RESUMEN
Herpes simplex virus type 1 (HSV-1) is a neurotropic alphaherpesvirus that establishes a lifelong infection in sensory neurons of infected individuals, accompanied with intermittent reactivation of latent virus causing (a)symptomatic virus shedding. Whereas acyclovir (ACV) is a safe and highly effective antiviral to treat HSV-1 infections, long-term usage can lead to emergence of ACV resistant (ACVR) HSV-1 and subsequently ACV refractory disease. Here, we isolated an HSV-1 strain from a patient with reactivated herpetic eye disease that did not respond to ACV treatment. The isolate carried a novel non-synonymous F289S mutation in the viral UL23 gene encoding the thymidine kinase (TK) protein. Because ACV needs conversion by viral TK and subsequently cellular kinases to inhibit HSV-1 replication, the UL23 gene is commonly mutated in ACVR HSV-1 strains. The potential role of the F289S mutation causing ACVR was investigated using CRISPR/Cas9-mediated HSV-1 genome editing. Reverting the F289S mutation in the original clinical isolate to the wild-type sequence S289F resulted in an ACV-sensitive (ACVS) phenotype, and introduction of the F289S substitution in an ACVS HSV-1 reference strain led to an ACVR phenotype. In summary, we identified a new HSV-1 TK mutation in the eye of a patient with ACV refractory herpetic eye disease, which was identified as the causative ACVR mutation with the aid of CRISPR/Cas9-mediated genome engineering technology. Direct editing of clinical HSV-1 isolates by CRISPR/Cas9 is a powerful strategy to assess whether single residue substitutions are causative to a clinical ACVR phenotype.
Asunto(s)
Aciclovir , Antivirales , Sistemas CRISPR-Cas , Farmacorresistencia Viral , Edición Génica , Herpesvirus Humano 1 , Mutación , Timidina Quinasa , Timidina Quinasa/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/enzimología , Humanos , Farmacorresistencia Viral/genética , Aciclovir/farmacología , Aciclovir/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , Herpes Simple/virología , Herpes Simple/tratamiento farmacológicoRESUMEN
Herpes simplex virus 1 (HSV-1), a double-stranded DNA virus, replicates using seven essential proteins encoded by its genome. Among these, the UL30 DNA polymerase, complexed with the UL42 processivity factor, orchestrates leading and lagging strand replication of the 152 kb viral genome. UL30 polymerase is a prime target for antiviral therapy, and resistance to current drugs can arise in immunocompromised individuals. Using electron cryo-microscopy (cryo-EM), we unveil the dynamic changes of the UL30/UL42 complex with DNA in three distinct states. First, a pre-translocation state with an open fingers domain ready for nucleotide incorporation. Second, a halted elongation state where the fingers close, trapping dATP in the dNTP pocket. Third, a DNA-editing state involving significant conformational changes to allow DNA realignment for exonuclease activity. Additionally, the flexible UL30 C-terminal domain interacts with UL42, forming an extended positively charged surface binding to DNA, thereby enhancing processive synthesis. These findings highlight substantial structural shifts in the polymerase and its DNA interactions during replication, offering insights for future antiviral drug development.
Asunto(s)
Microscopía por Crioelectrón , ADN Viral , ADN Polimerasa Dirigida por ADN , Herpesvirus Humano 1 , Proteínas Virales , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , Proteínas Virales/metabolismo , Proteínas Virales/química , Proteínas Virales/ultraestructura , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/genética , ADN Viral/metabolismo , ADN Viral/biosíntesis , Replicación del ADN , Holoenzimas/química , Holoenzimas/metabolismo , Modelos Moleculares , Replicación Viral , Unión Proteica , ExodesoxirribonucleasasRESUMEN
Herpes simplex virus 1 (HSV-1) encodes a family B DNA polymerase (Pol) capable of exonucleolytic proofreading whose functions have been extensively studied in the past. Early studies on the in vitro activity of purified Pol protein found that the enzymatic functions of the holoenzyme are largely separate. Consequently, exonuclease activity can be reduced or abolished by certain point mutations within catalytically important regions, with no or only minor effects on polymerase activity. Despite unimpaired polymerase activity, the recovery of HSV-1 mutants with a catalytically inactive exonuclease has been so far unsuccessful. Hence, mutations such as D368A, which abolish exonuclease activity, are believed to be lethal. Here, we show that HSV-1 can be recovered in the absence of Pol intrinsic exonuclease activity and demonstrate that a lack of proofreading causes the rapid accumulation of likely detrimental mutations. Although mutations that abolish exonuclease activity do not appear to be lethal, the lack of proofreading yields viruses with a suicidal phenotype that cease to replicate within few passages following reconstitution. Hence, we conclude that high replication fidelity conferred by proofreading is essential to maintain HSV-1 genome integrity and that a lack of exonuclease activity produces an initially viable but rapidly suicidal phenotype. However, stably replicating viruses with reduced exonuclease activity and therefore elevated mutation rates can be generated by mutating a catalytically less important site located within a conserved exonuclease domain. IMPORTANCE Recovery of fully exonuclease-deficient herpes simplex virus 1 (HSV-1) DNA polymerase mutants has been so far unsuccessful. However, exonuclease activity is not known to be directly essential for virus replication, and the lethal phenotype of certain HSV-1 polymerase mutants is thus attributed to factors other than exonuclease activity. Here, we showed that the recovery of a variety of exonuclease-deficient HSV-1 polymerase mutants is possible and that these mutants are initially replication competent. We, however, observed a progressive loss of mutant viability upon cell culture passaging, which coincided with the rapid accumulation of mutations in exonuclease-deficient viruses. We thus concluded that a lack of DNA proofreading in exonuclease-deficient viruses causes an initially viable but rapidly suicidal hypermutator phenotype and, consequently, the extinction of mutant viruses within few generations following recovery. This would make the absence of exonuclease activity the primary reason for the long-reported difficulties in culturing exonuclease-deficient HSV-1 mutants.
Asunto(s)
Herpesvirus Humano 1 , Replicación del ADN/genética , Exonucleasas/genética , Exonucleasas/metabolismo , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Mutación , FenotipoRESUMEN
Herpes simplex virus type 1 (HSV-1) is a prevalent human pathogen primarily transmitted through skin-to-skin contact, especially on and around mucosal surfaces where there is contact with contaminated saliva during periods of viral shedding. It is estimated that 90% of adults worldwide have HSV-1 antibodies. Cutaneous HSV-1 infections are characterized by a sensation of tingling or numbness at the initial infection site followed by an eruption of vesicles and then painful ulcers with crusting. These symptoms can take ten days to several weeks to heal, leading to significant morbidity. Histologically, infections cause ballooning degeneration of keratinocytes and formation of multinucleated giant cells, ultimately resulting in a localized immune response. Commonly prescribed treatments against HSV-1 infections are nucleoside analogs, such as acyclovir (ACV). However, the emergence of ACV-resistant HSV (ACVR-HSV) clinical isolates has created an urgent need for the development of compounds to control symptoms of cutaneous infections. RLS-0071, also known as peptide inhibitor of complement C1 (PIC1), is a 15-amino-acid anti-inflammatory peptide that inhibits classical complement pathway activation and modulates neutrophil activation. It has been previously shown to aid in the healing of chronic diabetic wounds by inhibiting the excessive activation of complement component C1 and infiltration of leukocytes. Here, we report that treatment of cutaneous infections of HSV-1 and ACVR-HSV-1 in BALB/cJ mice with RLS-0071 significantly reduced the rate of mortality, decreased zosteriform spread, and enhanced the healing of the infection-associated lesions compared to control-treated animals. Therefore, RLS-0071 may work synergistically with other antiviral drugs to aid in wound healing of HSV-1 cutaneous infection and may potentially aid in rapid wound healing of other pathology not limited to HSV-1.
Asunto(s)
Inactivadores del Complemento/uso terapéutico , Herpes Simple/tratamiento farmacológico , Herpesvirus Humano 1/efectos de los fármacos , Péptidos/uso terapéutico , Aciclovir/farmacología , Animales , Antivirales/farmacología , Inactivadores del Complemento/farmacología , Farmacorresistencia Viral , Herpes Simple/patología , Herpes Simple/virología , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/genética , Ratones , Ratones Endogámicos BALB C , Péptidos/farmacología , Timidina Quinasa/genéticaRESUMEN
The herpes simplex virus (HSV)-1 protein pUL21 is essential for efficient virus replication and dissemination. While pUL21 has been shown to promote multiple steps of virus assembly and spread, the molecular basis of its function remained unclear. Here we identify that pUL21 is a virus-encoded adaptor of protein phosphatase 1 (PP1). pUL21 directs the dephosphorylation of cellular and virus proteins, including components of the viral nuclear egress complex, and we define a conserved non-canonical linear motif in pUL21 that is essential for PP1 recruitment. In vitro evolution experiments reveal that pUL21 antagonises the activity of the virus-encoded kinase pUS3, with growth and spread of pUL21 PP1-binding mutant viruses being restored in adapted strains where pUS3 activity is disrupted. This study shows that virus-directed phosphatase activity is essential for efficient herpesvirus assembly and spread, highlighting the fine balance between kinase and phosphatase activity required for optimal virus replication.
Asunto(s)
Herpes Simple/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/fisiología , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Virales/metabolismo , Ensamble de Virus , Replicación Viral , Animales , Chlorocebus aethiops , Células HEK293 , Herpesvirus Humano 1/enzimología , Humanos , Monoéster Fosfórico Hidrolasas/genética , Células Vero , Proteínas Virales/genética , Liberación del VirusRESUMEN
Herpes simplex virus type 1 (HSV-1) is one of the nine herpesviruses that infect humans. HSV-1 encodes seven proteins to replicate its genome in the hijacked human cell. Among these are the herpes virus DNA helicase and primase that are essential components of its replication machinery. In the HSV-1 replisome, the helicase-primase complex is composed of three components including UL5 (helicase), UL52 (primase) and UL8 (non-catalytic subunit). UL5 and UL52 subunits are functionally interdependent, and the UL8 component is required for the coordination of UL5 and UL52 activities proceeding in opposite directions with respect to the viral replication fork. Anti-viral compounds currently under development target the functions of UL5 and UL52. Here, we review the structural and functional properties of the UL5/UL8/UL52 complex and highlight the gaps in knowledge to be filled to facilitate molecular characterization of the structure and function of the helicase-primase complex for development of alternative anti-viral treatments.
Asunto(s)
ADN Helicasas/química , ADN Helicasas/metabolismo , ADN Primasa/química , ADN Primasa/metabolismo , Herpesvirus Humano 1/enzimología , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Animales , Antivirales/farmacología , ADN Helicasas/genética , ADN Primasa/genética , Desarrollo de Medicamentos , Herpes Simple/tratamiento farmacológico , Herpes Simple/virología , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/genética , Humanos , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
Retinoic acid-inducible gene I (RIG-I) is a sensor that recognizes cytosolic double-stranded RNA derived from microbes to induce host immune response. Viruses, such as herpesviruses, deploy diverse mechanisms to derail RIG-I-dependent innate immune defense. In this study, we discovered that mouse RIG-I is intrinsically resistant to deamidation and evasion by herpes simplex virus 1 (HSV-1). Comparative studies involving human and mouse RIG-I indicate that N495 of human RIG-I dictates species-specific deamidation by HSV-1 UL37. Remarkably, deamidation of the other site, N549, hinges on that of N495, and it is catalyzed by cellular phosphoribosylpyrophosphate amidotransferase (PPAT). Specifically, deamidation of N495 enables RIG-I to interact with PPAT, leading to subsequent deamidation of N549. Collaboration between UL37 and PPAT is required for HSV-1 to evade RIG-I-mediated antiviral immune response. This work identifies an immune regulatory role of PPAT in innate host defense and establishes a sequential deamidation event catalyzed by distinct deamidases in immune evasion.IMPORTANCE Herpesviruses are ubiquitous pathogens in human and establish lifelong persistence despite host immunity. The ability to evade host immune response is pivotal for viral persistence and pathogenesis. In this study, we investigated the evasion, mediated by deamidation, of species-specific RIG-I by herpes simplex virus 1 (HSV-1). Our findings uncovered a collaborative and sequential action between viral deamidase UL37 and a cellular glutamine amidotransferase, phosphoribosylpyrophosphate amidotransferase (PPAT), to inactivate RIG-I and mute antiviral gene expression. PPAT catalyzes the rate-limiting step of the de novo purine synthesis pathway. This work describes a new function of cellular metabolic enzymes in host defense and viral immune evasion.
Asunto(s)
Amidofosforribosiltransferasa/metabolismo , Proteína 58 DEAD Box/metabolismo , Herpes Simple/enzimología , Herpesvirus Humano 1/enzimología , Proteínas Estructurales Virales/metabolismo , Replicación Viral , Amidofosforribosiltransferasa/genética , Secuencias de Aminoácidos , Animales , Proteína 58 DEAD Box/química , Proteína 58 DEAD Box/genética , Herpes Simple/genética , Herpes Simple/virología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno , Humanos , Ratones , Unión Proteica , Especificidad de la Especie , Proteínas Estructurales Virales/genéticaRESUMEN
This study aimed at understanding the impact of different substitutions at codon 715 localized in the region II of the palm domain of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) DNA polymerases (pol). Here, we report a new theoretical mutation V715S that confers resistance of HSV-1 to foscarnet/acyclovir (5.6- and 9.2-fold increases EC50 values compared to wild type, respectively) and of HCMV to foscarnet/ganciclovir (2.8- and 2.9-fold increases in EC50 values compared to wild type, respectively). To further analyze the importance of this amino acid, we investigated the impact of the already known mutations V715M and V715G on the replicative capacities and drug susceptibilities of both viruses as well as on the activity and drug inhibition of the DNA pol. The V715G recombinant HSV-1 mutant was resistant to foscarnet and acyclovir (3.4- and 4.6-fold EC50 increase, respectively) whereas the V715M mutant was susceptible to foscarnet and resistant to acyclovir (3.4-fold EC50 increase). The V715G recombinant HCMV mutant did not grow and the V715M mutant was resistant to foscarnet (3.7-fold EC50 increase) and susceptible to ganciclovir. Finally, we showed by three-dimensional modeling that the differential impact of these mutations on the viral replicative capacity and drug resistance profile was related to different hydrophobic local environments for V715 in the DNA pol of the two viruses. Furthermore, we hypothesize that the DNA pol of HSV-1 is more tolerant to changes at this residue compared to that of HCMV because of a more hydrophobic environment stabilizing the region.
Asunto(s)
Antivirales/farmacología , Codón , Citomegalovirus/genética , ADN Polimerasa Dirigida por ADN/genética , Exodesoxirribonucleasas/genética , Herpesvirus Humano 1/genética , Mutación Missense , Proteínas Virales/genética , Aciclovir/farmacología , Animales , Chlorocebus aethiops , Citomegalovirus/efectos de los fármacos , Citomegalovirus/enzimología , Citomegalovirus/fisiología , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Foscarnet/farmacología , Ganciclovir/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/fisiología , Interacciones Hidrofóbicas e Hidrofílicas , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Células Vero , Proteínas Virales/química , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
BACKGROUND AND AIM: The chronicity of hepatitis B virus (HBV) infection is the result of impaired HBV-specific immune responses that cannot eliminate or cure the infected hepatocytes efficiently. Previous studies have used immunodeficient mice such as herpes simplex virus type 1 thymidine kinase NOD/Scid/IL2Rrnull (HSV-TK-NOG) mice. However, it is difficult to analyze the immune response in the previous models. In the present study, we established a novel HBV infection model using herpes simplex virus type 1 thymidine kinase (HSV-TK) mice in which the host immune system was not impaired. METHODS: Herpes simplex virus type 1 thymidine kinase mice were injected intraperitoneally with ganciclovir (GCV). Seven days after GCV injection, GCV-treated mice were transplanted with 1 × 106 hepatocytes from HBV-transgenic (HBV-Tg) mice. RESULTS: Serum alanine aminotransferase levels in HSV-TK mice increased 1 and 2 weeks after GCV injection. The number and viability of hepatocytes from the whole liver of HBV-Tg mice significantly increased using digestion medium containing liberase. Hepatitis B surface antigen (HBsAg)-positive areas in the liver tissue were observed for at least 20 weeks after HBsAg-positive hepatocyte transplantation. In addition, we measured HBsAg in the serum after transplantation. HBsAg levels in HBV-Tg hepatocyte-replaced mice increased 4 weeks after transplantation. Furthermore, we examined the immune response in HSV-TK mice. The increase in hepatitis B surface antibody levels in replaced mice was maintained for 20 weeks. Also, interferon-γ-producing cells were increased in non-replaced mice. CONCLUSIONS: A novel HBV infection mouse model will help to understand the mechanisms of HBV tolerance similar to human chronic HBV-infected patients and can be used to develop a new strategy to treat chronic HBV infection.
Asunto(s)
Modelos Animales de Enfermedad , Hepatitis B Crónica , Herpesvirus Humano 1/enzimología , Ratones Transgénicos , Timidina Quinasa/genética , Animales , Ganciclovir/administración & dosificación , Antígenos de Superficie de la Hepatitis B , Hepatitis B Crónica/inmunología , Hepatocitos/trasplante , Inyecciones Intraperitoneales , Interferón gamma/metabolismo , Hígado/inmunologíaRESUMEN
PURPOSE: [18F]FHBG has been used as a positron emission tomography (PET) imaging tracer for the monitoring of herpes simplex virus type 1 thymidine kinase (HSV1-tk), a reporter gene for cell and gene therapy in humans. However, this tracer shows inadequate blood-brain barrier (BBB) penetration and, therefore, would be limited for accurate quantification of reporter gene expression in the brain. Here, we report the synthesis and evaluation of 9-(4-[18F]fluoro-3-(hydroxymethyl)butyl)-2(phenylthio)-6-oxopurine ([18F]FHBT) as a new PET tracer for imaging reporter gene expression of HSV1-tk and its mutant HSV1-sr39tk, with the aim of improved BBB penetration. PROCEDURES: [18F]FHBT was prepared by using a tosylate precursor and [18F]KF. The cellular uptake of [18F]FHBT was performed in HSV1-sr39tk-positive (+) or HSV1-sr39tk-negative (-) MDA-MB-231 breast cancer cells. The specificity of [18F]FHBT to assess HSV1-sr39tk expression was evaluated by in vitro blocking studies using 1 mM of ganciclovir (GCV). Penetration of [18F]FHBT and [18F]FHBG across the BBB was assessed by dynamic PET imaging studies in normal mice. RESULTS: The tosylate precursor reacted with [18F]KF using Kryptofix2.2.2 followed by deprotection to give [18F]FHBT in 10 % radiochemical yield (decay-corrected). The uptake of [18F]FHBT in HSV1-sr39tk (+) cells was significantly higher than that of HSV1-sr39tk (-) cells. In the presence of GCV (1 mM), the uptake of [18F]FHBT was significantly decreased, indicating that [18F]FHBT serves as a selective substrate of HSV1-sr39TK. PET images and time-activity curves of [18F]FHBT in the brain regions showed similar initial brain uptakes (~ 12.75 min) as [18F]FHBG (P > 0.855). Slower washout of [18F]FHBT was observed at the later time points (17.75 - 57.75 min, P > 0.207). CONCLUSIONS: Although [18F]FHBT showed no statistically significant improvement of BBB permeability compared with [18F]FHBG, we have demonstrated that the 2-(phenylthio)-6-oxopurine backbone can serve as a novel scaffold for developing HSV1-tk/HSV1-sr39tk reporter gene imaging agents for additional research in the future.
Asunto(s)
Genes Reporteros , Herpesvirus Humano 1/enzimología , Mutación/genética , Tomografía de Emisión de Positrones , Purinas/síntesis química , Timidina Quinasa/genética , Animales , Línea Celular Tumoral , Endocitosis , Femenino , Ganciclovir , Humanos , Ratones Endogámicos BALB C , Purinas/químicaRESUMEN
Together, medulloblastoma (MB) and atypical teratoid/rhabdoid tumors (AT/RT) represent two of the most prevalent pediatric brain malignancies. Current treatment involves radiation, which has high risks of developmental sequelae for patients under the age of three. New safer and more effective treatment modalities are needed. Cancer gene therapy is a promising alternative, but there are challenges with using viruses in pediatric patients. We developed a library of poly(beta-amino ester) (PBAE) nanoparticles and evaluated their efficacy for plasmid delivery of a suicide gene therapy to pediatric brain cancer models-specifically herpes simplex virus type I thymidine kinase (HSVtk), which results in controlled apoptosis of transfected cells. In vivo, PBAE-HSVtk treated groups had a greater median overall survival in mice implanted with AT/RT (Pâ¯=â¯0.0083 vs. control) and MB (Pâ¯<â¯0.0001 vs. control). Our data provide proof of principle for using biodegradable PBAE nanoparticles as a safe and effective nanomedicine for treating pediatric CNS malignancies.
Asunto(s)
Neoplasias Encefálicas , Terapia Genética , Herpesvirus Humano 1 , Nanopartículas , Timidina Quinasa , Proteínas Virales , Animales , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/terapia , Línea Celular Tumoral , Niño , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/genética , Humanos , Masculino , Ratones , Ratones Desnudos , Nanopartículas/química , Nanopartículas/uso terapéutico , Timidina Quinasa/biosíntesis , Timidina Quinasa/genética , Proteínas Virales/biosíntesis , Proteínas Virales/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite its low transfer efficiency, suicide gene therapy with HSV-TK is known for its bystander killing effect. The connexin-based gap junction is believed to mediate the bystander effect. Recently, we found that resveratrol, a polyphenol compound, increased the expression of Cx26 and Cx43, which are connexins and important constituents of gap junctions, in murine hepatoma cells. Hypothetically, the resveratrol-induced upregulation of gap junctions may improve the bystander effect that HSV-TK/GCV has on hepatoma cells. Our present investigation revealed that resveratrol could enhance intercellular communication at the gap junctions in CBRH7919 hepatoma cells and thereby enhance the bystander killing effect of GCV on CBRH7919TK cells. However, inhibition of gap junction using its long-term inhibitor alpha-glycyrrhetinic acid had a negative influence on the bystander effect of gene therapy with HSV-TK/GCV. In addition, combined resveratrol and GCV treatment in tumor-bearing mice with CBRH7919TK and CBRH7919WT cells at a ratio of 2:3 resulted in a significant decrease in the volume and weight of the tumor in comparison to GCV or only resveratrol. The present results demonstrate that resveratrol can enhance the bystander effect exerted by the HSV-TK/GCV system by enhancing connexin-mediated gap junctional communication.
Asunto(s)
Antioxidantes/farmacología , Ganciclovir/farmacología , Terapia Genética/métodos , Neoplasias Hepáticas Experimentales/terapia , Resveratrol/farmacología , Proteínas del Envoltorio Viral/farmacología , Animales , Sinergismo Farmacológico , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/genética , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , TransfecciónRESUMEN
Herpesviruses, including herpes simplex virus-1, encode and express a DNA polymerase that is required for replication of their dsDNA genomes. The catalytic subunit of this enzyme contains a 3'-5' exonuclease that is involved in proofreading during replication. Although certain mutations that severely impair exonuclease activity are not lethal to the virus, it was reported that virus containing the substitution of alanine for aspartate 368 (D368A), which ablates exonuclease activity, could not be recovered, raising the possibility that this activity is essential for viral replication. To investigate this issue, we produced virus containing this mutation (D368A Pol) using a complementing cell line. D368A Pol virus was unable to form plaques on non-complementing cells. Viral DNA synthesis and polymerase activity were severely inhibited in D368A-infected cells, as was expression of the enzyme, suggesting that effects on polymerase expression rather than on exonuclease activity per se largely explain the lethal phenotype of this mutation.
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ADN Viral/biosíntesis , ADN Polimerasa Dirigida por ADN/metabolismo , Exodesoxirribonucleasas/deficiencia , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/crecimiento & desarrollo , Proteínas Mutantes/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Sustitución de Aminoácidos , ADN Polimerasa Dirigida por ADN/genética , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Expresión Génica , Proteínas Mutantes/genética , Mutación Missense , Ensayo de Placa Viral , Proteínas Virales/genéticaRESUMEN
Many cellular processes pertinent for viral infection are regulated by the addition of small ubiquitin-like modifiers (SUMO) to key regulatory proteins, making SUMOylation an important mechanism by which viruses can commandeer cellular pathways. Epstein-Barr virus (EBV) is a master at manipulating of cellular processes, which enables life-long infection but can also lead to the induction of a variety of EBV-associated cancers. To identify new mechanisms by which EBV proteins alter cells, we screened a library of 51 EBV proteins for global effects on cellular SUMO1 and SUMO2 modifications (SUMOylation), identifying several proteins not previously known to manipulate this pathway. One EBV protein (BRLF1) globally induced the loss of SUMOylated proteins, in a proteasome-dependent manner, as well as the loss of promeylocytic leukemia nuclear bodies. However, unlike its homologue (Rta) in Kaposi's sarcoma associated herpesvirus, it did not appear to have ubiquitin ligase activity. In addition we identified the EBV SM protein as globally upregulating SUMOylation and showed that this activity was conserved in its homologues in herpes simplex virus 1 (HSV1 UL54/ICP27) and cytomegalovirus (CMV UL69). All three viral homologues were shown to bind SUMO and Ubc9 and to have E3 SUMO ligase activity in a purified system. These are the first SUMO E3 ligases discovered for EBV, HSV1 and CMV. Interestingly the homologues had different specificities for SUMO1 and SUMO2, with SM and UL69 preferentially binding SUMO1 and inducing SUMO1 modifications, and UL54 preferentially binding SUMO2 and inducing SUMO2 modifications. The results provide new insights into the function of this family of conserved herpesvirus proteins, and the conservation of this SUMO E3 ligase activity across diverse herpesviruses suggests the importance of this activity for herpesvirus infections.
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Citomegalovirus/enzimología , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 4/enzimología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Virales/metabolismo , Línea Celular , Estudio de Asociación del Genoma Completo , Humanos , SumoilaciónRESUMEN
The herpes simplex virus 1 (HSV-1) virion host shutoff (vhs) protein is an endoribonuclease that binds to the cellular translation initiation machinery and degrades associated mRNAs, resulting in the shutoff of host protein synthesis. Hence, its unrestrained activity is considered lethal, and it has been proposed that vhs is regulated by two other virus proteins, VP22 and VP16. We have found that during infection, translation of vhs requires VP22 but not the VP22-VP16 complex. Moreover, in the absence of VP22, vhs is not overactive against cellular or viral transcripts. In transfected cells, vhs was also poorly translated, correlating with the aberrant localization of its mRNA. Counterintuitively, vhs mRNA was predominantly nuclear in cells where vhs protein was detected. Likewise, transcripts from cotransfected plasmids were also retained in the same nuclei where vhs mRNA was located, while poly(A) binding protein (PABP) was relocalized to the nucleus in a vhs-dependent manner, implying a general block to mRNA export. Coexpression of VP16 and VP22 rescued the cytoplasmic localization of vhs mRNA but failed to rescue vhs translation. We identified a 230-nucleotide sequence in the 5' region of vhs that blocked its translation and, when transferred to a heterologous green fluorescent protein transcript, reduced translation without altering mRNA levels or localization. We propose that expression of vhs is tightly regulated by a combination of inherent untranslatability and autoinduced nuclear retention of its mRNA that results in a negative feedback loop, with nuclear retention but not translation of vhs mRNA being the target of rescue by the vhs-VP16-VP22 complex.IMPORTANCE A myriad of gene expression strategies has been discovered through studies carried out on viruses. This report concerns the regulation of the HSV-1 vhs endoribonuclease, a virus factor that is important for counteracting host antiviral responses by degrading their mRNAs but that must be regulated during infection to ensure that it does not act against and inhibit the virus itself. We show that regulation of vhs involves multifaceted posttranscriptional cellular and viral processes, including aberrant mRNA localization and a novel, autoregulated negative feedback loop to target its own and coexpressed mRNAs for nuclear retention, an activity that is relieved by coexpression of two other virus proteins, VP22 and VP16. These studies reveal the interplay of strategies by which multiple virus-encoded factors coordinate gene expression at the time that they are needed. These findings are broadly relevant to both virus and cellular gene expression.
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Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Herpesvirus Humano 1/enzimología , Ribonucleasas/genética , Proteínas Virales/genética , Proteínas Estructurales Virales/metabolismo , Regiones no Traducidas 5' , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Viral de la Expresión Génica , Herpesvirus Humano 1/genética , Sistemas de Lectura Abierta , Procesamiento Postranscripcional del ARN , Ribonucleasas/química , Ribonucleasas/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
All members of the Alphaherpesvirinae subfamily encode a serine/threonine kinase, designated Us3, which is not conserved in the other subfamilies. Us3 is a significant virulence factor for herpes simplex virus type 1 (HSV-1), which is one of the best-characterized members of the Alphaherpesvirinae family. Accumulating evidence indicates that HSV-1 Us3 is a multifunctional protein that plays various roles in the viral life cycle by phosphorylating a number of viral and cellular substrates. Therefore, the identification of Us3 substrates is directly connected to understanding Us3 functions and mechanisms. To date, more than 23 phosphorylation events upregulated by HSV-1 Us3 have been reported. However, few of these have been shown to be both physiological substrates of Us3 in infected cells and directly linked with Us3 functions in infected cells. In this chapter, we summarize the 12 physiological substrates of Us3 and the Us3-mediated functions. Furthermore, based on the identified phosphorylation sites of Us3 or Us3 homolog physiological substrates, we reverified consensus phosphorylation target sequences on the physiological substrates of Us3 and Us3 homologs in vitro and in infected cells. This information might aid the further identification of novel Us3 substrates and as yet unidentified Us3 functions.
Asunto(s)
Herpes Simple/virología , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Virales/metabolismo , Animales , Herpes Simple/genética , Herpes Simple/metabolismo , Herpesvirus Humano 1/genética , Interacciones Huésped-Patógeno , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Virales/química , Proteínas Virales/genética , Replicación ViralRESUMEN
In response to virus-induced shutoff host protein synthesis, dynamic aggregates containing mRNA, RNA-binding proteins and translation factors termed stress granules (SGs) often accumulate within the cytoplasm. SGs typically form following phosphorylation and inactivation of the eukaryotic translation initiation factor 2α (eIF2α), a substrate of the double-stranded RNA (dsRNA)-activated kinase protein kinase R (PKR). The detection of innate immune sensors and effectors like PKR at SGs suggests a role in pathogen nucleic acid sensing. However, the functional importance of SGs in host innate responses is unclear and has primarily been examined in response to infection with select RNA viruses. During infection with the DNA virus herpes simplex virus 1 (HSV-1), the virus-encoded virion host shutoff (VHS) endoribonuclease is required to restrict interferon production, PKR activation, and SG formation, although the relationship between these activities remains incompletely understood. Here, we show that in cells infected with a VHS-deficient HSV-1 (ΔVHS) dsRNA accumulated and localized to SGs. Surprisingly, formation of dsRNA and its concentration at SGs was not required for beta interferon mRNA induction, indicating that suppression of type I interferon induction by VHS does not stem from its control of dsRNA accumulation. Instead, STING signaling downstream of cGMP-AMP synthase (cGAS)-dependent DNA sensing is required for beta interferon induction. In contrast, significantly less PKR activation is observed when SG assembly is disrupted by ISRIB, an inhibitor of phosphorylated eIF2α-mediated translation repression, or depleting SG scaffolding proteins G3BP1 or TIA1. This demonstrates that PKR activation is intimately linked to SG formation and that SGs form important hubs to potentiate PKR activation during infection.IMPORTANCE Formation of cytoplasmic stress granules that are enriched for innate immune sensors and effectors is suppressed during many viral infections. It is unclear, however, to what extent this is a side effect of viral efforts to maintain protein synthesis or intentional disruption of a hub for innate immune sensing. In this study, we utilize a herpes simplex virus 1 mutant lacking the RNA nuclease VHS which upon infection induces SGs, PKR activation, and beta interferon to address this question. We show that dsRNA is localized to SGs and that SGs can function to promote PKR activation in the context of a DNA virus infection, but we find no evidence to support their importance for interferon induction during HSV-1 infection.
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Gránulos Citoplasmáticos/inmunología , Fibroblastos/inmunología , Herpesvirus Humano 1/inmunología , Inmunidad Innata , Ribonucleasas/inmunología , Transducción de Señal/inmunología , Proteínas Virales/inmunología , Células Cultivadas , Gránulos Citoplasmáticos/enzimología , Gránulos Citoplasmáticos/genética , Fibroblastos/metabolismo , Fibroblastos/virología , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/genética , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Nucleotidiltransferasas/metabolismo , ARN Bicatenario/genética , ARN Bicatenario/inmunología , ARN Bicatenario/metabolismo , ARN Viral/genética , ARN Viral/inmunología , ARN Viral/metabolismo , Ribonucleasas/genética , Ribonucleasas/metabolismo , Transducción de Señal/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Ion Torrent next-generation sequencing (NGS) technology was applied to study the mode of emergence of acyclovir (ACV)-resistant (ACVr) herpes simplex virus type 1 (HSV-1) in patients with hematopoietic stem cell transplantation (HSCT) by quantitatively detecting mutations in the viral thymidine kinase (vTK) gene in the HSV-1 isolates recovered from HSCT patients. All of the mutations detected with the Sanger sequencing method in the vTK genes of HSV-1 isolates were also detected with the NGS assay. Furthermore, different mutations, which conferred ACV resistance and were not detected with the Sanger sequencing method, were also detected in a quantitative manner by using the NGS assay. The approach described here is applicable to studying the emergence process of vTK gene mutation-associated ACVr HSV-1 more in detail than the Sanger method. The NGS assay makes it possible to make a diagnosis of vTK gene mutation-associated ACVr HSV-1 infections at the early stage, which the ratio of ACVr HSV-1 is much lower than that of ACV-sensitive (ACVs) HSV-1.
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Aciclovir/farmacología , Antivirales/farmacología , Farmacorresistencia Viral , Técnicas de Genotipaje/métodos , Herpesvirus Humano 1/efectos de los fármacos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Pruebas de Sensibilidad Microbiana/métodos , Trasplante de Células Madre Hematopoyéticas , Herpes Simple/virología , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/genética , Humanos , Mutación , Timidina Quinasa/genéticaRESUMEN
The catalytic subunit (Pol) of herpes simplex virus 1 (HSV-1) DNA polymerase has been extensively studied both as a model for other family B DNA polymerases and for its differences from these enzymes as an antiviral target. Among the activities of HSV-1 Pol is an intrinsic RNase H activity that cleaves RNA from RNA-DNA hybrids. There has long been a controversy regarding whether this activity is due to the 3'-to-5' exonuclease of Pol or whether it is a separate activity, possibly acting on 5' RNA termini. To investigate this issue, we compared wild-type HSV-1 Pol and a 3'-to-5' exonuclease-deficient mutant, D368A Pol, for DNA polymerase activity, 3'-to-5' exonuclease activity, and RNase H activity in vitro Additionally, we assessed the RNase H activity using differentially end-labeled templates with 5' or 3' RNA termini. The mutant enzyme was at most modestly impaired for DNA polymerase activity but was drastically impaired for 3'-to-5' exonuclease activity, with no activity detected even at high enzyme-to-DNA substrate ratios. Importantly, the mutant showed no detectable ability to excise RNA with either a 3' or 5' terminus, while the wild-type HSV-1 Pol was able to cleave RNA from the annealed RNA-DNA hairpin template, but only detectably with a 3' RNA terminus in a 3'-to-5' direction and at a rate lower than that of the exonuclease activity. These results suggest that HSV-1 Pol does not have an RNase H separable from its 3'-to-5' exonuclease activity and that this activity prefers DNA degradation over degradation of RNA from RNA-DNA hybrids.IMPORTANCE Herpes simplex virus 1 (HSV-1) is a member of the Herpesviridae family of DNA viruses, several of which cause morbidity and mortality in humans. Although the HSV-1 DNA polymerase has been studied for decades and is a crucial target for antivirals against HSV-1 infection, several of its functions remain to be elucidated. A hypothesis suggesting the existence of a 5'-to-3' RNase H activity intrinsic to this enzyme that could remove RNA primers from Okazaki fragments has been particularly controversial. In this study, we were unable to identify RNase H activity of HSV-1 DNA polymerase on RNA-DNA hybrids with 5' RNA termini. We detected RNase H activity on hybrids with 3' termini, but this was due to the 3'-to-5' exonuclease. Thus, HSV-1 is unlikely to use this method to remove RNA primers during DNA replication but may use pathways similar to those used in eukaryotic Okazaki fragment maturation.
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Dominio Catalítico , ADN Polimerasa Dirigida por ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Exonucleasas/metabolismo , Herpesvirus Humano 1/enzimología , Ribonucleasa H/metabolismo , Proteínas Virales/metabolismo , ADN , Replicación del ADN , Exonucleasas/genética , Herpesvirus Humano 1/genética , Mutación , ARN , Moldes GenéticosRESUMEN
Infection with herpes simplex virus-1 (HSV-1) brings numerous changes in cellular gene expression. Levels of most host mRNAs are reduced, limiting synthesis of host proteins, especially those involved in antiviral defenses. The impact of HSV-1 on host microRNAs (miRNAs), an extensive network of short non-coding RNAs that regulate mRNA stability/translation, remains largely unexplored. Here we show that transcription of the miR-183 cluster (miR-183, miR-96, and miR-182) is selectively induced by HSV-1 during productive infection of primary fibroblasts and neurons. ICP0, a viral E3 ubiquitin ligase expressed as an immediate-early protein, is both necessary and sufficient for this induction. Nuclear exclusion of ICP0 or removal of the RING (really interesting new gene) finger domain that is required for E3 ligase activity prevents induction. ICP0 promotes the degradation of numerous host proteins and for the most part, the downstream consequences are unknown. Induction of the miR-183 cluster can be mimicked by depletion of host transcriptional repressors zinc finger E-box binding homeobox 1 (ZEB1)/-crystallin enhancer binding factor 1 (δEF1) and zinc finger E-box binding homeobox 2 (ZEB2)/Smad-interacting protein 1 (SIP1), which we establish as new substrates for ICP0-mediated degradation. Thus, HSV-1 selectively stimulates expression of the miR-183 cluster by ICP0-mediated degradation of ZEB transcriptional repressors.