing a comparative genomics analysis of 224 jumbo phages, we suggest an objective inclusion criterion based on genome size distributions and present a synthetic overview of their manifold adaptations across major biological systems. By means of clustering and principal component analysis of the phyletic patterns of conserved genes, all known jumbo phages can be classified into three higher-order groups, which include both myoviral and siphoviral morphologies indicating multiple independent origins from smaller predecessors. Our study uncovers several under-appreciated or unreported aspects of the DNA replication, recombination, transcription and virion maturation systems. Leveraging sensitive sequence analysis methods, we identify novel protein-modifying enzymes that might help hijack the host-machinery. Focusing on host–virus conflicts, we detect strategies used to counter different wings of the bacterial immune system, such as cyclic nucleotide- and NAD+-dependent effector-activation, and prevention of superinfection during pseudolysogeny. We reconstruct the RNA-repair systems of jumbo phages that counter the consequences of RNA-targeting host effectors. These findings also suggest that several jumbo phage proteins provide a snapshot of the systems found in ancient replicons preceding the last universal ancestor of cellular life.
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Affiliation(s)
- Lakshminarayan M. Iyer
- National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA; (L.M.I.); (V.A.); (A.M.B.)
| | - Vivek Anantharaman
- National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA; (L.M.I.); (V.A.); (A.M.B.)
| | - Arunkumar Krishnan
- Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Berhampur, Odisha 760010, India;
| | - A. Maxwell Burroughs
- National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA; (L.M.I.); (V.A.); (A.M.B.)
| | - L. Aravind
- National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA; (L.M.I.); (V.A.); (A.M.B.)
- Correspondence:
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12
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Matía A, Lorenzo MM, Blasco R. Tools for the targeted genetic modification of poxvirus genomes. Curr Opin Virol 2020; 44:183-190. [PMID: 33242829 DOI: 10.1016/j.coviro.2020.10.006] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2020] [Revised: 10/27/2020] [Accepted: 10/27/2020] [Indexed: 12/14/2022]
Abstract
The potential of viruses as biotechnology platforms is becoming more appealing due to technological advances in synthetic biology techniques and to the increasing accessibility of means to manipulate virus genomes. Among viral systems, poxviruses, and their prototype member Vaccinia Virus, are one of the outstanding choices for different biotechnological and medical applications based on heterologous gene expression, recombinant vaccines or oncolytic viruses. The refinement of genetic engineering methods on Vaccinia Virus over the last decades have contributed to facilitate the manipulation of the genomes of poxviruses, and may aid in the improvement of virus variants designed for different goals through reverse genetic approaches. Targeted genetic changes are usually performed by homologous recombination with the viral genome. In addition to the classic approach, recent methodological advances that may assist new strategies for the mutation or edition of poxvirus genomes are reviewed.
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Affiliation(s)
- Alejandro Matía
- Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (I.N.I.A.), Ctra. La Coruña km 7.5, E-28040 Madrid, Spain
| | - María M Lorenzo
- Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (I.N.I.A.), Ctra. La Coruña km 7.5, E-28040 Madrid, Spain
| | - Rafael Blasco
- Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (I.N.I.A.), Ctra. La Coruña km 7.5, E-28040 Madrid, Spain.
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13
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Rapid poxvirus engineering using CRISPR/Cas9 as a selection tool. Commun Biol 2020; 3:643. [PMID: 33144673 PMCID: PMC7641209 DOI: 10.1038/s42003-020-01374-6] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2020] [Accepted: 10/01/2020] [Indexed: 02/08/2023] Open
Abstract
In standard uses of CRISPR/Cas9 technology, the cutting of genomes and their efficient repair are considered to go hand-in-hand to achieve desired genetic changes. This includes the current approach for engineering genomes of large dsDNA viruses. However, for poxviruses we show that Cas9-guide RNA complexes cut viral genomes soon after their entry into cells, but repair of these breaks is inefficient. As a result, Cas9 targeting makes only modest, if any, improvements to basal rates of homologous recombination between repair constructs and poxvirus genomes. Instead, Cas9 cleavage leads to inhibition of poxvirus DNA replication thereby suppressing virus spread in culture. This unexpected outcome allows Cas9 to be used as a powerful tool for selecting conventionally generated poxvirus recombinants, which are otherwise impossible to separate from a large background of parental virus without the use of marker genes. This application of CRISPR/Cas9 greatly speeds up the generation of poxvirus-based vaccines, making this platform considerably more attractive in the context of personalised cancer vaccines and emerging disease outbreaks. Gowripalan, Smith et al. use CRISPR/Cas9 technology to rapidly select recombinant poxviruses without using selectable marker genes. They find that Cas9 cleavage inhibits poxvirus DNA replication, suppressing virus spread in culture. This application makes poxviruses more attractive vector platforms for fighting cancer and emerging disease outbreaks.
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14
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Liu SB, Qiu XQ, Guo WQ, Li JL, Su Q, Du JH, Hu HJ, Wang XX, Song YH, Lou X, Xu XB. Transcriptome Analysis of FEN1 Knockdown HEK293T Cell Strain Reveals Alteration in Nucleic Acid Metabolism, Virus Infection, Cell Morphogenesis and Cancer Development. Comb Chem High Throughput Screen 2020; 22:379-386. [PMID: 31272350 DOI: 10.2174/1386207322666190704095602] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2019] [Revised: 06/04/2019] [Accepted: 06/10/2019] [Indexed: 12/24/2022]
Abstract
AIM AND OBJECTIVE Flap endonuclease-1 (FEN1) plays a central role in DNA replication and DNA damage repair process. In mammals, FEN1 functional sites variation is related to cancer and chronic inflammation, and supports the role of FEN1 as a tumor suppressor. However, FEN1 is overexpressed in multiple types of cancer cells and is associated with drug resistance, supporting its role as an oncogene. Hence, it is vital to explore the multi-functions of FEN1 in normal cell metabolic process. This study was undertaken to examine how the gene expression profile changes when FEN1 is downregulated in 293T cells. MATERIALS AND METHODS Using the RNA sequencing and real-time PCR approaches, the transcript expression profile of FEN1 knockdown HEK293T cells have been detected for the next step evaluation, analyzation, and validation. RESULTS Our results confirmed that FEN1 is important for cell viability. We showed that when FEN1 downregulation led to the interruption of nucleic acids related metabolisms, cell cycle related metabolisms are significantly interrupted. FEN1 may also participate in non-coding RNA processing, ribosome RNA processing, transfer RNA processing, ribosome biogenesis, virus infection and cell morphogenesis. CONCLUSION These findings provide insight into how FEN1 nuclease might regulate a wide variety of biological processes, and laid the foundation for understanding the role of other RAD2 family nucleases in cell growth and metabolism.
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Affiliation(s)
- Song-Bai Liu
- Suzhou Key Laboratory for Medical Biotechnology, Suzhou Vocational Health College, Suzhou 215009, China
| | - Xiu-Qin Qiu
- Suzhou Key Laboratory for Medical Biotechnology, Suzhou Vocational Health College, Suzhou 215009, China
| | - Wei-Qiang Guo
- School of Chemistry, Biology and Materials Engineering, Suzhou University of Science and Technology, Suzhou 215009, China
| | - Jin-Li Li
- Department of Radiation Oncology, The Affiliated Hospital of Soochow University, Suzhou 215006, China
| | - Qian Su
- Suzhou Key Laboratory for Medical Biotechnology, Suzhou Vocational Health College, Suzhou 215009, China
| | - Jia-Hui Du
- Suzhou Key Laboratory for Medical Biotechnology, Suzhou Vocational Health College, Suzhou 215009, China
| | - He-Juan Hu
- Suzhou Key Laboratory for Medical Biotechnology, Suzhou Vocational Health College, Suzhou 215009, China
| | - Xiao-Xiao Wang
- Suzhou Key Laboratory for Medical Biotechnology, Suzhou Vocational Health College, Suzhou 215009, China
| | - Yao-Hua Song
- Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, Soochow University, Suzhou 215006, China
| | - Xiao Lou
- 307 Hospital of Chinese People's Liberation Army,The Fifth Medical Center of Chinese PLA General Hospital, Beijing 100071, China
| | - Xiang-Bin Xu
- College of Food Science and Technology, Hainan University, Haikou 570228, China
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15
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Moss B. Investigating Viruses During the Transformation of Molecular Biology: Part II. Annu Rev Virol 2020; 7:15-36. [PMID: 32392458 DOI: 10.1146/annurev-virology-021020-100558] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
My scientific career started at an extraordinary time, shortly after the discoveries of the helical structure of DNA, the central dogma of DNA to RNA to protein, and the genetic code. Part I of this series emphasizes my education and early studies highlighted by the isolation and characterization of numerous vaccinia virus enzymes, determination of the cap structure of messenger RNA, and development of poxviruses as gene expression vectors for use as recombinant vaccines. Here I describe a shift in my research focus to combine molecular biology and genetics for a comprehensive understanding of poxvirus biology. The dominant paradigm during the early years was to select a function, isolate the responsible proteins, and locate the corresponding gene, whereas later the common paradigm was to select a gene, make a mutation, and determine the altered function. Motivations, behind-the-scenes insights, importance of new technologies, and the vital roles of trainees and coworkers are emphasized.
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Affiliation(s)
- Bernard Moss
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA;
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16
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MicroRNA-140 impedes DNA repair by targeting FEN1 and enhances chemotherapeutic response in breast cancer. Oncogene 2019; 39:234-247. [PMID: 31471584 DOI: 10.1038/s41388-019-0986-0] [Citation(s) in RCA: 65] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2019] [Revised: 05/29/2019] [Accepted: 06/15/2019] [Indexed: 01/18/2023]
Abstract
An increased DNA repair capacity is associated with drug resistance and limits the efficacy of chemotherapy in breast cancers. Flap endonuclease 1 (FEN1) participates in various DNA repair pathways and contributes to cancer progression and drug resistance in chemotherapy. Inhibition of FEN1 serves as a potent strategy for cancer therapy. Here, we demonstrate that microRNA-140 (miR-140) inhibits FEN1 expression via directly binding to its 3' untranslated region, leading to impaired DNA repair and repressed breast cancer progression. Overexpression of miR-140 sensitizes breast cancer cells to chemotherapeutic agents and overcomes drug resistance in breast cancer. Notably, ectopic expression of FEN1 abates the effects of miR-140 on DNA damage and the chemotherapy response in breast cancer cells. Furthermore, the transcription factor/repressor Ying Yang 1 (YY1) directly binds to the miR-140 promoter and activates miR-140 expression, which is attenuated in doxorubicin resistance. Our results demonstrate that miR-140 acts as a tumor suppressor in breast cancer by inhibiting FEN1 to repress DNA damage repair and reveal miR-140 to be a new anti-tumorigenesis factor for adjunctive breast cancer therapy. This novel mechanism will enhance the treatment effect of chemotherapy in breast cancer.
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17
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Huttunen M, Mercer J. Quantitative PCR-Based Assessment of Vaccinia Virus RNA and DNA in Infected Cells. Methods Mol Biol 2019; 2023:189-208. [PMID: 31240679 DOI: 10.1007/978-1-4939-9593-6_12] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/09/2023]
Abstract
Quantitative PCR-based methods have proven to be easy-to-use, cost-effective procedures for the quantification of viral gene expression and viral genome numbers. Quantitative PCR (qPCR) and quantitative reverse transcriptase-PCR (qRT-PCR) are rapid and sensitive approaches that can be used to pinpoint defects in viral DNA replication and transcriptional activity, respectively. Due to the significant nucleotide overlap between Poxviridae these methods can be employed across a wide range of viruses from this family. Here we provide methods for the quantification of vaccinia DNA replication by qPCR and quantification of the three classes of vaccinia gene transcription by qRT-PCR.
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Affiliation(s)
- Moona Huttunen
- MRC-Laboratory for Molecular Cell Biology, University College London, London, UK.
| | - Jason Mercer
- MRC-Laboratory for Molecular Cell Biology, University College London, London, UK.
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18
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Luteijn RD, Drexler I, Smith GL, Lebbink RJ, Wiertz EJHJ. Mutagenic repair of double-stranded DNA breaks in vaccinia virus genomes requires cellular DNA ligase IV activity in the cytosol. J Gen Virol 2018; 99:790-804. [PMID: 29676720 PMCID: PMC7614823 DOI: 10.1099/jgv.0.001034] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Poxviruses comprise a group of large dsDNA viruses that include members relevant to human and animal health, such as variola virus, monkeypox virus, cowpox virus and vaccinia virus (VACV). Poxviruses are remarkable for their unique replication cycle, which is restricted to the cytoplasm of infected cells. The independence from the host nucleus requires poxviruses to encode most of the enzymes involved in DNA replication, transcription and processing. Here, we use the CRISPR/Cas9 genome engineering system to induce DNA damage to VACV (strain Western Reserve) genomes. We show that targeting CRISPR/Cas9 to essential viral genes limits virus replication efficiently. Although VACV is a strictly cytoplasmic pathogen, we observed extensive viral genome editing at the target site; this is reminiscent of a non-homologous end-joining DNA repair mechanism. This pathway was not dependent on the viral DNA ligase, but critically involved the cellular DNA ligase IV. Our data show that DNA ligase IV can act outside of the nucleus to allow repair of dsDNA breaks in poxvirus genomes. This pathway might contribute to the introduction of mutations within the genome of poxviruses and may thereby promote the evolution of these viruses.
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Affiliation(s)
- Rutger David Luteijn
- Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.,Present address: Department of Molecular and Cell Biology, University of California, Berkeley, USA
| | - Ingo Drexler
- Institute for Virology, University Hospital Düsseldorf, Düsseldorf, Germany
| | | | - Robert Jan Lebbink
- Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Emmanuel J H J Wiertz
- Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands
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19
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Isolation and Characterization of vΔI3 Confirm that Vaccinia Virus SSB Plays an Essential Role in Viral Replication. J Virol 2018; 92:JVI.01719-17. [PMID: 29093092 DOI: 10.1128/jvi.01719-17] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2017] [Accepted: 10/20/2017] [Indexed: 01/11/2023] Open
Abstract
Vaccinia virus is unusual among DNA viruses in replicating exclusively in the cytoplasm of infected cells. The single-stranded DNA (ssDNA) binding protein (SSB) I3 is among the replication machinery encoded by the 195-kb genome, although direct genetic analysis of I3 has been lacking. Herein, we describe a complementing cell line (CV1-I3) that fully supports the replication of a null virus (vΔI3) lacking the I3 open reading frame (ORF). In noncomplementing CV1-CAT cells, vΔI3 shows a severe defect in the production of infectious virus (≥200-fold reduction). Early protein synthesis and core disassembly occur normally. However, DNA replication is profoundly impaired (≤0.2% of wild-type [WT] levels), and late proteins do not accumulate. When several other noncomplementing cell lines are infected with vΔI3, the yield of infectious virus is also dramatically reduced (168- to 1,776-fold reduction). Surprisingly, the residual levels of DNA accumulation vary from 1 to 12% in the different cell lines (CV1-CAT < A549 < BSC40 < HeLa); however, any nascent DNA that can be detected is subgenomic in size. Although this subgenomic DNA supports late protein expression, it does not support the production of infectious virions. Electron microscopy (EM) analysis of vΔI3-infected BSC40 cells reveals that immature virions are abundant but no mature virions are observed. Aberrant virions characteristic of a block to genome encapsidation are seen instead. Finally, we demonstrate that a CV1 cell line encoding a previously described I3 variant with impaired ssDNA binding activity is unable to complement vΔI3. This report provides definitive evidence that the vaccinia virus I3 protein is the replicative SSB and is essential for productive viral replication.IMPORTANCE Poxviruses are of historical and contemporary importance as infectious agents, vaccines, and oncolytic therapeutics. The cytoplasmic replication of poxviruses is unique among DNA viruses of mammalian cells and necessitates that the double-stranded DNA (dsDNA) genome encode the viral replication machinery. This study focuses on the I3 protein. As a ssDNA binding protein (SSB), I3 has been presumed to play essential roles in genome replication, recombination, and repair, although genetic analysis has been lacking. Herein, we report the characterization of an I3 deletion virus. In the absence of I3 expression, DNA replication is severely compromised and viral yield profoundly decreased. The production of infectious virus can be restored in a cell line expressing WT I3 but not in a cell line expressing an I3 mutant that is defective in ssDNA binding activity. These data show conclusively that I3 is an essential viral protein and functions as the viral replicative SSB.
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20
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Carulei O, Douglass N, Williamson AL. Comparative analysis of avian poxvirus genomes, including a novel poxvirus from lesser flamingos (Phoenicopterus minor), highlights the lack of conservation of the central region. BMC Genomics 2017; 18:947. [PMID: 29207949 PMCID: PMC5718139 DOI: 10.1186/s12864-017-4315-0] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2017] [Accepted: 11/17/2017] [Indexed: 11/17/2022] Open
Abstract
BACKGROUND Avian poxviruses are important pathogens of both wild and domestic birds. To date, seven isolates from subclades A and B and one from proposed subclade E, have had their genomes completely sequenced. The genomes of these isolates have been shown to exhibit typical poxvirus genome characteristics with conserved central regions and more variable terminal regions. Infection with avian poxviruses (APVs) has been reported in three species of captive flamingo, as well as a free-living, lesser flamingo at Kamfers dam, near Kimberley, South Africa. This study was undertaken to further characterise this virus which may have long term effects on this important and vulnerable, breeding population. RESULTS Gene content and synteny as well as percentage identities between conserved orthologues was compared between Flamingopox virus (FGPV) and the other sequenced APV genomes. Dotplot comparisons revealed major differences in central regions that have been thought to be conserved. Further analysis revealed five regions of difference, of differing lengths, spread across the central, conserved regions of the various genomes. Although individual gene identities at the nucleotide level did not vary greatly, gene content and synteny between isolates/species at these identified regions were more divergent than expected. CONCLUSION Basic comparative genomics revealed the expected similarities in genome architecture but an in depth, comparative, analysis showed all avian poxvirus genomes to differ from other poxvirus genomes in fundamental and unexpected ways. The reasons for these large genomic rearrangements in regions of the genome that were thought to be relatively conserved are yet to be elucidated. Sequencing and analysis of further avian poxvirus genomes will help characterise this complex genus of poxviruses.
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Affiliation(s)
- Olivia Carulei
- Division of Medical Virology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
| | - Nicola Douglass
- Division of Medical Virology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
| | - Anna-Lise Williamson
- Division of Medical Virology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
- Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa
- National Health Laboratory Service, Cape Town, South Africa
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21
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Matelska D, Steczkiewicz K, Ginalski K. Comprehensive classification of the PIN domain-like superfamily. Nucleic Acids Res 2017; 45:6995-7020. [PMID: 28575517 PMCID: PMC5499597 DOI: 10.1093/nar/gkx494] [Citation(s) in RCA: 60] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2017] [Accepted: 05/24/2017] [Indexed: 12/21/2022] Open
Abstract
PIN-like domains constitute a widespread superfamily of nucleases, diverse in terms of the reaction mechanism, substrate specificity, biological function and taxonomic distribution. Proteins with PIN-like domains are involved in central cellular processes, such as DNA replication and repair, mRNA degradation, transcription regulation and ncRNA maturation. In this work, we identify and classify the most complete set of PIN-like domains to provide the first comprehensive analysis of sequence–structure–function relationships within the whole PIN domain-like superfamily. Transitive sequence searches using highly sensitive methods for remote homology detection led to the identification of several new families, including representatives of Pfam (DUF1308, DUF4935) and CDD (COG2454), and 23 other families not classified in the public domain databases. Further sequence clustering revealed relationships between individual sequence clusters and showed heterogeneity within some families, suggesting a possible functional divergence. With five structural groups, 70 defined clusters, over 100,000 proteins, and broad biological functions, the PIN domain-like superfamily constitutes one of the largest and most diverse nuclease superfamilies. Detailed analyses of sequences and structures, domain architectures, and genomic contexts allowed us to predict biological function of several new families, including new toxin-antitoxin components, proteins involved in tRNA/rRNA maturation and transcription/translation regulation.
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Affiliation(s)
- Dorota Matelska
- University of Warsaw, CeNT, Laboratory of Bioinformatics and Systems Biology, Zwirki i Wigury 93, 02-089 Warsaw, Poland
| | - Kamil Steczkiewicz
- University of Warsaw, CeNT, Laboratory of Bioinformatics and Systems Biology, Zwirki i Wigury 93, 02-089 Warsaw, Poland
| | - Krzysztof Ginalski
- University of Warsaw, CeNT, Laboratory of Bioinformatics and Systems Biology, Zwirki i Wigury 93, 02-089 Warsaw, Poland
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22
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Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry. J Virol 2017; 91:JVI.01015-17. [PMID: 28747503 DOI: 10.1128/jvi.01015-17] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2017] [Accepted: 07/19/2017] [Indexed: 12/22/2022] Open
Abstract
Poxviruses replicate within the cytoplasm and encode proteins for DNA and mRNA synthesis. To investigate poxvirus replication and transcription from a new perspective, we incorporated 5-ethynyl-2'-deoxyuridine (EdU) into nascent DNA in cells infected with vaccinia virus (VACV). The EdU-labeled DNA was conjugated to fluor- or biotin-azide and visualized by confocal, superresolution, and transmission electron microscopy. Nuclear labeling decreased dramatically after infection, accompanied by intense labeling of cytoplasmic foci. The nascent DNA colocalized with the VACV single-stranded DNA binding protein I3 in multiple puncta throughout the interior of factories, which were surrounded by endoplasmic reticulum. Complexes containing EdU-biotin-labeled DNA cross-linked to proteins were captured on streptavidin beads. After elution and proteolysis, the peptides were analyzed by mass spectrometry to identify proteins associated with nascent DNA. The known viral replication proteins, a telomere binding protein, and a protein kinase were associated with nascent DNA, as were the DNA-dependent RNA polymerase and intermediate- and late-stage transcription initiation and elongation factors, plus the capping and methylating enzymes. These results suggested that the replicating pool of DNA is transcribed and that few if any additional viral proteins directly engaged in replication and transcription remain to be discovered. Among the host proteins identified by mass spectrometry, topoisomerases IIα and IIβ and PCNA were noteworthy. The association of the topoisomerases with nascent DNA was dependent on expression of the viral DNA ligase, in accord with previous proteomic studies. Further investigations are needed to determine possible roles for PCNA and other host proteins detected.IMPORTANCE Poxviruses, unlike many well-characterized animal DNA viruses, replicate entirely within the cytoplasm of animal cells, raising questions regarding the relative roles of viral and host proteins. We adapted newly developed procedures for click chemistry and iPOND (Isolation of proteins on nascent DNA) to investigate vaccinia virus (VACV), the prototype poxvirus. Nuclear DNA synthesis ceased almost immediately following VACV infection, followed swiftly by the synthesis of viral DNA within discrete cytoplasmic foci. All viral proteins known from genetic and proteomic studies to be required for poxvirus DNA replication were identified in the complexes containing nascent DNA. The additional detection of the viral DNA-dependent RNA polymerase and intermediate and late transcription factors provided evidence for a temporal coupling of replication and transcription. Further studies are needed to assess the potential roles of host proteins, including topoisomerases IIα and IIβ and PCNA, which were found associated with nascent DNA.
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23
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Inhibition of Poxvirus Gene Expression and Genome Replication by Bisbenzimide Derivatives. J Virol 2017; 91:JVI.00838-17. [PMID: 28659488 PMCID: PMC5571260 DOI: 10.1128/jvi.00838-17] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2017] [Accepted: 06/22/2017] [Indexed: 12/19/2022] Open
Abstract
Virus infection of humans and livestock can be devastating for individuals and populations, sometimes resulting in large economic and societal impact. Prevention of virus disease by vaccination or antiviral agents is difficult to achieve. A notable exception was the eradication of human smallpox by vaccination over 30 years ago. Today, humans and animals remain susceptible to poxvirus infections, including zoonotic poxvirus transmission. Here we identified a small molecule, bisbenzimide (bisbenzimidazole), and its derivatives as potent agents against prototypic poxvirus infection in cell culture. We show that bisbenzimide derivatives, which preferentially bind the minor groove of double-stranded DNA, inhibit vaccinia virus infection by blocking viral DNA replication and abrogating postreplicative intermediate and late gene transcription. The bisbenzimide derivatives are potent against vaccinia virus and other poxviruses but ineffective against a range of other DNA and RNA viruses. The bisbenzimide derivatives are the first inhibitors of their class, which appear to directly target the viral genome without affecting cell viability. IMPORTANCE Smallpox was one of the most devastating diseases in human history until it was eradicated by a worldwide vaccination campaign. Due to discontinuation of routine vaccination more than 30 years ago, the majority of today's human population remains susceptible to infection with poxviruses. Here we present a family of bisbenzimide (bisbenzimidazole) derivatives, known as Hoechst nuclear stains, with high potency against poxvirus infection. Results from a variety of assays used to dissect the poxvirus life cycle demonstrate that bisbenzimides inhibit viral gene expression and genome replication. These findings can lead to the development of novel antiviral drugs that target viral genomes and block viral replication.
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24
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Dobson BM, Tscharke DC. Redundancy complicates the definition of essential genes for vaccinia virus. J Gen Virol 2016; 96:3326-3337. [PMID: 26290187 DOI: 10.1099/jgv.0.000266] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Vaccinia virus (VACV) genes are characterized as either essential or non-essential for growth in culture. It seems intuitively obvious that if a gene can be deleted without imparting a growth defect in vitro it does not have a function related to basic replication or spread. However, this interpretation relies on the untested assumption that there is no redundancy across the genes that have roles in growth in cell culture. First, we provide a comprehensive summary of the literature that describes the essential genes of VACV. Next, we looked for interactions between large blocks of non-essential genes located at the ends of the genome by investigating sets of VACVs with large deletions at the genomic termini. Viruses with deletions at either end of the genome behaved as expected, exhibiting only mild or host-range defects. In contrast, combining deletions at both ends of the genome for the VACV Western Reserve (WR) strain caused a devastating growth defect on all cell lines tested. Unexpectedly, we found that the well-studied VACV growth factor homologue encoded by C11R has a role in growth in vitro that is exposed when 42 genes are absent from the left end of the VACV WR genome. These results demonstrate that some non-essential genes contribute to basic viral growth, but redundancy means these functions are not revealed by single-gene-deletion mutants.
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Affiliation(s)
- Bianca M Dobson
- Division of Biomedical Science and Biochemistry, Research School of Biology, John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia
| | - David C Tscharke
- Department of Immunology and Infectious Disease, John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia.,Division of Biomedical Science and Biochemistry, Research School of Biology, John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia
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25
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Harrison ML, Desaulniers MA, Noyce RS, Evans DH. The acidic C-terminus of vaccinia virus I3 single-strand binding protein promotes proper assembly of DNA-protein complexes. Virology 2016; 489:212-22. [PMID: 26773382 DOI: 10.1016/j.virol.2015.12.020] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2015] [Revised: 08/24/2015] [Accepted: 12/28/2015] [Indexed: 11/25/2022]
Abstract
The vaccinia virus I3L gene encodes a single-stranded DNA binding protein (SSB) that is essential for virus DNA replication and is conserved in all Chordopoxviruses. The I3 protein contains a negatively charged C-terminal tail that is a common feature of SSBs. Such acidic tails are critical for SSB-dependent replication, recombination and repair. We cloned and purified variants of the I3 protein, along with a homolog from molluscum contagiosum virus, and tested how the acidic tail affected DNA-protein interactions. Deleting the C terminus of I3 enhanced the affinity for single-stranded DNA cellulose and gel shift analyses showed that it also altered the migration of I3-DNA complexes in agarose gels. Microinjecting an antibody against I3 into vaccinia-infected cells also selectively inhibited virus replication. We suggest that this domain promotes cooperative binding of I3 to DNA in a way that would maintain an open DNA configuration around a replication site.
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Affiliation(s)
- Melissa L Harrison
- Department of Medical Microbiology & Immunology, Li Ka-Shing Institute for Virology, 6020 Katz Group Centre, University of Alberta, Edmonton, AB, Canada T6G 2E1
| | - Megan A Desaulniers
- Department of Medical Microbiology & Immunology, Li Ka-Shing Institute for Virology, 6020 Katz Group Centre, University of Alberta, Edmonton, AB, Canada T6G 2E1
| | - Ryan S Noyce
- Department of Medical Microbiology & Immunology, Li Ka-Shing Institute for Virology, 6020 Katz Group Centre, University of Alberta, Edmonton, AB, Canada T6G 2E1
| | - David H Evans
- Department of Medical Microbiology & Immunology, Li Ka-Shing Institute for Virology, 6020 Katz Group Centre, University of Alberta, Edmonton, AB, Canada T6G 2E1.
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26
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Genetic Confirmation that the H5 Protein Is Required for Vaccinia Virus DNA Replication. J Virol 2015; 89:6312-27. [PMID: 25855734 DOI: 10.1128/jvi.00445-15] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2015] [Accepted: 03/28/2015] [Indexed: 11/20/2022] Open
Abstract
UNLABELLED The duplication of the poxvirus double-stranded DNA genome occurs in cytoplasmic membrane-delimited factories. This physical autonomy from the host nucleus suggests that poxvirus genomes encode the full repertoire of proteins committed for genome replication. Biochemical and genetic analyses have confirmed that six viral proteins are required for efficient DNA synthesis; indirect evidence has suggested that the multifunctional H5 protein may also have a role. Here we show that H5 localizes to replication factories, as visualized by immunofluorescence and immunoelectron microscopy, and can be retrieved upon purification of the viral polymerase holoenzyme complex. The temperature-sensitive (ts) mutant Dts57, which was generated by chemical mutagenesis and has a lesion in H5, exhibits defects in DNA replication and morphogenesis under nonpermissive conditions, depending upon the experimental protocol. The H5 variant encoded by the genome of this mutant is ts for function but not stability. For a more precise investigation of how H5 contributes to DNA synthesis, we placed the ts57 H5 allele in an otherwise wild-type viral background and also performed small interfering RNA-mediated depletion of H5. Finally, we generated a complementing cell line, CV-1-H5, which allowed us to generate a viral recombinant in which the H5 open reading frame was deleted and replaced with mCherry (vΔH5). Analysis of vΔH5 allowed us to demonstrate conclusively that viral DNA replication is abrogated in the absence of H5. The loss of H5 does not compromise the accumulation of other early viral replication proteins or the uncoating of the virion core, suggesting that H5 plays a direct and essential role in facilitating DNA synthesis. IMPORTANCE Variola virus, the causative agent of smallpox, is the most notorious member of the Poxviridae family. Poxviruses are unique among DNA viruses that infect mammalian cells, in that their replication is restricted to the cytoplasm of the cell. This physical autonomy from the nucleus has both cell biological and genetic ramifications. Poxviruses must establish cytoplasmic niches that support replication, and the genomes must encode the repertoire of proteins necessary for genome synthesis. Here we focus on H5, a multifunctional and abundant viral protein. We confirm that H5 associates with the DNA polymerase holoenzyme and localizes to the sites of DNA synthesis. By generating an H5-expressing cell line, we were able to isolate a deletion virus that lacks the H5 gene and show definitively that genome synthesis does not occur in the absence of H5. These data support the hypothesis that H5 is a crucial participant in cytoplasmic poxvirus genome replication.
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Moussatche N, Condit RC. Fine structure of the vaccinia virion determined by controlled degradation and immunolocalization. Virology 2014; 475:204-18. [PMID: 25486587 DOI: 10.1016/j.virol.2014.11.020] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2014] [Accepted: 11/18/2014] [Indexed: 10/24/2022]
Abstract
The vaccinia virion is a membraned, slightly flattened, barrel-shaped particle, with a complex internal structure featuring a biconcave core flanked by lateral bodies. Although the architecture of the purified mature virion has been intensely characterized by electron microscopy, the distribution of the proteins within the virion has been examined primarily using biochemical procedures. Thus, it has been shown that non-ionic and ionic detergents combined or not with a sulfhydryl reagent can be used to disrupt virions and, to a limited degree, separate the constituent proteins in different fractions. Applying a controlled degradation technique to virions adsorbed on EM grids, we were able to immuno-localize viral proteins within the virion particle. Our results show after NP40 and DTT treatment, membrane proteins are removed from the virion surface revealing proteins that are associated with the lateral bodies and the outer layer of the core wall. Combined treatment using high salt and high DTT removed lateral body proteins and exposed proteins of the internal core wall. Cores treated with proteases could be disrupted and the internal components were exposed. Cts8, a mutant in the A3 protein, produces aberrant virus that, when treated with NP-40 and DTT, releases to the exterior the virus DNA associated with other internal core proteins. With these results, we are able to propose a model for the structure the vaccinia virion.
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Affiliation(s)
- Nissin Moussatche
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA.
| | - Richard C Condit
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA
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Leão TL, da Fonseca FG. Subversion of cellular stress responses by poxviruses. World J Clin Infect Dis 2014; 4:27-40. [DOI: 10.5495/wjcid.v4.i4.27] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/29/2014] [Revised: 07/26/2014] [Accepted: 09/10/2014] [Indexed: 02/06/2023] Open
Abstract
Cellular stress responses are powerful mechanisms that prevent and cope with the accumulation of macromolecular damage in the cells and also boost host defenses against pathogens. Cells can initiate either protective or destructive stress responses depending, to a large extent, on the nature and duration of the stressing stimulus as well as the cell type. The productive replication of a virus within a given cell places inordinate stress on the metabolism machinery of the host and, to assure the continuity of its replication, many viruses have developed ways to modulate the cell stress responses. Poxviruses are among the viruses that have evolved a large number of strategies to manipulate host stress responses in order to control cell fate and enhance their replicative success. Remarkably, nearly every step of the stress responses that is mounted during infection can be targeted by virally encoded functions. The fine-tuned interactions between poxviruses and the host stress responses has aided virologists to understand specific aspects of viral replication; has helped cell biologists to evaluate the role of stress signaling in the uninfected cell; and has tipped immunologists on how these signals contribute to alert the cells against pathogen invasion and boost subsequent immune responses. This review discusses the diverse strategies that poxviruses use to subvert host cell stress responses.
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Nicholls TJ, Zsurka G, Peeva V, Schöler S, Szczesny RJ, Cysewski D, Reyes A, Kornblum C, Sciacco M, Moggio M, Dziembowski A, Kunz WS, Minczuk M. Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease. Hum Mol Genet 2014; 23:6147-62. [PMID: 24986917 PMCID: PMC4222359 DOI: 10.1093/hmg/ddu336] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
MGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete processing of 5′ ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nuclease S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an additional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA.
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Affiliation(s)
| | - Gábor Zsurka
- Department of Epileptology, Life and Brain Center and
| | | | | | - Roman J Szczesny
- Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland and
| | - Dominik Cysewski
- Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland and
| | - Aurelio Reyes
- Mitochondrial Biology Unit, Medical Research Council, Cambridge, UK
| | - Cornelia Kornblum
- Department of Neurology, University of Bonn Medical Center, Bonn, Germany
| | - Monica Sciacco
- Neuromuscular Unit, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Centro Dino Ferrari, University of Milan, Milan, Italy
| | - Maurizio Moggio
- Neuromuscular Unit, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Centro Dino Ferrari, University of Milan, Milan, Italy
| | - Andrzej Dziembowski
- Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland and
| | | | - Michal Minczuk
- Mitochondrial Biology Unit, Medical Research Council, Cambridge, UK,
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Human antibody responses to the polyclonal Dryvax vaccine for smallpox prevention can be distinguished from responses to the monoclonal replacement vaccine ACAM2000. CLINICAL AND VACCINE IMMUNOLOGY : CVI 2014; 21:877-85. [PMID: 24759651 DOI: 10.1128/cvi.00035-14] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Dryvax (Wyeth Laboratories, Inc., Marietta, PA) is representative of the vaccinia virus preparations that were previously used for preventing smallpox. While Dryvax was highly effective, the national supply stocks were depleted, and there were manufacturing concerns regarding sterility and the clonal heterogeneity of the vaccine. ACAM2000 (Acambis, Inc./Sanofi-Pasteur Biologics Co., Cambridge, MA), a single-plaque-purified vaccinia virus derivative of Dryvax, recently replaced the polyclonal smallpox vaccine for use in the United States. A substantial amount of sequence heterogeneity exists within the polyclonal proteome of Dryvax, including proteins that are missing from ACAM2000. Reasoning that a detailed comparison of antibody responses to the polyclonal and monoclonal vaccines may be useful for identifying unique properties of each antibody response, we utilized a protein microarray comprised of approximately 94% of the vaccinia poxvirus proteome (245 proteins) to measure protein-specific antibody responses of 71 individuals receiving a single vaccination with ACAM2000 or Dryvax. We observed robust antibody responses to 21 poxvirus proteins in vaccinated individuals, including 11 proteins that distinguished Dryvax responses from ACAM2000. Analysis of protein sequences from Dryvax clones revealed amino acid level differences in these 11 antigenic proteins and suggested that sequence variation and clonal heterogeneity may contribute to the observed differences between Dryvax and ACAM2000 antibody responses.
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Matson J, Chou W, Ngo T, Gershon PD. Static and dynamic protein phosphorylation in the Vaccinia virion. Virology 2014; 452-453:310-23. [PMID: 24606709 DOI: 10.1016/j.virol.2014.01.012] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2013] [Revised: 12/02/2013] [Accepted: 01/17/2014] [Indexed: 11/19/2022]
Abstract
To the best of our knowledge, two phosphorylation sites have been reported previously, among 11 known Vaccinia virus phosphoproteins. Here, via phosphopeptide mass spectrometry, up to 189 phosphorylation sites were identified among 48 proteins in preparations of purified Vaccinia mature virus (MV). 8.5% of phospho-residues were pTyr. Viral phosphoproteins were found in diverse functional classes, including structural proteins, membrane proteins and RNA polymerase subunits. Among the nine identified membrane phosphoproteins, the sites in just one, namely A14L, were deduced to be internal with respect to the accompanying membrane. Examination of sites in known substrates of the Vaccinia-encoded protein kinase VPK2, indicated VPK2 to be a proline-dependent kinase. The MV phosphoproteome was enriched in potential substrates of cellular kinases belonging to the CDK2/CDK3, CK2, and p38 groups. Quantitative mass spectrometry identified several sites that became phosphorylated during intravirion kinase activation in vitro, each showing one of two distinct pH-dependency profiles.
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Affiliation(s)
- J Matson
- University of North Carolina, Chapel Hill, NC, United States
| | - W Chou
- Department of Molecular Biology and Biochemistry, UC-Irvine, Irvine, CA 92697, United States
| | - T Ngo
- Department of Molecular Biology and Biochemistry, UC-Irvine, Irvine, CA 92697, United States
| | - P D Gershon
- Department of Molecular Biology and Biochemistry, UC-Irvine, Irvine, CA 92697, United States.
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Redrejo-Rodríguez M, Salas ML. Repair of base damage and genome maintenance in the nucleo-cytoplasmic large DNA viruses. Virus Res 2013; 179:12-25. [PMID: 24184318 DOI: 10.1016/j.virusres.2013.10.017] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2013] [Revised: 10/21/2013] [Accepted: 10/21/2013] [Indexed: 11/27/2022]
Abstract
Among the DNA viruses, the so-called nucleo-cytoplasmic large DNA viruses (NCLDV) constitute a monophyletic group that currently consists of seven families of viruses infecting a very broad variety of eukaryotes, from unicellular marine protists to humans. Many recent papers have analyzed the sequence and structure of NCLDV genomes and their phylogeny, providing detailed analysis about their genomic structure and evolutionary history and proposing their inclusion in a new viral order named Megavirales that, according to some authors, should be considered as a fourth domain of life, aside from Bacteria, Archaea and Eukarya. The maintenance of genetic information protected from environmental attacks and mutations is essential not only for the survival of cellular organisms but also viruses. In cellular organisms, damaged DNA bases are removed in two major repair pathways: base excision repair (BER) and nucleotide incision repair (NIR) that constitute the major pathways responsible for repairing most endogenous base lesions and abnormal bases in the genome by precise repair procedures. Like cells, many NCLDV encode proteins that might constitute viral DNA repair pathways that would remove damages through BER/NIR pathways. However, the molecular mechanisms and, specially, the biological roles of those viral repair pathways have not been deeply addressed in the literature so far. In this paper, we review viral-encoded BER proteins and the genetic and biochemical data available about them. We propose and discuss probable viral-encoded DNA repair mechanisms and pathways, as compared with the functional and molecular features of known homologs proteins.
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Affiliation(s)
- Modesto Redrejo-Rodríguez
- Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, 28049 Madrid, Spain.
| | - María L Salas
- Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, 28049 Madrid, Spain
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The D10 decapping enzyme of vaccinia virus contributes to decay of cellular and viral mRNAs and to virulence in mice. J Virol 2013; 88:202-11. [PMID: 24155373 DOI: 10.1128/jvi.02426-13] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Posttranscriptional mechanisms are important for regulation of cellular and viral gene expression. The presence of the 5' cap structure m(7)G(5')ppp(5')Nm is a general feature of mRNAs that provides protection from exoribonuclease digestion and enhances translation. Vaccinia virus and other poxviruses encode enzymes for both cap synthesis and decapping. Decapping is mediated by two related enzymes, D9 and D10, which are synthesized before and after viral DNA replication, respectively. The timing of D10 synthesis correlates better with the shutdown of host gene expression, and deletion of this gene has been shown to cause persistence of host and viral mRNAs in infected cells. Here, we constructed specific mutant viruses in which translation of D10 was prevented by stop codons or activity of D10 was abrogated by catalytic site mutations, without other genomic alterations. Both mutants formed plaques of normal size and replicated to similar extents as the parental virus in monkey epithelial cells and mouse embryonic fibroblasts. The synthesis of viral proteins was slightly delayed, and cellular and viral mRNAs persisted longer in cells infected with the mutants compared to either the parental virus or clonal revertant. Despite the mild effects in vitro, both mutants were more attenuated than the revertants in intranasal and intraperitoneal mouse models, and less infectious virus was recovered from organs. In addition, there was less lung histopathology following intranasal infection with mutant viruses. These data suggest that the D10 decapping enzyme may help restrict antiviral responses by accelerating host mRNA degradation during poxvirus infection.
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Abstract
Poxviruses are large, enveloped viruses that replicate in the cytoplasm and encode proteins for DNA replication and gene expression. Hairpin ends link the two strands of the linear, double-stranded DNA genome. Viral proteins involved in DNA synthesis include a 117-kDa polymerase, a helicase-primase, a uracil DNA glycosylase, a processivity factor, a single-stranded DNA-binding protein, a protein kinase, and a DNA ligase. A viral FEN1 family protein participates in double-strand break repair. The DNA is replicated as long concatemers that are resolved by a viral Holliday junction endonuclease.
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Affiliation(s)
- Bernard Moss
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
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Abstract
The A19 protein of vaccinia virus (VACV) is conserved among chordopoxviruses, expressed late in infection, packaged in the virus core, and required for a late step in morphogenesis. Multiple-sequence alignments of A19 homologs indicated conservation of a series of lysines and arginines, which could represent a nuclear localization or nucleic acid binding motif, and a pair of CXXC motifs that suggested a zinc finger or redox active sites. The importance of the CXXC motif was confirmed by cysteine-to-serine substitutions, which rendered the altered protein unable to trans-complement infectivity of a null mutant. Nevertheless, the cysteines were not required for function of the poxvirus-specific redox pathway. Epitope-tagged A19 proteins were detected in the nucleus and cytoplasm in both infected and uninfected cells, but this distribution was unaffected by alanine substitutions of the arginine residues, which only partially reduced the ability of the mutated protein to trans-complement infectivity. Viral proteins specifically associated with affinity-purified A19 were identified by mass spectrometry as components of the transcription complex, including RNA polymerase subunits, RAP94 (RNA polymerase-associated protein 94), early transcription factors, capping enzyme, and nucleoside triphosphate phosphohydrolase I, and two core proteins required for morphogenesis. Further studies suggested that the interaction of A19 with the RNA polymerase did not require RAP94 or other intermediate or late viral proteins but was reduced by mutation of cysteines in the putative zinc finger domain. Although A19 was not required for incorporation of the transcription complex in virus particles, the transcriptional activity of A19-deficient virus particles was severely reduced.
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Vaccinia virus A19 protein participates in the transformation of spherical immature particles to barrel-shaped infectious virions. J Virol 2013; 87:10700-9. [PMID: 23885081 DOI: 10.1128/jvi.01258-13] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The A19L open reading frame of vaccinia virus encodes a 9-kDa protein that is conserved in all sequenced chordopoxviruses, yet until now it has not been specifically characterized in any species. We appended an epitope tag after the start codon of the A19L open reading frame without compromising infectivity. The protein was synthesized after viral DNA replication and was phosphorylated independently of the vaccinia virus F10 kinase. The A19 protein was present in purified virions and was largely resistant to nonionic detergent extraction, suggesting a location within the core. A conditional lethal mutant virus was constructed by placing the A19 open reading frame under the control of the Escherichia coli lac repressor system. A19 synthesis and infectious virus formation were dependent on inducer. In the absence of inducer, virion morphogenesis was interrupted, and spherical dense particles that had greatly reduced amounts of the D13 scaffold accumulated in place of barrel-shaped mature virions. The infectivity of purified A19-deficient particles was more than 2 log units less than that of A19-containing virions. Nevertheless, the A19-deficient particles contained DNA, and except for the absence of A19 and decreased core protein processing, they appeared to have a similar protein composition as A19-containing virions. Thus, the A19 protein participates in the maturation of immature vaccinia virus virions to infectious particles.
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Abstract
In recent years, there have been numerous unprecedented technological advances in the field of molecular biology; these include DNA sequencing, mass spectrometry of proteins, and microarray analysis of mRNA transcripts. Perhaps, however, it is the area of genomics, which has now generated the complete genome sequences of more than 100 poxviruses, that has had the greatest impact on the average virology researcher because the DNA sequence data is in constant use in many different ways by almost all molecular virologists. As this data resource grows, so does the importance of the availability of databases and software tools to enable the bench virologist to work with and make use of this (valuable/expensive) DNA sequence information. Thus, providing researchers with intuitive software to first select and reformat genomics data from large databases, second, to compare/analyze genomics data, and third, to view and interpret large and complex sets of results has become pivotal in enabling progress to be made in modern virology. This chapter is directed at the bench virologist and describes the software required for a number of common bioinformatics techniques that are useful for comparing and analyzing poxvirus genomes. In a number of examples, we also highlight the Viral Orthologous Clusters database system and integrated tools that we developed for the management and analysis of complete viral genomes.
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Affiliation(s)
- Melissa Da Silva
- Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada
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Yutin N, Koonin EV. Hidden evolutionary complexity of Nucleo-Cytoplasmic Large DNA viruses of eukaryotes. Virol J 2012; 9:161. [PMID: 22891861 PMCID: PMC3493329 DOI: 10.1186/1743-422x-9-161] [Citation(s) in RCA: 123] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2012] [Accepted: 08/08/2012] [Indexed: 11/27/2022] Open
Abstract
Background The Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) constitute an apparently monophyletic group that consists of at least 6 families of viruses infecting a broad variety of eukaryotic hosts. A comprehensive genome comparison and maximum-likelihood reconstruction of the NCLDV evolution revealed a set of approximately 50 conserved, core genes that could be mapped to the genome of the common ancestor of this class of eukaryotic viruses. Results We performed a detailed phylogenetic analysis of these core NCLDV genes and applied the constrained tree approach to show that the majority of the core genes are unlikely to be monophyletic. Several of the core genes have been independently acquired from different sources by different NCLDV lineages whereas for the majority of these genes displacement by homologs from cellular organisms in one or more groups of the NCLDV was demonstrated. Conclusions A detailed study of the evolution of the genomic core of the NCLDV reveals substantial complexity and diversity of evolutionary scenarios that was largely unsuspected previously. The phylogenetic coherence between the core genes is sufficient to validate the hypothesis on the evolution of all NCLDV from a common ancestral virus although the set of ancestral genes might be smaller than previously inferred from patterns of gene presence-absence.
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Affiliation(s)
- Natalya Yutin
- National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA
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Abstract
Vaccinia virus transcription is regulated in three stages. An intermediate transcription factor, comprised of virus-encoded polypeptides A8 and A23, was previously identified by in vitro analyses. To investigate its role, we engineered cells that stably expressed both subunits and complemented the replication of A8 and A23 deletion mutant viruses. Without A8 or A23, viral early gene expression and DNA replication occurred but intermediate and late gene expression and resolution of genome concatemers were not detected.
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Molecular genetic and biochemical characterization of the vaccinia virus I3 protein, the replicative single-stranded DNA binding protein. J Virol 2012; 86:6197-209. [PMID: 22438556 DOI: 10.1128/jvi.00206-12] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Vaccinia virus, the prototypic poxvirus, efficiently and faithfully replicates its ∼200-kb DNA genome within the cytoplasm of infected cells. This intracellular localization dictates that vaccinia virus encodes most, if not all, of its own DNA replication machinery. Included in the repertoire of viral replication proteins is the I3 protein, which binds to single-stranded DNA (ssDNA) with great specificity and stability and has been presumed to be the replicative ssDNA binding protein (SSB). We substantiate here that I3 colocalizes with bromodeoxyuridine (BrdU)-labeled nascent viral genomes and that these genomes accumulate in cytoplasmic factories that are delimited by membranes derived from the endoplasmic reticulum. Moreover, we report on a structure/function analysis of I3 involving the isolation and characterization of 10 clustered charge-to-alanine mutants. These mutants were analyzed for their biochemical properties (self-interaction and DNA binding) and biological competence. Three of the mutant proteins, encoded by the I3 alleles I3-4, -5, and -7, were deficient in self-interaction and unable to support virus viability, strongly suggesting that the multimerization of I3 is biologically significant. Mutant I3-5 was also deficient in DNA binding. Additionally, we demonstrate that small interfering RNA (siRNA)-mediated depletion of I3 causes a significant decrease in the accumulation of progeny genomes and that this reduction diminishes the yield of infectious virus.
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Vaccinia virus A6 is essential for virion membrane biogenesis and localization of virion membrane proteins to sites of virion assembly. J Virol 2012; 86:5603-13. [PMID: 22398288 DOI: 10.1128/jvi.00330-12] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Poxvirus acquires its primary envelope through a process that is distinct from those of other enveloped viruses. The molecular mechanism of this process is poorly understood, but several poxvirus proteins essential for the process have been identified in studies of vaccinia virus (VACV), the prototypical poxvirus. Previously, we identified VACV A6 as an essential factor for virion morphogenesis by studying a temperature-sensitive mutant with a lesion in A6. Here, we further studied A6 by constructing and characterizing an inducible virus (iA6) that could more stringently repress A6 expression. When A6 expression was induced by the inducer isopropyl-β-D-thiogalactoside (IPTG), iA6 replicated normally, and membrane proteins of mature virions (MVs) predominantly localized in viral factories where virions were assembled. However, when A6 expression was repressed, electron microscopy of infected cells showed the accumulation of large viroplasm inclusions containing virion core proteins but no viral membranes. Immunofluorescence and cell fractionation studies showed that the major MV membrane proteins A13, A14, D8, and H3 did not localize to viral factories but instead accumulated in the secretory compartments, including the endoplasmic reticulum. Overall, our results show that A6 is an additional VACV protein that participates in an early step of virion membrane biogenesis. Furthermore, A6 is required for MV membrane protein localization to sites of virion assembly, suggesting that MV membrane proteins or precursors of MV membranes are trafficked to sites of virion assembly through an active, virus-mediated process that requires A6.
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Yoshida T, Claverie JM, Ogata H. Mimivirus reveals Mre11/Rad50 fusion proteins with a sporadic distribution in eukaryotes, bacteria, viruses and plasmids. Virol J 2011; 8:427. [PMID: 21899737 PMCID: PMC3175470 DOI: 10.1186/1743-422x-8-427] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2011] [Accepted: 09/07/2011] [Indexed: 11/28/2022] Open
Abstract
BACKGROUND The Mre11/Rad50 complex and the homologous SbcD/SbcC complex in bacteria play crucial roles in the metabolism of DNA double-strand breaks, including DNA repair, genome replication, homologous recombination and non-homologous end-joining in cellular life forms and viruses. Here we investigated the amino acid sequence of the Mimivirus R555 gene product, originally annotated as a Rad50 homolog, and later shown to have close homologs in marine microbial metagenomes. RESULTS Our bioinformatics analysis revealed that R555 protein sequence is constituted from the fusion of an N-terminal Mre11-like domain with a C-terminal Rad50-like domain. A systematic database search revealed twelve additional cases of Mre11/Rad50 (or SbcD/SbcC) fusions in a wide variety of unrelated organisms including unicellular and multicellular eukaryotes, the megaplasmid of a bacterium associated to deep-sea hydrothermal vents (Deferribacter desulfuricans) and the plasmid of Clostridium kluyveri. We also showed that R555 homologs are abundant in the metagenomes from different aquatic environments and that they most likely belong to aquatic viruses. The observed phyletic distribution of these fusion proteins suggests their recurrent creation and lateral gene transfers across organisms. CONCLUSIONS The existence of the fused version of protein sequences is consistent with known functional interactions between Mre11 and Rad50, and the gene fusion probably enhanced the opportunity for lateral transfer. The abundance of the Mre11/Rad50 fusion genes in viral metagenomes and their sporadic phyletic distribution in cellular organisms suggest that viruses, plasmids and transposons played a crucial role in the formation of the fusion proteins and their propagation into cellular genomes.
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Affiliation(s)
- Takashi Yoshida
- Laboratory of Marine Microbiology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
| | - Jean-Michel Claverie
- Structural and Genomic Information Laboratory, CNRS-UPR 2589, Aix-Marseille University, Mediterranean Institute of Microbiology, 163 Avenue de Luminy, Case 934, 13288 Marseille Cedex 9, France
| | - Hiroyuki Ogata
- Structural and Genomic Information Laboratory, CNRS-UPR 2589, Aix-Marseille University, Mediterranean Institute of Microbiology, 163 Avenue de Luminy, Case 934, 13288 Marseille Cedex 9, France
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Molecular characterization of the host defense activity of the barrier to autointegration factor against vaccinia virus. J Virol 2011; 85:11588-600. [PMID: 21880762 DOI: 10.1128/jvi.00641-11] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The barrier to autointegration factor (BAF) is an essential cellular protein with functions in mitotic nuclear reassembly, retroviral preintegration complex stability, and transcriptional regulation. Molecular properties of BAF include the ability to bind double-stranded DNA in a sequence-independent manner, homodimerize, and bind proteins containing a LEM domain. These capabilities allow BAF to compact DNA and assemble higher-order nucleoprotein complexes, the nature of which is poorly understood. Recently, it was revealed that BAF also acts as a potent host defense against poxviral DNA replication in the cytoplasm. Here, we extend these observations by examining the molecular mechanism through which BAF acts as a host defense against vaccinia virus replication and cytoplasmic DNA in general. Interestingly, BAF rapidly relocalizes to transfected DNA from a variety of sources, demonstrating that BAF's activity as a host defense factor is not limited to poxviral infection. BAF's relocalization to cytoplasmic foreign DNA is highly dependent upon its DNA binding and dimerization properties but does not appear to require its LEM domain binding activity. However, the LEM domain protein emerin is recruited to cytoplasmic DNA in a BAF-dependent manner during both transfection and vaccinia virus infection. Finally, we demonstrate that the DNA binding and dimerization capabilities of BAF are essential for its function as an antipoxviral effector, while the presence of emerin is not required. Together, these data provide further mechanistic insight into which of BAF's molecular properties are employed by cells to impair the replication of poxviruses or respond to foreign DNA in general.
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Boyle KA, Stanitsa ES, Greseth MD, Lindgren JK, Traktman P. Evaluation of the role of the vaccinia virus uracil DNA glycosylase and A20 proteins as intrinsic components of the DNA polymerase holoenzyme. J Biol Chem 2011; 286:24702-13. [PMID: 21572084 PMCID: PMC3137046 DOI: 10.1074/jbc.m111.222216] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2011] [Revised: 05/09/2011] [Indexed: 01/04/2023] Open
Abstract
The vaccinia virus DNA polymerase is inherently distributive but acquires processivity by associating with a heterodimeric processivity factor comprised of the viral A20 and D4 proteins. D4 is also an enzymatically active uracil DNA glycosylase (UDG). The presence of an active repair protein as an essential component of the polymerase holoenzyme is a unique feature of the replication machinery. We have shown previously that the A20-UDG complex has a stoichiometry of ∼1:1, and our data suggest that A20 serves as a bridge between polymerase and UDG. Here we show that conserved hydrophobic residues in the N' terminus of A20 are important for its binding to UDG. Our data argue against the assembly of D4 into higher order multimers, suggesting that the processivity factor does not form a toroidal ring around the DNA. Instead, we hypothesize that the intrinsic, processive DNA scanning activity of UDG tethers the holoenzyme to the DNA template. The inclusion of UDG as an essential holoenzyme component suggests that replication and base excision repair may be coupled. Here we show that the DNA polymerase can utilize dUTP as a substrate in vitro. Moreover, uracil moieties incorporated into the nascent strand during holoenzyme-mediated DNA synthesis can be excised by the viral UDG present within this holoenzyme, leaving abasic sites. Finally, we show that the polymerase stalls upon encountering an abasic site in the template strand, indicating that, like many replicative polymerases, the poxviral holoenzyme cannot perform translesion synthesis across an abasic site.
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Affiliation(s)
- Kathleen A. Boyle
- From the Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
| | - Eleni S. Stanitsa
- From the Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
| | - Matthew D. Greseth
- From the Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
| | - Jill K. Lindgren
- From the Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
| | - Paula Traktman
- From the Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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Maruri-Avidal L, Domi A, Weisberg AS, Moss B. Participation of vaccinia virus l2 protein in the formation of crescent membranes and immature virions. J Virol 2011; 85:2504-11. [PMID: 21228235 PMCID: PMC3067936 DOI: 10.1128/jvi.02505-10] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2010] [Accepted: 12/30/2010] [Indexed: 11/20/2022] Open
Abstract
Morphogenesis of vaccinia virus begins with the appearance of crescent-shaped membrane precursors of immature virions in cytoplasmic factories. During the initial characterization of the product of the L2R reading frame, we discovered that it plays an important role in crescent formation. The L2 protein was expressed early in infection and was associated with the detergent-soluble membrane fraction of mature virions, consistent with two potential membrane-spanning domains. All chordopoxviruses have L2 homologs, suggesting an important function. Indeed, we were unable to isolate an infectious L2R deletion mutant. Consequently, we constructed an inducible mutant with a conditional lethal phenotype. When L2 expression was repressed, proteolytic processing of the major core proteins and the A17 protein, which is an essential component of the immature virion membrane, failed to occur, suggesting an early block in viral morphogenesis. At 8 h after infection in the presence of inducer, immature and mature virions were abundantly seen by electron microscopy. In contrast, those structures were rare in the absence of inducer and were replaced by large, dense aggregates of viroplasm. A minority of these aggregates had short spicule-coated membranes, which resembled the beginnings of crescent formation, at their periphery. These short membrane segments at the edge of the dense viroplasm increased in number at later times, and some immature virions were seen. Although the L2 protein was not detected under nonpermissive conditions, minute amounts could account for stunted and delayed viral membrane formation. These findings suggested that L2 is required for the formation or elongation of crescent membranes.
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Affiliation(s)
- Liliana Maruri-Avidal
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-3210
| | - Arban Domi
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-3210
| | - Andrea S. Weisberg
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-3210
| | - Bernard Moss
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-3210
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Weitzman MD, Lilley CE, Chaurushiya MS. Genomes in conflict: maintaining genome integrity during virus infection. Annu Rev Microbiol 2010; 64:61-81. [PMID: 20690823 DOI: 10.1146/annurev.micro.112408.134016] [Citation(s) in RCA: 148] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The cellular surveillance network for sensing and repairing damaged DNA prevents an array of human diseases, and when compromised it can lead to genomic instability and cancer. The carefully maintained cellular response to DNA damage is challenged during viral infection, when foreign DNA is introduced into the cell. The battle between virus and host generates a genomic conflict. The host attempts to limit viral infection and protect its genome, while the virus deploys tactics to eliminate, evade, or exploit aspects of the cellular defense. Studying this conflict has revealed that the cellular DNA damage response machinery comprises part of the intrinsic cellular defense against viral infection. In this review we examine recent advances in this emerging field. We identify common themes used by viruses in their attempts to commandeer or circumvent the host cell's DNA repair machinery, and highlight potential outcomes of the conflict for both virus and host.
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Affiliation(s)
- Matthew D Weitzman
- Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
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