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1.
Nucleic Acids Res ; 2021 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-34671803

RESUMO

Eukaryotic cells recognize intracellular pathogens through pattern recognition receptors, including sensors of aberrant nucleic acid structures. Sensors of double-stranded RNA (dsRNA) are known to detect replication intermediates of RNA viruses. It has long been suggested that annealing of mRNA from symmetrical transcription of both top and bottom strands of DNA virus genomes can produce dsRNA during infection. Supporting this hypothesis, nearly all DNA viruses encode inhibitors of dsRNA-recognition pathways. However, direct evidence that DNA viruses produce dsRNA is lacking. Contrary to dogma, we show that the nuclear-replicating DNA virus adenovirus (AdV) does not produce detectable levels of dsRNA during infection. In contrast, abundant dsRNA is detected within the nucleus of cells infected with AdV mutants defective for viral RNA processing. In the presence of nuclear dsRNA, the cytoplasmic dsRNA sensor PKR is relocalized and activated within the nucleus. Accumulation of viral dsRNA occurs in the late phase of infection, when unspliced viral transcripts form intron/exon base pairs between top and bottom strand transcripts. We propose that DNA viruses actively limit dsRNA formation by promoting efficient splicing and mRNA processing, thus avoiding detection and restriction by host innate immune sensors of pathogenic nucleic acids.

2.
mSystems ; : e0046821, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34463575

RESUMO

Viral infections are associated with extensive remodeling of the cellular proteome. Viruses encode gene products that manipulate host proteins to redirect cellular processes or subvert antiviral immune responses. Adenovirus (AdV) encodes proteins from the early E4 region which are necessary for productive infection. Some cellular antiviral proteins are known to be targeted by AdV E4 gene products, resulting in their degradation or mislocalization. However, the full repertoire of host proteome changes induced by viral E4 proteins has not been defined. To identify cellular proteins and processes manipulated by viral products, we developed a global, unbiased proteomics approach to analyze changes to the host proteome during infection with adenovirus serotype 5 (Ad5) virus. We used whole-cell proteomics to measure total protein abundances in the proteome during Ad5 infection. Since host antiviral proteins can antagonize viral infection by associating with viral genomes and inhibiting essential viral processes, we used Isolation of Proteins on Nascent DNA (iPOND) proteomics to identify proteins associated with viral genomes during infection with wild-type Ad5 or an E4 mutant virus. By integrating these proteomics data sets, we identified cellular factors that are degraded in an E4-dependent manner or are associated with the viral genome in the absence of E4 proteins. We further show that some identified proteins exert inhibitory effects on Ad5 infection. Our systems-level analysis reveals cellular processes that are manipulated during Ad5 infection and points to host factors counteracted by early viral proteins as they remodel the host proteome to promote efficient infection. IMPORTANCE Viral infections induce myriad changes to the host cell proteome. As viruses harness cellular processes and counteract host defenses, they impact abundance, post-translational modifications, interactions, or localization of cellular proteins. Elucidating the dynamic changes to the cellular proteome during viral replication is integral to understanding how virus-host interactions influence the outcome of infection. Adenovirus encodes early gene products from the E4 genomic region that are known to alter host response pathways and promote replication, but the full extent of proteome modifications they mediate is not known. We used an integrated proteomics approach to quantitate protein abundance and protein associations with viral DNA during virus infection. Systems-level analysis identifies cellular proteins and processes impacted in an E4-dependent manner, suggesting ways that adenovirus counteracts potentially inhibitory host defenses. This study provides a global view of adenovirus-mediated proteome remodeling, which can serve as a model to investigate virus-host interactions of DNA viruses.

3.
EMBO Rep ; 22(9): e52145, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34347354

RESUMO

The APOBEC3 cytidine deaminases are implicated as the cause of a prevalent somatic mutation pattern found in cancer genomes. The APOBEC3 enzymes act as viral restriction factors by mutating viral genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes, although the regulatory measures for APOBEC3 expression and activity remain undefined. It is therefore difficult to predict circumstances that enable APOBEC3 interaction with cellular DNA that leads to mutagenesis. The APOBEC3A (A3A) enzyme is the most potent deaminase of the family. Using proteomics, we evaluate protein interactors of A3A to identify potential regulators. We find that A3A interacts with the chaperonin-containing TCP-1 (CCT) complex, a cellular machine that assists in protein folding and function. Importantly, depletion of CCT results in A3A-induced DNA damage and cytotoxicity. Evaluation of cancer genomes demonstrates an enrichment of A3A mutational signatures in cancers with silencing mutations in CCT subunit genes. Together, these data suggest that the CCT complex interacts with A3A, and that disruption of CCT function results in increased A3A mutational activity.


Assuntos
Chaperonina com TCP-1 , Citidina Desaminase , Chaperonina com TCP-1/genética , Citidina Desaminase/genética , Mutagênese , Proteínas/genética
4.
mBio ; 12(1)2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33593981

RESUMO

An unusual feature of papillomaviruses is that their genomes are packaged into virions along with host histones. Viral minichromosomes were visualized as "beads on a string" by electron microscopy in the 1970s but, to date, little is known about the posttranslational modifications of these histones. To investigate this, we analyzed the histone modifications in HPV16/18 quasivirions, wart-derived bovine papillomavirus (BPV1), and wart-derived human papillomavirus type 1 (HPV1) using quantitative mass spectrometry. The chromatin from all three virion samples had abundant posttranslational modifications (acetylation, methylation, and phosphorylation). These histone modifications were verified by acid urea polyacrylamide electrophoresis and immunoblot analysis. Compared to matched host cell controls, the virion minichromosome was enriched in histone modifications associated with active chromatin and depleted for those commonly found in repressed chromatin. We propose that the viral minichromosome acquires specific histone modifications late in infection that are coupled to the mechanisms of viral replication, late gene expression, and encapsidation. We predict that, in turn, these same modifications benefit early stages of infection by helping to evade detection, promoting localization of the viral chromosome to beneficial regions of the nucleus, and promoting early transcription and replication.IMPORTANCE A relatively unique feature of papillomaviruses is that the viral genome is associated with host histones inside the virion. However, little is known about the nature of the epigenome within papillomavirions or its biological relevance to the infectious viral cycle. Here, we define the epigenetic signature of the H3 and H4 histones from HPV16 virions generated in cell culture and native human papillomavirus type 1 (HPV1) and bovine papillomavirus 1 (BPV1) virions isolated from bovine and human wart tissue. We show that native virions are enriched in posttranslational modifications associated with active chromatin and depleted with those associated with repressed chromatin compared to cellular chromatin. Native virions were also enriched in the histone variant H3.3 compared to the canonical histone H3.1. We propose that the composition of virion-packaged chromatin reflects the late stages of the viral life cycle and promotes the early stages of infection by being primed for viral transcription.


Assuntos
Cromossomos/metabolismo , Código das Histonas , Histonas/metabolismo , Papillomaviridae/genética , Papillomaviridae/metabolismo , Vírion/genética , Vírion/metabolismo , Animais , Bovinos , Cromossomos/genética , Células HEK293 , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/metabolismo , Humanos , Queratinócitos/virologia , Metilação , Processamento de Proteína Pós-Traducional , Replicação Viral
5.
Nat Microbiol ; 6(2): 234-245, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33432153

RESUMO

Intrinsic antiviral host factors confer cellular defence by limiting virus replication and are often counteracted by viral countermeasures. We reasoned that host factors that inhibit viral gene expression could be identified by determining proteins bound to viral DNA (vDNA) in the absence of key viral antagonists. Herpes simplex virus 1 (HSV-1) expresses E3 ubiquitin-protein ligase ICP0 (ICP0), which functions as an E3 ubiquitin ligase required to promote infection. Cellular substrates of ICP0 have been discovered as host barriers to infection but the mechanisms for inhibition of viral gene expression are not fully understood. To identify restriction factors antagonized by ICP0, we compared proteomes associated with vDNA during HSV-1 infection with wild-type virus and a mutant lacking functional ICP0 (ΔICP0). We identified the cellular protein Schlafen family member 5 (SLFN5) as an ICP0 target that binds vDNA during HSV-1 ΔICP0 infection. We demonstrated that ICP0 mediates ubiquitination of SLFN5, which leads to its proteasomal degradation. In the absence of ICP0, SLFN5 binds vDNA to repress HSV-1 transcription by limiting accessibility of RNA polymerase II to viral promoters. These results highlight how comparative proteomics of proteins associated with viral genomes can identify host restriction factors and reveal that viral countermeasures can overcome SLFN antiviral activity.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Regulação Viral da Expressão Gênica , Herpes Simples/virologia , Interações Hospedeiro-Patógeno , Simplexvirus/genética , Transcrição Genética , Animais , Proteínas de Ciclo Celular/genética , Chlorocebus aethiops , DNA Viral/metabolismo , Células HEK293 , Células HeLa , Herpes Simples/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Regiões Promotoras Genéticas , Proteômica , RNA Polimerase II/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Células Vero
6.
Nat Commun ; 11(1): 6016, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-33243990

RESUMO

Adenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of N6-methyladenosine (m6A) to mRNAs. While m6A-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m6A mapping using short-read sequencing, here we profile m6A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m6A detection. Although both early and late viral transcripts contain m6A, depletion of m6A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m6A discovery within individual transcripts at nucleotide resolution, and highlight the role of m6A in regulating splicing of a viral pathogen.


Assuntos
Adenosina/análogos & derivados , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/genética , Splicing de RNA , RNA Viral/metabolismo , Células A549 , Adenosina/metabolismo , Adenovírus Humanos/patogenicidade , DNA Viral/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , Análise de Sequência de RNA , Replicação Viral
7.
Nat Cell Biol ; 22(10): 1197-1210, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32989251

RESUMO

Alveolar epithelial regeneration is essential for recovery from devastating lung diseases. This process occurs when type II alveolar pneumocytes (AT2 cells) proliferate and transdifferentiate into type I alveolar pneumocytes (AT1 cells). We used genome-wide analysis of chromatin accessibility and gene expression following acute lung injury to elucidate repair mechanisms. AT2 chromatin accessibility changed substantially following injury to reveal STAT3 binding motifs adjacent to genes that regulate essential regenerative pathways. Single-cell transcriptome analysis identified brain-derived neurotrophic factor (Bdnf) as a STAT3 target gene with newly accessible chromatin in a unique population of regenerating AT2 cells. Furthermore, the BDNF receptor tropomyosin receptor kinase B (TrkB) was enriched on mesenchymal alveolar niche cells (MANCs). Loss or blockade of AT2-specific Stat3, Bdnf or mesenchyme-specific TrkB compromised repair and reduced Fgf7 expression by niche cells. A TrkB agonist improved outcomes in vivo following lung injury. These data highlight the biological and therapeutic importance of the STAT3-BDNF-TrkB axis in orchestrating alveolar epithelial regeneration.


Assuntos
Células Epiteliais Alveolares/citologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Lesão Pulmonar/prevenção & controle , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor trkB/metabolismo , Regeneração , Fator de Transcrição STAT3/metabolismo , Células Epiteliais Alveolares/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Feminino , Humanos , Lesão Pulmonar/etiologia , Lesão Pulmonar/patologia , Masculino , Glicoproteínas de Membrana/genética , Proteínas Tirosina Quinases/genética , Receptor trkB/genética , Fator de Transcrição STAT3/genética
8.
mBio ; 11(4)2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843550

RESUMO

Paramyxoviruses are negative-sense single-stranded RNA viruses that comprise many important human and animal pathogens, including human parainfluenza viruses. These viruses bud from the plasma membrane of infected cells after the viral ribonucleoprotein complex (vRNP) is transported from the cytoplasm to the cell membrane via Rab11a-marked recycling endosomes. The viral proteins that are critical for mediating this important initial step in viral assembly are unknown. Here, we used the model paramyxovirus, murine parainfluenza virus 1, or Sendai virus (SeV), to investigate the roles of viral proteins in Rab11a-driven virion assembly. We previously reported that infection with SeV containing high levels of copy-back defective viral genomes (DVGs) (DVG-high SeV) generates heterogenous populations of cells. Cells enriched in full-length (FL) virus produce viral particles containing standard or defective viral genomes, while cells enriched in DVGs do not, despite high levels of defective viral genome replication. Here, we took advantage of this heterogenous cell phenotype to identify proteins that mediate interaction of vRNPs with Rab11a. We examined the roles of matrix protein and nucleoprotein and determined that their presence is not sufficient to drive interaction of vRNPs with recycling endosomes. Using a combination of mass spectrometry and comparative analyses of protein abundance and localization in DVG-high and FL-virus-high (FL-high) cells, we identified viral polymerase complex component protein L and, specifically, its cofactor C as interactors with Rab11a. We found that accumulation of L and C proteins within the cell is the defining feature that differentiates cells that proceed to viral egress from cells containing viruses that remain in replication phases.IMPORTANCE Paramyxoviruses are members of a family of viruses that include a number of pathogens imposing significant burdens on human health. In particular, human parainfluenza viruses are an important cause of pneumonia and bronchiolitis in children for which there are no vaccines or directly acting antivirals. These cytoplasmic replicating viruses bud from the plasma membrane and co-opt cellular endosomal recycling pathways to traffic viral ribonucleoprotein complexes from the cytoplasm to the membrane of infected cells. The viral proteins required for viral engagement with the recycling endosome pathway are still not known. Here, we used the model paramyxovirus Sendai virus, or murine parainfluenza virus 1, to investigate the role of viral proteins in this initial step of viral assembly. We found that the viral polymerase components large protein L and accessory protein C are necessary for engagement with recycling endosomes. These findings are important in identifying viral proteins as potential targets for development of antivirals.


Assuntos
Endossomos/virologia , Ribonucleoproteínas/metabolismo , Vírus Sendai/fisiologia , Montagem de Vírus , Células A549 , Linhagem Celular , Humanos , Ribonucleoproteínas/genética , Vírus Sendai/enzimologia , Vírus Sendai/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
9.
Nat Microbiol ; 5(10): 1217-1231, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32661314

RESUMO

Viruses promote infection by hijacking the ubiquitin machinery of the host to counteract or redirect cellular processes. Adenovirus encodes two early proteins, E1B55K and E4orf6, that together co-opt a cellular ubiquitin ligase complex to overcome host defences and promote virus production. Adenovirus mutants lacking E1B55K or E4orf6 display defects in viral RNA processing and protein production, but previously identified substrates of the redirected ligase do not explain these phenotypes. Here, we used a quantitative proteomics approach to identify substrates of E1B55K/E4orf6-mediated ubiquitination that facilitate RNA processing. While all currently known cellular substrates of E1B55K and E4orf6 are degraded by the proteasome, we uncovered RNA-binding proteins as high-confidence substrates that are not decreased in overall abundance. We focused on two RNA-binding proteins, RALY and hnRNP-C, which we confirm are ubiquitinated without degradation. Knockdown of RALY and hnRNP-C increased levels of viral RNA splicing, protein abundance and progeny production during infection with E1B55K-deleted virus. Furthermore, infection with E1B55K-deleted virus resulted in an increased interaction of hnRNP-C with viral RNA and attenuation of viral RNA processing. These data suggest that viral-mediated ubiquitination of RALY and hnRNP-C relieves a restriction on viral RNA processing and reveal an unexpected role for non-degradative ubiquitination in the manipulation of cellular processes during virus infection.


Assuntos
Infecções por Adenoviridae/virologia , Adenoviridae/fisiologia , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Infecções por Adenoviridae/metabolismo , Sequência de Bases , Sítios de Ligação , Biologia Computacional/métodos , Humanos , Motivos de Nucleotídeos , Ligação Proteica , Proteoma , Proteômica/métodos , Processamento Pós-Transcricional do RNA , Splicing de RNA , Ubiquitinação
10.
Virus Res ; 285: 198015, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32416261

RESUMO

Herpes simplex virus 1 (HSV-1) hijacks ubiquitination machinery to modify the cellular proteome to create an environment permissive for virus replication. HSV-1 encodes its own RING-finger E3 ubiquitin (Ub) ligase, Infected Cell Protein 0 (ICP0), that directly interfaces with component proteins of the Ub pathway to inactivate host immune defences and cellular processes that restrict the progression of HSV-1 infection. Consequently, ICP0 plays a critical role in the infectious cycle of HSV-1 that is required to promote the efficient onset of lytic infection and productive reactivation of viral genomes from latency. This review will describe the current knowledge regarding the biochemical properties and known substrates of ICP0 during HSV-1 infection. We will highlight the gaps in the characterization of ICP0 function and propose future areas of research required to understand fully the biological properties of this important HSV-1 regulatory protein.


Assuntos
Herpes Simples/virologia , Herpesvirus Humano 1/patogenicidade , Proteínas Imediatamente Precoces/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Interações entre Hospedeiro e Microrganismos , Humanos
11.
Hum Gene Ther ; 31(9-10): 499-511, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32303138

RESUMO

Recombinant adeno-associated virus has emerged as one of the most promising gene therapy delivery vectors. Development of these vectors took advantage of key features of the wild-type adeno-associated virus (AAV), enabled by basic studies of the underlying biology and requirements for transcription, replication, and packaging of the viral genome. Each step in generating and utilizing viral vectors involves numerous molecular interactions that together determine the efficiency of vector production and gene delivery. Once delivered into the cell, interactions with host proteins will determine the fate of the viral genome, and these will impact the intended goal of gene delivery. Here, we provide an overview of known interactions of the AAV genome with viral and cellular proteins involved in its amplification, packaging, and expression. Further appreciation of how the AAV genome interacts with host factors will enhance how this simple virus can be harnessed for an array of vector purposes that benefit human health.


Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos , Interações entre Hospedeiro e Microrganismos , Transdução Genética , Replicação Viral , Animais , Técnicas de Transferência de Genes , Genoma Viral , Vírus Auxiliares/fisiologia , Humanos
12.
Cancer Discov ; 10(4): 552-567, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32001516

RESUMO

Primary resistance to CD19-directed chimeric antigen receptor T-cell therapy (CART19) occurs in 10% to 20% of patients with acute lymphoblastic leukemia (ALL); however, the mechanisms of this resistance remain elusive. Using a genome-wide loss-of-function screen, we identified that impaired death receptor signaling in ALL led to rapidly progressive disease despite CART19 treatment. This was mediated by an inherent resistance to T-cell cytotoxicity that permitted antigen persistence and was subsequently magnified by the induction of CAR T-cell functional impairment. These findings were validated using samples from two CAR T-cell clinical trials in ALL, where we found that reduced expression of death receptor genes was associated with worse overall survival and reduced T-cell fitness. Our findings suggest that inherent dysregulation of death receptor signaling in ALL directly leads to CAR T-cell failure by impairing T-cell cytotoxicity and promoting progressive CAR T-cell dysfunction. SIGNIFICANCE: Resistance to CART19 is a significant barrier to efficacy in the treatment of B-cell malignancies. This work demonstrates that impaired death receptor signaling in tumor cells causes failed CART19 cytotoxicity and drives CART19 dysfunction, identifying a novel mechanism of antigen-independent resistance to CAR therapy.See related commentary by Green and Neelapu, p. 492.


Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Morte Celular/metabolismo , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Transdução de Sinais
13.
Viruses ; 12(2)2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-32013091

RESUMO

DNA viruses that replicate in the nucleus encompass a range of ubiquitous and clinically important viruses, from acute pathogens to persistent tumor viruses. These viruses must co-opt nuclear processes for the benefit of the virus, whilst evading host processes that would otherwise attenuate viral replication. Accordingly, DNA viruses induce the formation of membraneless assemblies termed viral replication compartments (VRCs). These compartments facilitate the spatial organization of viral processes and regulate virus-host interactions. Here, we review advances in our understanding of VRCs. We cover their initiation and formation, their function as the sites of viral processes, and aspects of their composition and organization. In doing so, we highlight ongoing and emerging areas of research highly pertinent to our understanding of nuclear-replicating DNA viruses.


Assuntos
Núcleo Celular/virologia , Vírus de DNA/fisiologia , Interações entre Hospedeiro e Microrganismos , Compartimentos de Replicação Viral , Replicação Viral , Humanos , Proteínas Virais/genética
14.
Nat Commun ; 11(1): 23, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911620

RESUMO

Assembly of infectious influenza A viruses (IAV) is a complex process involving transport from the nucleus to the plasma membrane. Rab11A-containing recycling endosomes have been identified as a platform for intracellular transport of viral RNA (vRNA). Here, using high spatiotemporal resolution light-sheet microscopy (~1.4 volumes/second, 330 nm isotropic resolution), we quantify Rab11A and vRNA movement in live cells during IAV infection and report that IAV infection decreases speed and increases arrest of Rab11A. Unexpectedly, infection with respiratory syncytial virus alters Rab11A motion in a manner opposite to IAV, suggesting that Rab11A is a common host component that is differentially manipulated by respiratory RNA viruses. Using two-color imaging we demonstrate co-transport of Rab11A and IAV vRNA in infected cells and provide direct evidence that vRNA-associated Rab11A have altered transport. The mechanism of altered Rab11A movement is likely related to a decrease in dynein motors bound to Rab11A vesicles during IAV infection.


Assuntos
Dineínas/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Transporte Biológico , Dineínas/genética , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/virologia , RNA Viral/genética , RNA Viral/metabolismo , Proteínas rab de Ligação ao GTP/genética
15.
FEBS Lett ; 593(24): 3531-3550, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31764999

RESUMO

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.


Assuntos
Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/fisiologia , Núcleo Celular/virologia , Genoma Viral , Interações Hospedeiro-Patógeno , Humanos , Replicação Viral
16.
DNA Repair (Amst) ; 83: 102700, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31563041

RESUMO

The APOBEC3 family of cytosine deaminases are part of the innate immune response to viral infection, but also have the capacity to damage cellular DNA. Detection of mutational signatures consistent with APOBEC3 activity, together with elevated APOBEC3 expression in cancer cells, has raised the possibility that these enzymes contribute to oncogenesis. Genome deamination by APOBEC3 enzymes also elicits DNA damage response signaling and presents therapeutic vulnerabilities for cancer cells. Here, we discuss implications of APOBEC3 activity in cancer and the potential to exploit their mutagenic activity for targeted cancer therapies.


Assuntos
Antineoplásicos/farmacologia , Antivirais/farmacologia , Citidina Desaminase/metabolismo , Terapia de Alvo Molecular/métodos , Neoplasias/enzimologia , Animais , Citidina Desaminase/genética , Humanos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética
17.
Cell Rep ; 28(2): 434-448.e6, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31291579

RESUMO

Cellular SAMHD1 inhibits replication of many viruses by limiting intracellular deoxynucleoside triphosphate (dNTP) pools. We investigate the influence of SAMHD1 on human cytomegalovirus (HCMV). During HCMV infection, we observe SAMHD1 induction, accompanied by phosphorylation via viral kinase UL97. SAMHD1 depletion increases HCMV replication in permissive fibroblasts and conditionally permissive myeloid cells. We show this is due to enhanced gene expression from the major immediate-early (MIE) promoter and is independent of dNTP levels. SAMHD1 suppresses innate immune responses by inhibiting nuclear factor κB (NF-κB) activation. We show that SAMHD1 regulates the HCMV MIE promoter through NF-κB activation. Chromatin immunoprecipitation reveals increased RELA and RNA polymerase II on the HCMV MIE promoter in the absence of SAMHD1. Our studies reveal a mechanism of HCMV virus restriction by SAMHD1 and show how SAMHD1 deficiency activates an innate immune pathway that paradoxically results in increased viral replication through transcriptional activation of the HCMV MIE gene promoter.


Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/patogenicidade , NF-kappa B/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Humanos , Fosforilação , Regulação para Cima
19.
FASEB J ; 33(8): 9388-9403, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31107607

RESUMO

Homologous recombination (HR) is considered a major driving force of evolution because it generates and expands genetic diversity. Evidence of HR between coinfecting herpesvirus DNA genomes can be found frequently both in vitro and in clinical isolates. Each herpes simplex virus type 1 (HSV-1) replication compartment (RC) derives from a single incoming genome and maintains a specific territory within the nucleus. This raises intriguing questions about where and when coinfecting viral genomes interact. To study the spatiotemporal requirements for intergenomic recombination, we developed an assay with dual-color FISH that enables detection of HR between different pairs of coinfecting HSV-1 genomes. Our results revealed that HR increases intermingling of RCs derived from different genomes. Furthermore, inhibition of RC movement reduces the rate of HR events among coinfecting viruses. Finally, we observed correlation between nuclear size and the number of RCs per nucleus. Our findings suggest that both viral replication and recombination are subject to nuclear spatial constraints. Other DNA viruses and cellular DNA are likely to encounter similar restrictions.-Tomer, E., Cohen, E. M., Drayman, N., Afriat, A., Weitzman, M. D., Zaritsky, A., Kobiler, O. Coalescing replication compartments provide the opportunity for recombination between coinfecting herpesviruses.


Assuntos
Genoma Viral/genética , Herpesvirus Humano 1/genética , Replicação Viral/fisiologia , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Replicação do DNA/genética , Replicação do DNA/fisiologia , Feminino , Herpesvirus Humano 1/fisiologia , Humanos , Hibridização in Situ Fluorescente , Recombinação Genética/genética , Células Vero , Replicação Viral/genética
20.
mSphere ; 4(1)2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30814317

RESUMO

More than 14,000 neonates are infected with herpes simplex virus (HSV) annually. Approximately half display manifestations limited to the skin, eyes, or mouth (SEM disease). The rest develop invasive infections that spread to the central nervous system (CNS disease or encephalitis) or throughout the infected neonate (disseminated disease). Invasive HSV disease is associated with significant morbidity and mortality, but the viral and host factors that predispose neonates to these forms are unknown. To define viral diversity within the infected neonatal population, we evaluated 10 HSV-2 isolates from newborns with a range of clinical presentations. To assess viral fitness independently of host immune factors, we measured viral growth characteristics in cultured cells and found diverse in vitro phenotypes. Isolates from neonates with CNS disease were associated with larger plaque size and enhanced spread, with the isolates from cerebrospinal fluid (CSF) exhibiting the most robust growth. We sequenced complete viral genomes of all 10 neonatal viruses, providing new insights into HSV-2 genomic diversity in this clinical setting. We found extensive interhost and intrahost genomic diversity throughout the viral genome, including amino acid differences in more than 90% of the viral proteome. The genes encoding glycoprotein G (gG; US4), glycoprotein I (gI; US7), and glycoprotein K (gK; UL53) and viral proteins UL8, UL20, UL24, and US2 contained variants that were found in association with CNS isolates. Many of these viral proteins are known to contribute to cell spread and neurovirulence in mouse models of CNS disease. This report represents the first application of comparative pathogen genomics to neonatal HSV disease.IMPORTANCE Herpes simplex virus (HSV) causes invasive disease in half of infected neonates, resulting in significant mortality and permanent cognitive morbidity. The factors that contribute to invasive disease are not understood. This study revealed diversity among HSV isolates from infected neonates and detected the first associations between viral genetic variations and clinical disease manifestations. We found that viruses isolated from newborns with encephalitis showed enhanced spread in culture. These viruses contained protein-coding variations not found in viruses causing noninvasive disease. Many of these variations were found in proteins known to impact neurovirulence and viral spread between cells. This work advances our understanding of HSV diversity in the neonatal population and how it may impact disease outcome.


Assuntos
Variação Genética , Herpes Simples/virologia , Herpesvirus Humano 2/genética , Complicações Infecciosas na Gravidez/virologia , Linhagem Celular , Encefalite Viral/virologia , Feminino , Genoma Viral , Genômica , Genótipo , Idade Gestacional , Herpes Simples/complicações , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/isolamento & purificação , Herpesvirus Humano 2/patogenicidade , Humanos , Recém-Nascido , Masculino , Fenótipo , Gravidez , Proteínas Virais/genética
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