RESUMEN
Viruses exploit the host cell machinery to enable infection and propagation. This review discusses the complex landscape of DNA virus-host interactions, focusing primarily on herpesviruses and adenoviruses, which replicate in the nucleus of infected cells, and vaccinia virus, which replicates in the cytoplasm. We discuss experimental approaches used to discover and validate interactions of host proteins with viral genomes and how these interactions impact processes that occur during infection, including the host DNA damage response and viral genome replication, repair, and transcription. We highlight the current state of knowledge regarding virus-host protein interactions and also outline emerging areas and future directions for research.
Asunto(s)
ADN Viral , Genoma Viral , Interacciones Huésped-Patógeno , Replicación Viral , Humanos , ADN Viral/genética , ADN Viral/metabolismo , Virus ADN/genética , Animales , Proteínas Virales/metabolismo , Proteínas Virales/genética , Herpesviridae/genética , Herpesviridae/metabolismo , Herpesviridae/fisiología , Virus Vaccinia/genéticaRESUMEN
The movement of potyviruses, the largest genus of single-stranded, positive-sense RNA viruses responsible for serious diseases in crops, is very complex. As potyviruses developed strategies to hijack the host secretory pathway and plasmodesmata (PD) for their transport, the goal of this study was to identify membrane and/or PD-proteins that interact with the 6K2 protein, a potyviral protein involved in replication and cell-to-cell movement of turnip mosaic virus (TuMV). Using split-ubiquitin membrane yeast two-hybrid assays, we screened an Arabidopsis cDNA library for interactors of TuMV6K2. We isolated AtHVA22a (Hordeum vulgare abscisic acid responsive gene 22), which belongs to a multigenic family of transmembrane proteins, homologous to Receptor expression-enhancing protein (Reep)/Deleted in polyposis (DP1)/Yop1 family proteins in animal and yeast. HVA22/DP1/Yop1 family genes are widely distributed in eukaryotes, but the role of HVA22 proteins in plants is still not well known, although proteomics analysis of PD fractions purified from Arabidopsis suspension cells showed that AtHVA22a is highly enriched in a PD proteome. We confirmed the interaction between TuMV6K2 and AtHVA22a in yeast, as well as in planta by using bimolecular fluorescence complementation and showed that TuMV6K2/AtHVA22a interaction occurs at the level of the viral replication compartment during TuMV infection. Finally, we showed that the propagation of TuMV is increased when AtHVA22a is overexpressed in planta but slowed down upon mutagenesis of AtHVA22a by CRISPR-Cas9. Altogether, our results indicate that AtHVA22a plays an agonistic effect on TuMV propagation and that the C-terminal tail of the protein is important in this process.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Potyvirus , Potyvirus/patogenicidad , Potyvirus/fisiología , Arabidopsis/virología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Enfermedades de las Plantas/virología , Proteínas Virales/metabolismo , Proteínas Virales/genética , Replicación Viral , Nicotiana/virología , Nicotiana/genéticaRESUMEN
Adenoviruses are a group of double-stranded DNA viruses that can mainly cause respiratory, gastrointestinal, and eye infections in humans. In addition, adenoviruses are employed as vector vaccines for combatting viral infections, including SARS-CoV-2, and serve as excellent gene therapy vectors. These viruses have the ability to modulate the host cell machinery to their advantage and trigger significant restructuring of the nuclei of infected cells through the activity of viral proteins. One of those, the adenovirus DNA-binding protein (DBP), is a multifunctional non-structural protein that is integral to the reorganization processes. DBP is encoded in the E2A transcriptional unit and is highly abundant in infected cells. Its activity is unequivocally linked to the formation, structure, and integrity of virus-induced replication compartments, molecular hubs for the regulation of viral processes, and control of the infected cell. DBP also plays key roles in viral DNA replication, transcription, viral gene expression, and even host range specificity. Notably, post-translational modifications of DBP, such as SUMOylation and extensive phosphorylation, regulate its biological functions. DBP was first investigated in the 1970s, pioneering research on viral DNA-binding proteins. In this literature review, we provide an overview of DBP and specifically summarize key findings related to its complex structure, diverse functions, and significant role in the context of viral replication. Finally, we address novel insights and perspectives for future research.
Asunto(s)
Adenoviridae , Replicación del ADN , Proteínas de Unión al ADN , Proteínas Virales , Humanos , Adenoviridae/fisiología , Adenovirus Humanos/fisiología , ADN Viral/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprising the cognate viral DNA polymerase, vPOL, encoded by ORF9, and the viral DNA polymerase processivity factor, vPPF, encoded by ORF59. MHV68 vUNG co-localized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone or in combination. Lastly, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity. IMPORTANCE Gammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo. In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus in forming a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus-associated cancers.
Asunto(s)
Gammaherpesvirinae , Rhadinovirus , Animales , Ratones , Uracil-ADN Glicosidasa/genética , Uracil-ADN Glicosidasa/metabolismo , Replicación Viral , Replicación del ADN , ADN Viral/genética , Rhadinovirus/genética , Rhadinovirus/metabolismo , Gammaherpesvirinae/genética , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Uracilo , MamíferosRESUMEN
Subcellular organization is essential for life. Cells organize their functions into organelles to concentrate their machinery and supplies for optimal efficiency. Likewise, viruses organize their replication machinery into compartments or factories within their host cells for optimal replicative efficiency. In this review, we discuss how DNA viruses that infect both eukaryotic cells and bacteria assemble replication compartments for synthesis of progeny viral DNA and transcription of the viral genome. Eukaryotic DNA viruses assemble replication compartments in the nucleus of the host cell while DNA bacteriophages assemble compartments called phage nuclei in the bacterial cytoplasm. Thus, DNA viruses infecting host cells from different domains of life share common replication strategies.
Asunto(s)
Bacteriófagos , Virus , Bacterias/genética , Bacteriófagos/genética , Núcleo Celular , Virus ADN/genética , ADN Bacteriano , ADN Viral/genética , Eucariontes/genética , Células Eucariotas , Replicación Viral , Virus/genéticaRESUMEN
The human adenovirus type C5 (HAdV-C5) E1B-55K protein is a multifunctional regulator of HAdV-C5 replication, participating in many processes required for maximal virus production. Its multifunctional properties are primarily regulated by post-translational modifications (PTMs). The most influential E1B-55K PTMs are phosphorylation at highly conserved serine and threonine residues at the C-terminus, and SUMO conjugation to lysines 104 (K104) and 101 (K101) situated in the N-terminal region of the protein, which have been shown to regulate each other. Reversible SUMO conjugation provides a molecular switch that controls key functions of the viral protein, including intracellular trafficking and viral immune evasion. Interestingly, SUMOylation at SUMO conjugation site (SCS) K104 is negatively regulated by another multifunctional HAdV-C5 protein, E4orf6, which is known to form a complex with E1B-55K. To further evaluate the role of E4orf6 in the regulation of SUMO conjugation to E1B-55K, we analyzed different virus mutants expressing E1B-55K proteins with amino acid exchanges in both SCS (K101 and K104) in the presence or absence of E4orf6. We could exclude phosphorylation as factor for E4orf6-mediated reduction of E1B-55K SUMOylation. In fact, we demonstrate that a direct interaction between E1B-55K and E4orf6 is required to reduce E1B-55K SUMOylation. Additionally, we show that an E4orf6-mediated decrease of SUMO conjugation to K101 and K104 result in impaired co-localization of E1B-55K and SUMO in viral replication compartments. These findings indicate that E4orf6 inhibits E1B-55K SUMOylation, which could favor assembly of E4orf6-dependent E3 ubiquitin ligase complexes that are known to degrade a variety of host restriction factors by proteasomal degradation and, thereby, promote viral replication.
Asunto(s)
Infecciones por Adenovirus Humanos , Adenovirus Humanos , Adenoviridae/metabolismo , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Adenovirus Humanos/fisiología , Humanos , Sumoilación , Replicación ViralRESUMEN
Epstein-Barr virus (EBV) persistently infects people worldwide. Delivery of â¼170-kb EBV genomes to nuclei and use of nuclear membrane-less replication compartments (RCs) for their lytic cycle amplification necessitate evasion of intrinsic antiviral responses. Proteomics analysis indicates that, upon B cell infection or lytic reactivation, EBV depletes the cohesin SMC5/6, which has major roles in chromosome maintenance and DNA damage repair. The major tegument protein BNRF1 targets SMC5/6 complexes by a ubiquitin proteasome pathway dependent on calpain proteolysis and Cullin-7. In the absence of BNRF1, SMC5/6 associates with R-loop structures, including at the viral lytic origin of replication, and interferes with RC formation and encapsidation. CRISPR analysis identifies RC restriction roles of SMC5/6 components involved in DNA entrapment and SUMOylation. Our study highlights SMC5/6 as an intrinsic immune sensor and restriction factor for a human herpesvirus RC and has implications for the pathogenesis of EBV-associated cancers.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Proteínas del Envoltorio Viral , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Cromosomas/metabolismo , Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/fisiología , Humanos , Proteínas del Envoltorio Viral/genética , Replicación Viral/genética , CohesinasRESUMEN
Adenoviruses represent ubiquitous and clinically significant human pathogens, gene-delivery vectors, and oncolytic agents. The study of adenovirus-infected cells has long been used as an excellent model to investigate fundamental aspects of both DNA virus infection and cellular biology. While many key details supporting a well-established model of adenovirus replication have been elucidated over a period spanning several decades, more recent findings suggest that we have only started to appreciate the complex interplay between viral genome replication and cellular processes. Here, we present a concise overview of adenovirus DNA replication, including the biochemical process of replication, the spatial organization of replication within the host cell nucleus, and insights into the complex plethora of virus-host interactions that influence viral genome replication. Finally, we identify emerging areas of research relating to the replication of adenovirus genomes.