RESUMO
Science is humanity's best insurance against threats from nature, but it is a fragile enterprise that must be nourished and protected. The preponderance of scientific evidence indicates a natural origin for SARS-CoV-2. Yet, the theory that SARS-CoV-2 was engineered in and escaped from a lab dominates media attention, even in the absence of strong evidence. We discuss how the resulting anti-science movement puts the research community, scientific research, and pandemic preparedness at risk.
RESUMO
The dynamic nature of interactions between invading viral pathogens and their hosts has fascinated scientists for several decades. The well-known capacity of herpes simplex virus (HSV) to establish life-long infections in humans reflects a dynamic balance between maintaining a latent state in which viral genomes are silenced and re-entry into the lytic phase during reactivation. Silencing of the viral genome has been shown to be a function of innate immune signalling, intrinsic cellular antiviral mechanisms and epigenetic repression. Thus, although many important observations have been made identifying cellular processes that contribute to the repression of the viral genome and latency, the field has lacked an understanding of how these factors work together. In this issue of EMBO Reports, Suzich et al (2021) present convincing evidence that brings together individual observations into a cohesive model that explains many of these outstanding mysteries. Here, we will review the background data that lead to this outstanding piece of work.
Assuntos
Herpesvirus Humano 1 , Repressão Epigenética , Genoma Viral , Herpesvirus Humano 1/genética , Humanos , Latência Viral/genéticaRESUMO
Most DNA viruses that use recombination-dependent mechanisms to replicate their DNA encode a single-strand annealing protein (SSAP). The herpes simplex virus (HSV) single-strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. ICP8 is a classical replicative SSB and interacts physically and/or functionally with the other viral replication proteins. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a member of the SSAP family. The role of annealing during HSV infection has been difficult to assess in part, because it has not been possible to distinguish between the role of ICP8 as an SSAP from its role as a replicative SSB during viral replication. In this paper, we have characterized an ICP8 mutant, Q706A/F707A (QF), that lacks annealing activity but retains many other functions characteristic of replicative SSBs. Like WT ICP8, the QF mutant protein forms filaments in vitro, binds ssDNA cooperatively, and stimulates the activities of other replication proteins including the viral polymerase, helicase-primase complex, and the origin binding protein. Interestingly, the QF mutant does not complement an ICP8-null virus for viral growth, replication compartment formation, or DNA replication. Thus, we have been able to separate the activities of ICP8 as a replicative SSB from its annealing activity. Taken together, our data indicate that the annealing activity of ICP8 is essential for viral DNA replication in the context of infection and support the notion that HSV-1 uses recombination-dependent mechanisms during DNA replication.
Assuntos
Replicação do DNA/fisiologia , DNA Viral/biossíntese , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 1/fisiologia , Recombinação Genética/fisiologia , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Substituição de Aminoácidos , Animais , Chlorocebus aethiops , DNA de Cadeia Simples/biossíntese , DNA de Cadeia Simples/genética , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Mutação , Mutação de Sentido Incorreto , Células Vero , Proteínas Virais/genéticaRESUMO
The NIH Virtual SARS-CoV-2 Antiviral Summit, held on 6 November 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for coronavirus disease 2019 (COVID-19), including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small-molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.
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Antivirais/uso terapêutico , Tratamento Farmacológico da COVID-19 , SARS-CoV-2/efeitos dos fármacos , Antivirais/farmacologia , COVID-19/virologia , Desenvolvimento de Medicamentos , Humanos , National Institutes of Health (U.S.) , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Estados Unidos , Replicação Viral/efeitos dos fármacosRESUMO
Molecular chaperones and cochaperones are the most abundant cellular effectors of protein homeostasis, assisting protein folding and preventing aggregation of misfolded proteins. We have previously shown that herpes simplex virus 1 (HSV-1) infection results in the drastic spatial reorganization of the cellular chaperone Hsc70 into nuclear domains called VICE (Virus Induced Chaperone Enriched) domains and that this recruitment is dependent on the viral immediate early protein ICP22. Here, we present several lines of evidence supporting the notion that ICP22 functions as a virally encoded cochaperone (J-protein/Hsp40) functioning together with its Hsc70 partner to recognize and manage aggregated and misfolded proteins. We show that ICP22 results in (i) nuclear sequestration of nonnative proteins, (ii) reduction of cytoplasmic aggresomes in cells expressing aggregation-prone proteins, and (iii) thermoprotection against heat inactivation of firefly luciferase, and (iv) sequence homology analysis indicated that ICP22 contains an N-terminal J domain and a C-terminal substrate binding domain, similar to type II cellular J proteins. ICP22 may thus be functionally similar to J-protein/Hsp40 cochaperones that function together with their HSP70 partners to prevent aggregation of nonnative proteins. This is not the first example of a virus hijacking a function of a cellular chaperone, since simian immunodeficiency virus T antigen was previously shown to contain a J domain; however, this the first known example of the acquisition of a functional J-like protein by a virus and suggests that HSV has taken advantage of the adaptable nature of J proteins to evolve a multifunctional cochaperone that functions with Hsc70 to promote lytic infection.IMPORTANCE Viruses have evolved a variety of strategies to succeed in a hostile environment. The herpes simplex virus 1 (HSV-1) immediate early protein ICP22 plays several roles in the virus life cycle, including downregulation of cellular gene expression, upregulation of late viral gene expression, inhibition of apoptosis, prevention of aggregation of nonnative proteins, and the recruitment of a cellular heat shock protein, Hsc70, to nuclear domains. We present evidence that ICP22 functionally resembles a cellular J-protein/HSP40 family cochaperone, interacting specifically with Hsc70. We suggest that HSV has taken advantage of the adaptable nature of J proteins to evolve a multifunctional cochaperone that functions with Hsc70 to promote lytic infection.
Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Herpesvirus Humano 1/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Animais , Núcleo Celular/metabolismo , Chlorocebus aethiops , Células HEK293 , Herpes Simples/virologia , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 1/fisiologia , Humanos , Proteínas Imediatamente Precoces/genética , Chaperonas Moleculares/metabolismo , Fosforilação , Dobramento de Proteína , RNA Polimerase II/metabolismo , Células Vero , Proteínas Virais/metabolismoRESUMO
This study examined the microbicidal activity of 222-nm UV radiation (UV222), which is potentially a safer alternative to the 254-nm UV radiation (UV254) that is often used for surface decontamination. Spores and/or growing and stationary-phase cells of Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Staphylococcus aureus, and Clostridioides difficile and a herpesvirus were all killed or inactivated by UV222 and at lower fluences than with UV254B. subtilis spores and cells lacking the major DNA repair protein RecA were more sensitive to UV222, as were spores lacking their DNA-protective proteins, the α/ß-type small, acid-soluble spore proteins. The spore cores' large amount of Ca2+-dipicolinic acid (â¼25% of the core dry weight) also protected B. subtilis and C. difficile spores against UV222, while spores' proteinaceous coat may have given some slight protection against UV222 Survivors among B. subtilis spores treated with UV222 acquired a large number of mutations, and this radiation generated known mutagenic photoproducts in spore and cell DNA, primarily cyclobutane-type pyrimidine dimers in growing cells and an α-thyminyl-thymine adduct termed the spore photoproduct (SP) in spores. Notably, the loss of a key SP repair protein markedly decreased spore UV222 resistance. UV222-treated B. subtilis spores germinated relatively normally, and the generation of colonies from these germinated spores was not salt sensitive. The latter two findings suggest that UV222 does not kill spores by general protein damage, and thus, the new results are consistent with the notion that DNA damage is responsible for the killing of spores and cells by UV222IMPORTANCE Spores of a variety of bacteria are resistant to common decontamination agents, and many of them are major causes of food spoilage and some serious human diseases, including anthrax caused by spores of Bacillus anthracis Consequently, there is an ongoing need for efficient methods for spore eradication, in particular methods that have minimal deleterious effects on people or the environment. UV radiation at 254 nm (UV254) is sporicidal and commonly used for surface decontamination but can cause deleterious effects in humans. Recent work, however, suggests that 222-nm UV (UV222) may be less harmful to people than UV254 yet may still kill bacteria and at lower fluences than UV254 The present work has identified the damage by UV222 that leads to the killing of growing cells and spores of some bacteria, many of which are human pathogens, and UV222 also inactivates a herpesvirus.
Assuntos
Bacillus/efeitos da radiação , Clostridioides difficile/efeitos da radiação , Dano ao DNA , Simplexvirus/efeitos da radiação , Esporos Bacterianos/efeitos da radiação , Staphylococcus aureus/efeitos da radiação , Bacillus/fisiologia , Clostridioides difficile/fisiologia , Simplexvirus/fisiologia , Esporos Bacterianos/fisiologia , Staphylococcus aureus/fisiologia , Raios Ultravioleta/efeitos adversosRESUMO
The purpose of this review is to explore recombination strategies in DNA viruses. Homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. Recombination and DNA replication are interconnected, with recombination being essential for repairing DNA damage and supporting replication of the viral genome. Recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understanding viral origins as well as the dynamic nature of viral-host interactions. Both bacteriophage λ and herpes simplex virus (HSV) display high rates of recombination, both utilizing their own proteins and commandeering cellular proteins to promote recombination reactions. We focus primarily on λ and HSV, as they have proven amenable to both genetic and biochemical analysis and have recently been shown to exhibit some surprising similarities that will guide future studies.
Assuntos
Bacteriófago lambda/fisiologia , Vírus de DNA/genética , Recombinação Genética , Simplexvirus/genética , Vírus de DNA/fisiologia , Genoma Viral , Simplexvirus/fisiologiaRESUMO
During lytic infection, herpes simplex virus (HSV) DNA is replicated by a mechanism involving DNA recombination. For instance, replication of the HSV-1 genome produces X- and Y-branched structures, reminiscent of recombination intermediates. HSV-1's replication machinery includes a trimeric helicase-primase composed of helicase (UL5) and primase (UL52) subunits and a third subunit, UL8. UL8 has been reported to stimulate the helicase and primase activities of the complex in the presence of ICP8, an HSV-1 protein that functions as an annealase, a protein that binds complementary single-stranded DNA (ssDNA) and facilitates its annealing to duplex DNA. UL8 also influences the intracellular localization of the UL5/UL52 subunits, but UL8's catalytic activities are not known. In this study we used a combination of biochemical techniques and transmission electron microscopy. First, we report that UL8 alone forms protein filaments in solution. Moreover, we also found that UL8 binds to ssDNAs >50-nucletides long and promotes the annealing of complementary ssDNA to generate highly branched duplex DNA structures. Finally, UL8 has a very high affinity for replication fork structures containing a gap in the lagging strand as short as 15 nucleotides, suggesting that UL8 may aid in directing or loading the trimeric complex onto a replication fork. The properties of UL8 uncovered here suggest that UL8 may be involved in the generation of the X- and Y-branched structures that are the hallmarks of HSV replication.
Assuntos
DNA Helicases/metabolismo , DNA Primase/metabolismo , Replicação do DNA , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/genética , Proteínas Virais/metabolismo , Sequência de Bases , DNA de Cadeia Simples/biossíntese , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Herpesvirus Humano 1/ultraestrutura , Peso MolecularRESUMO
The herpes simplex virus (HSV) type I alkaline nuclease, UL12, has 5'-to-3' exonuclease activity and shares homology with nucleases from other members of the Herpesviridae family. We previously reported that a UL12-null virus exhibits a severe defect in viral growth. To determine whether the growth defect was a result of loss of nuclease activity or another function of UL12, we introduced an exonuclease-inactivating mutation into the viral genome. The recombinant virus, UL12 D340E (the D340E mutant), behaved identically to the null virus (AN-1) in virus yield experiments, exhibiting a 4-log decrease in the production of infectious virus. Furthermore, both viruses were severely defective in cell-to-cell spread and produced fewer DNA-containing capsids and more empty capsids than wild-type virus. In addition, DNA packaged by the viral mutants was aberrant, as determined by infectivity assays and pulsed-field gel electrophoresis. We conclude that UL12 exonuclease activity is essential for the production of viral DNA that can be packaged to produce infectious virus. This conclusion was bolstered by experiments showing that a series of natural and synthetic α-hydroxytropolones recently reported to inhibit HSV replication also inhibit the nuclease activity of UL12. Taken together, our results demonstrate that the exonuclease activity of UL12 is essential for the production of infectious virus and may be considered a target for development of antiviral agents.IMPORTANCE Herpes simplex virus is a major pathogen, and although nucleoside analogs such as acyclovir are highly effective in controlling HSV-1 or -2 infections in immunocompetent individuals, their use in immunocompromised patients is complicated by the development of resistance. Identification of additional proteins essential for viral replication is necessary to develop improved therapies. In this communication, we confirm that the exonuclease activity of UL12 is essential for viral replication through the analysis of a nuclease-deficient viral mutant. We demonstrate that the exonuclease activity of UL12 is essential for the production of viral progeny and thus provides an attractive, druggable enzymatic target.
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Desoxirribonucleases/metabolismo , Herpesvirus Humano 1/patogenicidade , Mutação , Proteínas Virais/metabolismo , Montagem de Vírus , Animais , Capsídeo/metabolismo , Chlorocebus aethiops , Replicação do DNA , Desoxirribonucleases/química , Desoxirribonucleases/genética , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/fisiologia , Humanos , Células Vero , Proteínas Virais/química , Proteínas Virais/genética , Replicação ViralRESUMO
Human ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that prevents protein degradation by removing polyubiquitin chains from its substrates. It regulates the stability of a number of human transcription factors and tumor suppressors and plays a critical role in the development of several types of cancer, including prostate and small cell lung cancer. In addition, human USP7 is targeted by several viruses of the Herpesviridae family and is required for effective herpesvirus infection. The USP7 C-terminal region (C-USP7) contains five ubiquitin-like domains (UBL1-5) that interact with several USP7 substrates. Although structures of the USP7 C terminus bound to its substrates could provide vital information for understanding USP7 substrate specificity, no such data has been available to date. In this work we have demonstrated that the USP7 ubiquitin-like domains can be studied in isolation by solution NMR spectroscopy, and we have determined the structure of the UBL1 domain. Furthermore, we have employed NMR and viral plaque assays to probe the interaction between the C-USP7 and HSV-1 immediate-early protein ICP0 (infected cell protein 0), which is essential for efficient lytic infection and virus reactivation from latency. We have shown that depletion of the USP7 in HFF-1 cells negatively affects the efficiency of HSV-1 lytic infection. We have also found that USP7 directly binds ICP0 via its C-terminal UBL1-2 domains and mapped the USP7-binding site for ICP0. Therefore, this study represents a first step toward understanding the molecular mechanism of C-USP7 specificity toward its substrates and may provide the basis for future development of novel antiviral and anticancer therapies.
Assuntos
Herpes Simples/metabolismo , Herpesvirus Humano 1/enzimologia , Proteínas Imediatamente Precoces/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular , Herpes Simples/genética , Herpesvirus Humano 1/química , Herpesvirus Humano 1/genética , Humanos , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/genética , Ligação Proteica , Estrutura Terciária de Proteína , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Peptidase 7 Específica de UbiquitinaRESUMO
UNLABELLED: Herpes simplex virus (HSV) dramatically reorganizes the infected-cell nucleus, leading to the formation of prereplicative sites and replication compartments. This process is driven by the essential viral single-stranded DNA (ssDNA) binding protein ICP8, which can form double-helical filaments in the absence of DNA. In this paper, we show that two conserved motifs, FNF (F1142, N1143, and F1144) and FW (F843 and W844), are essential for ICP8 self-interactions, and we propose that the FNF motif docks into the FW region during filament formation. Mammalian expression plasmids bearing mutations in these motifs (FNF and FW) were unable to complement an ICP8-null mutant for growth and replication compartment formation. Furthermore, FNF and FW mutants were able to inhibit wild-type (WT) virus plaque formation and filament formation, whereas a double mutant (FNF-FW) was not. These results suggest that single mutant proteins are incorporated into nonproductive ICP8 filaments, while the double mutant is unable to interact with WT ICP8 and does not interfere with WT growth. Cells transfected with WT ICP8 and the helicase-primase (H/P) complex exhibited punctate nuclear structures that resemble prereplicative sites; however, the FNF and FW mutants failed to do so. Taken together, these results suggest that the FNF and FW motifs are required for ICP8 self-interactions and that these interactions may be important for the formation of prereplicative sites and replication compartments. We propose that filaments or other higher-order structures of ICP8 may provide a scaffold onto which other proteins can be recruited to form prereplicative sites and replication compartments. IMPORTANCE: For nuclear viruses such as HSV, efficient DNA replication requires the formation of discrete compartments within the infected-cell nucleus in which replication proteins are concentrated and assembled into the HSV replisome. In this paper, we characterize the role of filament formation by the single-stranded DNA binding protein ICP8 in the formation of prereplicative sites and replication compartments. We propose that ICP8 protein filaments generate a protein scaffold for other cellular and viral proteins, resulting in a structure that concentrates both viral DNA and replication proteins. Replication compartments may be similar to other types of cellular membraneless compartments thought to be formed by phase separations caused by low-affinity, multivalent interactions involving proteins and nucleic acids within cells. ICP8 scaffolds could facilitate the formation of replication compartments by mediating interactions with other components of the replication machinery.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Multimerização Proteica , Simplexvirus/fisiologia , Proteínas Virais/metabolismo , Replicação Viral , Motivos de Aminoácidos , Animais , Chlorocebus aethiops , Análise Mutacional de DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Simplexvirus/crescimento & desenvolvimento , Células Vero , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
UNLABELLED: During DNA encapsidation, herpes simplex virus 1 (HSV-1) procapsids are converted to DNA-containing capsids by a process involving activation of the viral protease, expulsion of the scaffold proteins, and the uptake of viral DNA. Encapsidation requires six minor capsid proteins (UL6, UL15, UL17, UL25, UL28, and UL33) and one viral protein, UL32, not found to be associated with capsids. Although functions have been assigned to each of the minor capsid proteins, the role of UL32 in encapsidation has remained a mystery. Using an HSV-1 variant containing a functional hemagglutinin-tagged UL32, we demonstrated that UL32 was synthesized with true late kinetics and that it exhibited a previously unrecognized localization pattern. At 6 to 9 h postinfection (hpi), UL32 accumulated in viral replication compartments in the nucleus of the host cell, while at 24 hpi, it was additionally found in the cytoplasm. A newly generated UL32-null mutant was used to confirm that although B capsids containing wild-type levels of capsid proteins were synthesized, these procapsids were unable to initiate the encapsidation process. Furthermore, we showed that UL32 is redox sensitive and identified two highly conserved oxidoreductase-like C-X-X-C motifs that are essential for protein function. In addition, the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, were altered in the absence of UL32, suggesting that UL32 may act to modulate disulfide bond formation during procapsid assembly and maturation. IMPORTANCE: Although functions have been assigned to six of the seven required packaging proteins of HSV, the role of UL32 in encapsidation has remained a mystery. UL32 is a cysteine-rich viral protein that contains C-X-X-C motifs reminiscent of those in proteins that participate in the regulation of disulfide bond formation. We have previously demonstrated that disulfide bonds are required for the formation and stability of the viral capsids and are also important for the formation and stability of the UL6 portal ring. In this report, we demonstrate that the disulfide bond profiles of the viral proteins UL6, UL25, and VP19C and the viral protease, VP24, are altered in cells infected with a newly isolated UL32-null mutant virus, suggesting that UL32 acts as a chaperone capable of modulating disulfide bond formation. Furthermore, these results suggest that proper regulation of disulfide bonds is essential for initiating encapsidation.
Assuntos
Dissulfetos/metabolismo , Herpesvirus Humano 1/fisiologia , Proteínas Virais/metabolismo , Montagem de Vírus , Animais , Chlorocebus aethiops , Deleção de Genes , Perfilação da Expressão Gênica , Herpesvirus Humano 1/genética , Células Vero , Proteínas Virais/genéticaRESUMO
UNLABELLED: The herpes simplex virus 1 (HSV-1) virion DNA contains nicks and gaps, and in this study a novel assay for estimating the size and number of gaps in virion DNA was developed. Consistent with previous reports, we estimate that there are approximately 15 gaps per genome, and we calculate the average gap length to be approximately 30 bases. Virion DNA was isolated and treated with DNA-modifying enzymes in order to fill in the gaps and modify the ends. Interestingly, filling in gaps, blunting the ends, or adding random sequences to the 3' ends of DNA, producing 3' flaps, did not impair the infectivity of treated DNA following transfection of Vero cells. On the other hand, the formation of 5' flaps in the DNA following treatment resulted in a dramatic reduction (95 to 100%) in infectivity. Virion DNA stimulated DNA-PKcs activity in transfected cells, and DNA with 5' flaps stimulated a higher level of DNA-PKcs activity than that observed in cells transfected with untreated virion DNA. The infectivity of 5'-flapped DNA was restored in cells that do not express DNA-PKcs and in cells cotransfected with the immediate early protein ICP0, which degrades DNA-PKcs. These results are consistent with previous reports that DNA-dependent protein kinase (DNA-PK) and the nonhomologous end joining (NHEJ) repair pathway are intrinsically antiviral and that ICP0 can counteract this effect. We suggest that HSV-1 DNA with 5' flaps may induce an antiviral state due to the induction of a DNA damage response, primarily mediated by NHEJ, that renders the HSV-1 genome less efficient for lytic infection. IMPORTANCE: For productive lytic infection to occur, HSV-1 must counteract a variety of cellular intrinsic antiviral mechanisms, including the DNA damage response (DDR). DDR pathways have been associated with silencing of gene expression, cell cycle arrest, and induction of apoptosis. In addition, the fate of viral genomes is likely to play a role in whether viral genomes adopt a configuration suitable for lytic DNA replication. This study demonstrates that virion DNA activates the cellular DDR kinase, DNA-PK, and that this response is inhibitory to viral infection. Furthermore, we show that HSV-1 ubiquitin ligase, ICP0, plays an important role in counteracting the negative effects of DNA-PK activation. These findings support the notion that DNA-PK is antiviral and suggest that the fate of incoming viral DNA has important consequences for the progression of lytic infection. This study underscores the complex evolutionary relationships between HSV and its host.
Assuntos
Dano ao DNA , Reparo do DNA , DNA Viral/genética , Genoma Viral , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Resposta SOS em Genética , Animais , Quebras de DNA de Cadeia Simples , Deleção de Sequência , Transfecção , Células Vero , Replicação ViralRESUMO
Herpes Simplex Virus type 1 (HSV-1) has evolved to disable the cellular DNA damage response kinase, ATR. We have previously shown that HSV-1-infected cells are unable to phosphorylate the ATR substrate Chk1, even under conditions in which replication forks are stalled. Here we report that the HSV-1 single stranded DNA binding protein (ICP8), and the helicase/primase complex (UL8/UL5/UL52) form a nuclear complex in transfected cells that is necessary and sufficient to disable ATR signaling. This complex localizes to sites of DNA damage and colocalizes with ATR/ATRIP and RPA, but under these conditions, the Rad9-Rad1-Hus1 checkpoint clamp (9-1-1) do not. ATR is generally activated by substrates that contain ssDNA adjacent to dsDNA, and previous work from our laboratory has shown that ICP8 and helicase/primase also recognize this substrate. We suggest that these four viral proteins prevent ATR activation by binding to the DNA substrate and obstructing loading of the 9-1-1 checkpoint clamp. Exclusion of 9-1-1 prevents recruitment of TopBP1, the ATR kinase activator, and thus effectively disables ATR signaling. These data provide the first example of viral DNA replication proteins obscuring access to a DNA substrate that would normally trigger a DNA damage response and checkpoint signaling. This unusual mechanism used by HSV suggests that it may be possible to inhibit ATR signaling by preventing recruitment of the 9-1-1 clamp and TopBP1.
Assuntos
DNA Helicases/metabolismo , DNA Primase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 1/metabolismo , Transdução de Sinais , Proteínas Virais/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chlorocebus aethiops , DNA Helicases/genética , DNA Primase/genética , DNA Viral/genética , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Exonucleases/genética , Exonucleases/metabolismo , Células HeLa , Herpesvirus Humano 1/genética , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células Vero , Proteínas Virais/genéticaRESUMO
Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of the host cell and is known to interact with several components of the cellular DNA-damage-signaling machinery. We have previously reported that the DNA damage response kinase, ATR, is specifically inactivated in HSV-1-infected cells. On the other hand, we have also shown that ATR and its scaffolding protein, ATRIP, are recruited to viral replication compartments, where they play beneficial roles during HSV-1 replication. In order to better understand this apparent discrepancy, we tested the hypothesis that some of the components of the ATR pathway may exert an antiviral effect on infection. In fact, we learned that all 10 of the canonical ATR pathway proteins are stable in HSV-infected cells and are recruited to viral replication compartments; furthermore, short hairpin RNA (shRNA) knockdown shows that several, including ATRIP, RPA70, TopBP1, Claspin, and CINP, are required for efficient HSV-1 replication. We also determined that activation of the ATR kinase prior to infection did not affect virus yield but did result in reduced levels of recombination between coinfecting viruses. Together, these data suggest that ATR pathway proteins are not antiviral per se but that activation of ATR signaling may have negative consequences during viral replication, such as inhibiting recombination.
Assuntos
Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Transdução de Sinais , Replicação Viral , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Humanos , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Production of concatemeric DNA is an essential step during HSV infection, as the packaging machinery must recognize longer-than-unit-length concatemers; however, the mechanism by which they are formed is poorly understood. Although it has been proposed that the viral genome circularizes and rolling circle replication leads to the formation of concatemers, several lines of evidence suggest that HSV DNA replication involves recombination-dependent replication reminiscent of bacteriophages λ and T4. Similar to λ, HSV-1 encodes a 5'-to-3' exonuclease (UL12) and a single strand annealing protein [SSAP (ICP8)] that interact with each other and can perform strand exchange in vitro. By analogy with λ phage, HSV may utilize viral and/or cellular recombination proteins during DNA replication. At least four double strand break repair pathways are present in eukaryotic cells, and HSV-1 is known to manipulate several components of these pathways. Chromosomally integrated reporter assays were used to measure the repair of double strand breaks in HSV-infected cells. Single strand annealing (SSA) was increased in HSV-infected cells, while homologous recombination (HR), non-homologous end joining (NHEJ) and alternative non-homologous end joining (A-NHEJ) were decreased. The increase in SSA was abolished when cells were infected with a viral mutant lacking UL12. Moreover, expression of UL12 alone caused an increase in SSA, which was completely eliminated when a UL12 mutant lacking exonuclease activity was expressed. UL12-mediated stimulation of SSA was decreased in cells lacking the cellular SSAP, Rad52, and could be restored by coexpressing the viral SSAP, ICP8, indicating that an SSAP is also required. These results demonstrate that UL12 can specifically stimulate SSA and that either ICP8 or Rad52 can function as an SSAP. We suggest that SSA is the homology-mediated repair pathway utilized during HSV infection.
Assuntos
Replicação do DNA , DNA de Cadeia Simples/metabolismo , DNA Viral/biossíntese , Desoxirribonucleases/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 1/fisiologia , Recombinação Homóloga , Proteínas Virais/metabolismo , DNA de Cadeia Simples/genética , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases/genética , Células HEK293 , Herpes Simples/genética , Humanos , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas Virais/genéticaRESUMO
The promyelocytic leukemia protein, PML, plays a vital role in the cellular response to oxidative stress; however, the molecular mechanism of its action remains poorly understood. Here, we identify redox-sensitive sites of PML. A molecule of PML is cysteine-rich and contains three zinc-binding domains including RING, B-box1, and B-box2. Using in vitro assays, we have compared the sensitivity of the isolated RING and B-box1 domains and shown that B-box1 is more sensitive to oxidation. NMR studies of PML dynamics showed that one of the Zn-coordination sites within the B-box1 undergoes significant conformational exchange, revealing a hotspot for exposure of reactive cysteines. In agreement with the in vitro data, enhancement of the B-box1 Zn-coordination dynamics led to more efficient recruitment of PML into PML nuclear bodies in cells. Overall, our results suggest that the increased sensitivity of B-box1 to oxidative stress makes this domain an important redox-sensing component of PML.
Assuntos
Proteínas Nucleares , Zinco , Proteínas Nucleares/metabolismo , Zinco/metabolismo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Sítios de Ligação , OxirreduçãoRESUMO
The herpes simplex virus 1 (HSV-1) UL6 portal protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for the encapsidation of the viral genome. We have demonstrated previously that the leucine zipper region of UL6 is important for intersubunit interactions and stable ring formation (J. K. Nellissery, R. Szczepaniak, C. Lamberti, and S. K. Weller, J. Virol. 81:8868-8877, 2007). We now demonstrate that intersubunit disulfide bonds exist between monomeric subunits and contribute to portal ring formation and/or stability. Intersubunit disulfide bonds were detected in purified portal rings by SDS-PAGE under nonreducing conditions. Furthermore, the treatment of purified portal rings with dithiothreitol (DTT) resulted in the disruption of the rings, suggesting that disulfide bonds confer stability to this complex structure. The UL6 protein contains nine cysteines that were individually mutated to alanine. Two of these mutants, C166A and C254A, failed to complement a UL6 null mutant in a transient complementation assay. Furthermore, viral mutants bearing the C166A and C254A mutations failed to produce infectious progeny and were unable to cleave or package viral DNA. In cells infected with C166A or C254A, B capsids were produced which contained UL6 at reduced levels compared to those seen in wild-type capsids. In addition, C166A and C254A mutant proteins expressed in insect cells infected with recombinant baculovirus failed to form ring structures. Cysteines at positions 166 and 254 thus appear to be required for intersubunit disulfide bond formation. Taken together, these results indicate that disulfide bond formation is required for portal ring formation and/or stability and for the production of procapsids that are capable of encapsidation.
Assuntos
Dissulfetos/metabolismo , Herpesvirus Humano 1/fisiologia , Multimerização Proteica , Proteínas Virais/metabolismo , Montagem de Vírus , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Cisteína/genética , Eletroforese em Gel de Poliacrilamida , Deleção de Genes , Teste de Complementação Genética , Mutagênese Sítio-Dirigida , Estabilidade Proteica , Subunidades Proteicas/metabolismo , Proteínas Virais/químicaRESUMO
Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of its human host cell and is known to interact with many cellular DNA repair proteins. In this study, we examined the role of cellular mismatch repair (MMR) proteins in the virus life cycle. Both MSH2 and MLH1 are required for efficient replication of HSV-1 in normal human cells and are localized to viral replication compartments. In addition, a previously reported interaction between MSH6 and ICP8 was confirmed by coimmunoprecipitation and extended to show that UL12 is also present in this complex. We also report for the first time that MLH1 associates with ND10 nuclear bodies and that like other ND10 proteins, MLH1 is recruited to the incoming genome. Knockdown of MLH1 inhibits immediate-early viral gene expression. MSH2, on the other hand, which is generally thought to play a role in mismatch repair at a step prior to that of MLH1, is not recruited to incoming genomes and appears to act at a later step in the viral life cycle. Silencing of MSH2 appears to inhibit early gene expression. Thus, both MLH1 and MSH2 are required but appear to participate in distinct events in the virus life cycle. The observation that MLH1 plays an earlier role in HSV-1 infection than does MSH2 is surprising and may indicate a novel function for MLH1 distinct from its known MSH2-dependent role in mismatch repair.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Herpes Simples/virologia , Herpesvirus Humano 1/patogenicidade , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Nucleares/metabolismo , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Western Blotting , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Células Cultivadas , Chlorocebus aethiops , Imunofluorescência , Genes Precoces , Células HeLa , Herpes Simples/genética , Herpesvirus Humano 1/crescimento & desenvolvimento , Humanos , Imunoprecipitação , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genética , Células VeroRESUMO
The heterotrimeric helicase-primase complex of herpes simplex virus type I (HSV-1), consisting of UL5, UL8, and UL52, possesses 5' to 3' helicase, single-stranded DNA (ssDNA)-dependent ATPase, primase, and DNA binding activities. In this study we confirm that the UL5-UL8-UL52 complex has higher affinity for forked DNA than for ssDNA and fails to bind to fully annealed double-stranded DNA substrates. In addition, we show that a single-stranded overhang of greater than 6 nucleotides is required for efficient enzyme loading and unwinding. Electrophoretic mobility shift assays and surface plasmon resonance analysis provide additional quantitative information about how the UL5-UL8-UL52 complex associates with the replication fork. Although it has previously been reported that in the absence of DNA and nucleoside triphosphates the UL5-UL8-UL52 complex exists as a monomer in solution, we now present evidence that in the presence of forked DNA and AMP-PNP, higher-order complexes can form. Electrophoretic mobility shift assays reveal two discrete complexes with different mobilities only when helicase-primase is bound to DNA containing a single-stranded region, and surface plasmon resonance analysis confirms larger amounts of the complex bound to forked substrates than to single-overhang substrates. Furthermore, we show that primase activity exhibits a cooperative dependence on protein concentration while ATPase and helicase activities do not. Taken together, these data suggest that the primase activity of the helicase-primase requires formation of a dimer or higher-order structure while ATPase activity does not. Importantly, this provides a simple mechanism for generating a two-polymerase replisome at the replication fork.