RESUMEN
Varicella-zoster virus (VZV) is a medically important alphaherpesvirus that induces fusion of the virion envelope and the cell membrane during entry, and between cells to form polykaryocytes within infected tissues during pathogenesis. All members of the Herpesviridae, including VZV, have a conserved core fusion complex composed of glycoproteins, gB, gH and gL. The ectodomain of the primary fusogen, gB, has five domains, DI-V, of which DI contains the fusion loops needed for fusion function. We recently demonstrated that DIV is critical for fusion initiation, which was revealed by a 2.8Å structure of a VZV neutralizing mAb, 93k, bound to gB and mutagenesis of the gB-93k interface. To further assess the mechanism of mAb 93k neutralization, the binding site of a non-neutralizing mAb to gB, SG2, was compared to mAb 93k using single particle cryogenic electron microscopy (cryo-EM). The gB-SG2 interface partially overlapped with that of gB-93k but, unlike mAb 93k, mAb SG2 did not interact with the gB N-terminus, suggesting a potential role for the gB N-terminus in membrane fusion. The gB ectodomain structure in the absence of antibody was defined at near atomic resolution by single particle cryo-EM (3.9Å) of native, full-length gB purified from infected cells and by X-ray crystallography (2.4Å) of the transiently expressed ectodomain. Both structures revealed that the VZV gB N-terminus (aa72-114) was flexible based on the absence of visible structures in the cryo-EM or X-ray crystallography data but the presence of gB N-terminal peptides were confirmed by mass spectrometry. Notably, N-terminal residues 109KSQD112 were predicted to form a small α-helix and alanine substitution of these residues abolished cell-cell fusion in a virus-free assay. Importantly, transferring the 109AAAA112 mutation into the VZV genome significantly impaired viral propagation. These data establish a functional role for the gB N-terminus in membrane fusion broadly relevant to the Herpesviridae.
Asunto(s)
Herpesvirus Humano 3/fisiología , Melanoma/metabolismo , Fusión de Membrana , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Melanoma/virología , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Dominios Proteicos , Homología de Secuencia , Células Tumorales Cultivadas , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
Herpes simplex virus 1 (HSV-1) infects skin and mucosal epithelial cells and then travels along axons to establish latency in the neurones of sensory ganglia. Although viral gene expression is restricted during latency, the latency-associated transcript (LAT) locus encodes many RNAs, including a 2 kb intron known as the hallmark of HSV-1 latency. Here, we studied HSV-1 infection and the role of the LAT locus in human skin xenografts in vivo and in cultured explants. We sequenced the genomes of our stock of HSV-1 strain 17syn+ and seven derived viruses and found nonsynonymous mutations in many viral proteins that had no impact on skin infection. In contrast, deletions in the LAT locus severely impaired HSV-1 replication and lesion formation in skin. However, skin replication was not affected by impaired intron splicing. Moreover, although the LAT locus has been implicated in regulating gene expression in neurones, we observed only small changes in transcript levels that were unrelated to the growth defect in skin, suggesting that its functions in skin may be different from those in neurones. Thus, although the LAT locus was previously thought to be dispensable for lytic infection, we show that it is a determinant of HSV-1 virulence during lytic infection of human skin.
Asunto(s)
Herpes Simple/virología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidad , MicroARNs/genética , Piel/virología , Virulencia/genética , Animales , Xenoinjertos , Humanos , Ratones , Factores de Virulencia/genéticaRESUMEN
Members of the Herpesviridae, including the medically important alphaherpesvirus varicella-zoster virus (VZV), induce fusion of the virion envelope with cell membranes during entry, and between cells to form polykaryocytes in infected tissues. The conserved glycoproteins, gB, gH and gL, are the core functional proteins of the herpesvirus fusion complex. gB serves as the primary fusogen via its fusion loops, but functions for the remaining gB domains remain unexplained. As a pathway for biological discovery of domain function, our approach used structure-based analysis of the viral fusogen together with a neutralizing antibody. We report here a 2.8 Å cryogenic-electron microscopy structure of native gB recovered from VZV-infected cells, in complex with a human monoclonal antibody, 93k. This high-resolution structure guided targeted mutagenesis at the gB-93k interface, providing compelling evidence that a domain spatially distant from the gB fusion loops is critical for herpesvirus fusion, revealing a potential new target for antiviral therapies.
Asunto(s)
Anticuerpos Neutralizantes/química , Herpesvirus Humano 3/química , Proteínas del Envoltorio Viral/química , Internalización del Virus , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/ultraestructura , Microscopía por Crioelectrón , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformación Proteica en Lámina beta/genética , Dominios Proteicos/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/ultraestructuraRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Background: Global polio eradication efforts rely in part on molecular methods of detecting polioviruses, both wild and vaccine strains, from human and environmental samples. Previous assays used for detection of Sabin oral polio vaccine (OPV) in fecal samples have been labor and time intensive and vary in their sensitivity and specificity. Methods: We developed a high-throughput, multiplex reverse-transcription quantitative polymerase chain reaction assay able to detect all 3 OPV strains in fecal samples. The assay used a KingFisher Duo Prime system for viral RNA isolation and extraction. Positive samples were retested and Sanger sequenced for verification of Sabin serotype identity. Results: The 95% lower limit of detection was determined to be 3 copies per reaction for Sabin 1 and 3 and 4 copies per reaction for Sabin 2, with no cross-reactivity between the 3 serotypes and their primers. A total of 554 samples (3.6%) were positive, with 304 positive samples (54.9%) containing >1 serotype. Of the positive samples, 476 (85.9%) contained enough RNA to be sequenced, and of these all sequences were Sabin serotypes. The previous assay we used could process 48 samples in a 10-hour period, whereas the new assay processed >100 samples in 6 hours. Conclusions: The new high-throughput, multiplex reverse-transcription quantitative polymerase chain reaction assay allowed for sensitive and specific detection of OPV serotypes while greatly decreasing sample handling and processing time. We were able to sequence 72.4% of the 210 positive samples in the cycle threshold range of 35-37.
Asunto(s)
Reacción en Cadena de la Polimerasa Multiplex/métodos , Poliomielitis/transmisión , Poliovirus/aislamiento & purificación , Preescolar , Reacciones Cruzadas , Heces/virología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Límite de Detección , México/epidemiología , Poliomielitis/epidemiología , Poliomielitis/prevención & control , Poliomielitis/virología , Poliovirus/genética , Poliovirus/inmunología , Vacuna Antipolio Oral , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , SerogrupoRESUMEN
Background: Currently, the primary mechanism for poliovirus detection is acute flaccid paralysis (AFP) surveillance, with environmental sampling serving as a complement. However, as AFP cases drop, environmental surveillance will become increasingly critical for poliovirus detection. Mexico provides a natural environment to study oral polio vaccine (OPV) transmission, as it provides routine injected polio vaccine immunization and biannual OPV campaigns in February and May. Methods: As part of a study of OPV transmission in which 155 children were vaccinated with OPV, monthly sewage samples were collected from rivers leading from 3 indigenous Mexican villages (Capoluca, Campo Grande, and Tuxpanguillo) from February to May 2015. Samples were also collected from October 2015 to October 2017, during which time there were standard OPV campaigns. Samples were analyzed for the presence of OPV serotypes, using a real-time qualitative polymerase chain reaction assay capable of detecting as few as 9, 12, and 10 copies/100 µL of viral ribonucleic acid for OPV serotypes 1, 2, and 3 (OPV-1, -2, and -3), respectively. Included here are 54 samples, taken up to November 2016. Results: Of the 54 samples, 13 (24%) were positive for OPV. After the vaccination of 155 children in February 2015, OPV was found 2 months after vaccination. After unrestricted OPV administration in February 2016, OPV was detected in sewage up to 8 months after vaccination. OPV-3 was found in 11 of the 13 positive samples (85%), OPV-2 was found in 3 positive samples (23%), and OPV-1 was found in 1 sample (8%). Conclusions: OPV can be detected even when small amounts of the vaccine are introduced into a community, as shown by OPV-positive sewage samples even when only 155 children were vaccinated. When OPV vaccination was unrestricted, sewage samples were positive up to 8 months after vaccination, implying community OPV circulation for at least 8 months. OPV-3 was the serotype most found in these samples, indicating prolonged transmission of OPV-3 when compared to the other serotypes. Future work could compare the phylogenetic variance of OPV isolates from sewage after OPV vaccinations.
Asunto(s)
Monitoreo del Ambiente , Reacción en Cadena de la Polimerasa Multiplex/métodos , Poliomielitis/transmisión , Vacuna Antipolio Oral , Poliovirus/aislamiento & purificación , Vacunación , Humanos , México , Poliomielitis/inmunología , Poliomielitis/virología , Poliovirus/genética , Poliovirus/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Ríos/virología , Sensibilidad y Especificidad , Serogrupo , Aguas del Alcantarillado/virología , Esparcimiento de VirusRESUMEN
Background: The World Health Assembly 2012 Polio Eradication and Endgame Strategic Plan calls for the eventual cessation of all oral polio vaccines (OPVs), to be replaced with inactivated polio vaccine (IPV); however, IPV induces less robust mucosal immunity than OPV. This study characterized household and community OPV shedding and transmission after OPV vaccination within primarily IPV-vaccinated communities. Methods: Households in 3 IPV-vaccinated Mexican communities were randomized to receive 3 levels of OPV vaccination coverage (70%, 30%, or 10%). Ten stool samples were collected from all household members over 71 days. Analysis compared vaccinated subjects, household contacts of vaccinated subjects, and subjects in unvaccinated households. Logistic and Cox regression models were fitted to characterize transmission of OPV by coverage and household vaccination status. Results: Among 148 vaccinated children, 380 household contacts, and 1124 unvaccinated community contacts, 78%, 18%, and 7%, respectively, shed OPV. Community and household contacts showed no differences in transmission (odds ratio [OR], 0.67; 95% confidence interval [CI], .37-1.20), in shedding trajectory (OR, 0.61; 95% CI, .35-1.07), or in time to shedding (hazard ratio, 0.68; 95% CI, .39-1.19). Transmission began as quickly as 1 day after vaccination and persisted as long as 71 days after vaccination. Transmission within unvaccinated households differed significantly across vaccination coverage communities, with the 70% community experiencing the most transmissions (15%), and the 10% community experiencing the least (4%). These trends persisted over time and in the time to first shedding analyses. Conclusions: Transmission did not differ between household contacts of vaccinees and unvaccinated households. Understanding poliovirus transmission dynamics is important for postcertification control.
Asunto(s)
Poliomielitis/prevención & control , Vacuna Antipolio de Virus Inactivados/administración & dosificación , Vacuna Antipolio Oral/administración & dosificación , Poliovirus/inmunología , Cobertura de Vacunación , Vacunación , Adolescente , Adulto , Niño , Preescolar , Monitoreo Epidemiológico , Composición Familiar , Femenino , Humanos , Lactante , Estudios Longitudinales , Masculino , México/epidemiología , Poliomielitis/epidemiología , Poliomielitis/transmisión , Poliomielitis/virología , Poliovirus/fisiología , Esparcimiento de VirusRESUMEN
Knowledge about the function of varicella-zoster virus glycoprotein M is limited; the requirement of gM for skin and neural tropism are unknown. VZV gM contains two predicted YXXΦ trafficking motifs and a dileucine motif in the carboxyl-terminus. We constructed a recombinant VZV with gM truncated from the first YXXΦ and five additional viruses with YXXΦ tyrosine substitutions, alone and in combination with dileucine substitution. All recombinant viruses grew to high titer but mutation of the membrane-proximal YXXΦ motif reduced plaque size in cultured cells and altered gM localization. C-terminus truncation had a pronounced effect on virion morphogenesis and plaque size, but not on overall replication kinetics in vitro. Mutation of gM trafficking motifs and truncation attenuated replication in human skin xenografts in vivo; gM truncation did not alter neurotropism. Our results demonstrate that the gM C-terminus is dispensable for virus replication in cultured cells but is important for skin pathogenesis.
Asunto(s)
Ganglios Espinales/virología , Herpes Zóster/virología , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/patogenicidad , Piel/virología , Proteínas de la Matriz Viral/química , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Modelos Animales de Enfermedad , Ganglios Espinales/patología , Herpes Zóster/patología , Herpesvirus Humano 3/metabolismo , Xenoinjertos , Humanos , Masculino , Ratones , Dominios Proteicos , Transporte de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Piel/patología , Carga Viral , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Ensayo de Placa Viral , Tropismo Viral , Virulencia , Replicación ViralRESUMEN
VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This study mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication.
Asunto(s)
Herpesvirus Humano 3/genética , Proteínas Inmediatas-Precoces/genética , Señales de Localización Nuclear/genética , Virus Reordenados/genética , Transactivadores/genética , Proteínas del Envoltorio Viral/genética , Replicación Viral/genética , Línea Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/virología , Citoplasma/metabolismo , Citoplasma/virología , Expresión Génica , Genes Reporteros , Herpesvirus Humano 3/metabolismo , Humanos , Proteínas Inmediatas-Precoces/química , Proteínas Inmediatas-Precoces/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Mutación , Señales de Localización Nuclear/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Virus Reordenados/metabolismo , Transactivadores/química , Transactivadores/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismoRESUMEN
The VZV genome has two origins of DNA replication (oriS), each of which consists of an AT-rich sequence and three origin binding protein (OBP) sites called Box A, C and B. In these experiments, the mutation in the core sequence CGC of the Box A and C not only inhibited DNA replication but also inhibited both ORF62 and ORF63 expression in reporter gene assays. In contrast the Box B mutation did not influence DNA replication or flanking gene transcription. These results suggest that efficient DNA replication enhances ORF62 and ORF63 transcription. Recombinant viruses carrying these mutations in both sites and one with a deletion of the whole oriS were constructed. Surprisingly, the recombinant virus lacking both copies of oriS retained the capacity to replicate in melanoma and HELF cells suggesting that VZV has another origin of DNA replication.
Asunto(s)
Varicela/virología , Replicación del ADN , ADN Viral/genética , Herpesvirus Humano 3/genética , Origen de Réplica , Secuencia de Bases , Línea Celular , Genoma Viral , Herpesvirus Humano 3/fisiología , Humanos , Datos de Secuencia Molecular , Transcripción Genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Several cellular transcription factors have been shown to be involved in IE62-mediated activation. The YY1 cellular transcription factor has activating and repressive effects on gene transcription. Analysis of the VZV genome revealed 19 postulated YY1 binding sites located within putative promoters of 16 VZV genes. Electrophoretic mobility shift assays (EMSA) confirmed the binding of YY1 to ORF10, ORF28/29 and gI promoters and the mutation of these binding sites inhibited YY1 binding and the promoter activation by IE62 alone or following VZV infection. Mutation of the ORF28/29 YY1 site in the VZV genome displayed insignificant influence on virus growth in melanoma cells; but it inhibited the virus replication significantly at day 5 and 6 post infection in HELF cells. This work suggests a novel role for the cellular factor YY1 in VZV replication through the mediation of IE62 activation of viral gene expression.
Asunto(s)
Regulación Viral de la Expresión Génica , Herpes Zóster/metabolismo , Herpesvirus Humano 3/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Transactivadores/metabolismo , Activación Transcripcional , Proteínas del Envoltorio Viral/metabolismo , Factor de Transcripción YY1/metabolismo , Línea Celular Tumoral , Herpes Zóster/genética , Herpes Zóster/virología , Herpesvirus Humano 3/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Regiones Promotoras Genéticas , Unión Proteica , Transactivadores/genética , Proteínas del Envoltorio Viral/genética , Factor de Transcripción YY1/genéticaRESUMEN
The varicella zoster virus (VZV) immediate early 62 protein (IE62) activates most if not all identified promoters of VZV genes and also some minimum model promoters that contain only a TATA box element. Analysis of the DNA elements that function in IE62 activation of the VZV ORF3 promoter revealed that the 100 nucleotides before the translation start site of the ORF3 gene contains the promoter elements. This promoter lacks any functional TATA box element. Cellular transcription factors Sp1, Sp3 and YY1 bind to the promoter, and mutation of their binding sites inhibited ORF3 gene expression. VZV regulatory proteins, IE63 and ORF29, ORF61 and ORF10 proteins inhibited IE62-mediated activation of this promoter. Mutation of the Sp1/Sp3 binding site in the VZV genome did not alter VZV replication kinetics. This work suggests that Sp family proteins contribute to the activation of VZV promoters by IE62 in the absence of functional TATA box.
Asunto(s)
Regulación Viral de la Expresión Génica , Herpesvirus Humano 3/patogenicidad , Proteínas Inmediatas-Precoces/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismo , Transactivadores/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Factor de Transcripción YY1/metabolismo , Línea Celular , Interacciones Huésped-Patógeno , HumanosRESUMEN
The tegument proteins encoded by ORF11 and ORF9 of varicella-zoster virus (VZV) are conserved among all alphaherpesvirus. We previously demonstrated that the ORF9 gene is essential, whereas ORF11 is dispensable in vitro but its deletion severely impairs VZV infection of skin xenografts in the SCID mouse model in vivo. Here we report that ORF11 protein interacts with ORF9 protein in infected cells as well as in the absence of other viral proteins, and we have mapped the ORF11 protein domain involved in their interaction. Although ORF11 is an RNA binding protein, the interaction between ORF11 and ORF9 proteins was not mediated by RNA or DNA bridging. VZV recombinants with mutations preventing ORF11 protein binding to ORF9 protein had no effect on 6-day growth kinetics based on plaque numbers, but plaque sizes were reduced in vitro. However, disruption of the ORF11 and ORF9 protein interaction was associated with failure to replicate in skin xenografts in vivo. Further, we demonstrate that in the absence of their interaction, the ORF9 protein displays an identical cellular localization, accumulating in the trans-Golgi region, whereas the ORF11 protein exhibits aberrant localization, dispersing throughout the cytoplasm. Overall, our observations suggest that while complete tegument assembly may not be necessary for VZV replication in vitro, the interaction between the ORF11 and ORF9 proteins appears to be critical for the proper localization of ORF11 protein to the assembly complex and for production of infectious virus during VZV pathogenesis in skin.
Asunto(s)
Varicela/virología , Herpesvirus Humano 3/metabolismo , Sistemas de Lectura Abierta , Proteínas de Unión al ARN/metabolismo , Proteínas Estructurales Virales/metabolismo , Animales , Línea Celular , Herpesvirus Humano 3/genética , Humanos , Ratones , Ratones SCID , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas Estructurales Virales/genéticaRESUMEN
Herpesvirus entry functions of the conserved glycoproteins gB and gH-gL have been delineated, but their role in regulating cell-cell fusion is poorly understood. Varicella-zoster virus (VZV) infection provides a valuable model for investigating cell-cell fusion because of the importance of this process for pathogenesis in human skin and sensory ganglia. The present study identifies a canonical immunoreceptor tyrosine-based inhibition motif (ITIM) in the gB cytoplasmic domain (gBcyt) and demonstrates that the gBcyt is a tyrosine kinase substrate. Orbitrap mass spectrometry confirmed that Y881, central to the ITIM, is phosphorylated. To determine whether the gBcyt ITIM regulates gB/gH-gL-induced cell-cell fusion in vitro, tyrosine residues Y881 and Y920 in the gBcyt were substituted with phenylalanine separately or together. Recombinant viruses with these substitutions were generated to establish their effects on syncytia formation in replication in vitro and in the human skin xenograft model of VZV pathogenesis. The Y881F substitution caused significantly increased cell-cell fusion despite reduced cell-surface gB. Importantly, the Y881F or Y881/920F substitutions in VZV caused aggressive syncytia formation, reducing cell-cell spread. These in vitro effects of aggressive syncytia formation translated to severely impaired skin infection in vivo. In contrast, the Y920F substitution did not affect virus replication in vitro or in vivo. These observations suggest that gB modulates cell-cell fusion via an ITIM-mediated Y881 phosphorylation-dependent mechanism, supporting a unique concept that intracellular signaling through this gBcyt motif regulates VZV syncytia formation and is essential for skin pathogenesis.
Asunto(s)
Herpesvirus Humano 3/metabolismo , Motivo de Inhibición del Inmunorreceptor Basado en Tirosina , Piel/patología , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Células CHO , Fusión Celular , Línea Celular Tumoral , Células Cultivadas , Cricetinae , Cricetulus , Células Gigantes/ultraestructura , Células Gigantes/virología , Células HEK293 , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/fisiología , Humanos , Melanoma/patología , Melanoma/ultraestructura , Melanoma/virología , Microscopía Confocal , Microscopía Electrónica de Transmisión , Modelos Moleculares , Mutación , Fosforilación , Estructura Terciaria de Proteína , Piel/virología , Trasplante Heterólogo , Tirosina/genética , Tirosina/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
The varicella-zoster virus (VZV) ORF61 protein is necessary for normal replication in vitro and virulence in human skin xenografts in the severe combined immunodeficiency mouse model in vivo. These experiments identify a hydrophobic domain that mediates ORF61 self-interaction. While not needed to inhibit host cell defenses, disruption of this domain (residues 250 to 320) severely impairs VZV growth, transactivation of the immediate early 63 and glycoprotein E genes, and the pathogenesis of VZV skin infection in vivo.
Asunto(s)
Varicela/fisiopatología , Herpesvirus Humano 3/metabolismo , Piel/virología , Proteínas Virales/química , Proteínas Virales/metabolismo , Replicación Viral/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/patogenicidad , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Inmediatas-Precoces/metabolismo , Immunoblotting , Inmunoprecipitación , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Piel/patología , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/genética , Replicación Viral/genéticaRESUMEN
The distribution and orientation of origin-binding protein (OBP) sites are the main architectural contrasts between varicella-zoster virus (VZV) and herpes simplex virus (HSV) origins of DNA replication (oriS). One important difference is the absence of a downstream OBP site in VZV, raising the possibility that an alternative cis element may replace its function. Our previous work established that Sp1, Sp3, and YY1 bind to specific sites within the downstream region of VZV oriS; we hypothesize that one or both of these sites may be the alternative cis element(s). Here, we show that the mutation of the Sp1/Sp3 site decreases DNA replication and transcription from the adjacent ORF62 and ORF63 promoters following superinfection with VZV. In contrast, in the absence of DNA replication or in transfection experiments with ORF62, only ORF63 transcription is affected. YY1 site mutations had no significant effect on either process. Recombinant viruses containing these mutations were then constructed. The Sp1/Sp3 site mutant exhibited a significant decrease in virus growth in MeWo cells and in human skin xenografts, while the YY1 site mutant virus grew as well as the wild type in MeWo cells, even showing a late increase in VZV replication in skin xenografts following infection. These results suggest that the Sp1/Sp3 site plays an important role in both VZV origin-dependent DNA replication and ORF62 and ORF63 transcription and that, in contrast to HSV, these events are linked during virus replication.
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Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Herpesvirus Humano 3/fisiología , Piel/virología , Transcripción Genética/genética , Proteínas Virales/genética , Replicación Viral/genética , Animales , Sitios de Unión/genética , Línea Celular Tumoral , Cartilla de ADN/genética , Herpesvirus Humano 3/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/fisiología , Immunoblotting , Técnicas In Vitro , Ratones , Ratones SCID , Plásmidos/genética , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp3/genética , Transactivadores/genética , Transactivadores/fisiología , Transcripción Genética/fisiología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/fisiología , Factor de Transcripción YY1/genéticaRESUMEN
Enveloped viruses require membrane fusion for cell entry and replication. For herpesviruses, this event is governed by the multiprotein core complex of conserved glycoproteins (g)B and gH/gL. The recent crystal structures of gH/gL from herpes simplex virus 2, pseudorabies virus, and Epstein-Barr virus revealed distinct domains that, surprisingly, do not resemble known viral fusogens. Varicella-zoster virus (VZV) causes chicken pox and shingles. VZV is an α-herpesvirus closely related to herpes simplex virus 2, enabling prediction of the VZV gH structure by homology modeling. We have defined specific roles for each gH domain in VZV replication and pathogenesis using structure-based site-directed mutagenesis of gH. The distal tip of domain (D)I was important for skin tropism, entry, and fusion. DII helices and a conserved disulfide bond were essential for gH structure and VZV replication. An essential (724)CXXC(727) motif was critical for DIII structural stability and membrane fusion. This assignment of domain-dependent mechanisms to VZV gH links elements of the glycoprotein structure to function in herpesvirus replication and virulence.
Asunto(s)
Herpesvirus Humano 3/fisiología , Fusión de Membrana/fisiología , Piel/virología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/fisiología , Tropismo Viral/fisiología , Herpesvirus Humano 3/patogenicidad , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Relación Estructura-Actividad , Proteínas del Envoltorio Viral/genética , Virulencia , Replicación ViralRESUMEN
Varicella zoster virus (VZV) ORF25 is a 156 amino acid protein belonging to the approximately 40 core proteins that are conserved throughout the Herpesviridae. By analogy to its functional orthologue UL33 in Herpes simplex virus 1 (HSV-1), ORF25 is thought to be a component of the terminase complex. To investigate how cleavage and encapsidation of viral DNA links to the nuclear egress of mature capsids in VZV, we tested 10 VZV proteins that are predicted to be involved in either of the two processes for protein interactions against each other using three independent protein-protein interaction (PPI) detection systems: the yeast-two-hybrid (Y2H) system, a luminescence based MBP pull-down interaction screening assay (LuMPIS), and a bioluminescence resonance energy transfer (BRET) assay. A set of 20 interactions was consistently detected by at least 2 methods and resulted in a dense interaction network between proteins associated in encapsidation and nuclear egress. The results indicate that the terminase complex in VZV consists of ORF25, ORF30, and ORF45/42 and support a model in which both processes are closely linked to each other. Consistent with its role as a central hub for protein interactions, ORF25 is shown to be essential for VZV replication.
Asunto(s)
Genes Virales , Herpesvirus Humano 3/química , Mapeo de Interacción de Proteínas/métodos , Proteínas Virales/química , Animales , Secuencia de Bases , Transferencia de Energía por Resonancia de Bioluminiscencia , Cápside/química , Núcleo Celular/química , Clonación Molecular/métodos , Cósmidos/química , Cósmidos/genética , ADN Viral/química , ADN Viral/genética , Escherichia coli/química , Escherichia coli/metabolismo , Eliminación de Gen , Células HeLa , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/fisiología , Humanos , Sueros Inmunes/química , Sistemas de Lectura Abierta , Estructura Terciaria de Proteína , Conejos , Transfección , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genética , Replicación ViralRESUMEN
Promyelocytic leukemia protein (PML) has antiviral functions and many viruses encode gene products that disrupt PML nuclear bodies (PML NBs). However, evidence of the relevance of PML NB modification for viral pathogenesis is limited and little is known about viral gene functions required for PML NB disruption in infected cells in vivo. Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes cutaneous lesions during primary and recurrent infection. Here we show that VZV disrupts PML NBs in infected cells in human skin xenografts in SCID mice and that the disruption is achieved by open reading frame 61 (ORF61) protein via its SUMO-interacting motifs (SIMs). Three conserved SIMs mediated ORF61 binding to SUMO1 and were required for ORF61 association with and disruption of PML NBs. Mutation of the ORF61 SIMs in the VZV genome showed that these motifs were necessary for PML NB dispersal in VZV-infected cells in vitro. In vivo, PML NBs were highly abundant, especially in basal layer cells of uninfected skin, whereas their frequency was significantly decreased in VZV-infected cells. In contrast, mutation of the ORF61 SIMs reduced ORF61 association with PML NBs, most PML NBs remained intact and importantly, viral replication in skin was severely impaired. The ORF61 SIM mutant virus failed to cause the typical VZV lesions that penetrate across the basement membrane into the dermis and viral spread in the epidermis was limited. These experiments indicate that VZV pathogenesis in skin depends upon the ORF61-mediated disruption of PML NBs and that the ORF61 SUMO-binding function is necessary for this effect. More broadly, our study elucidates the importance of PML NBs for the innate control of a viral pathogen during infection of differentiated cells within their tissue microenvironment in vivo and the requirement for a viral protein with SUMO-binding capacity to counteract this intrinsic barrier.
Asunto(s)
Herpesvirus Humano 3/genética , Herpesvirus Humano 3/patogenicidad , Cuerpos de Inclusión Intranucleares/metabolismo , Piel/virología , Proteínas Virales/genética , Animales , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Clonación Molecular , Genes Virales , Herpesvirus Humano 3/fisiología , Humanos , Cuerpos de Inclusión Intranucleares/virología , Leucemia Promielocítica Aguda , Ratones , Ratones SCID , Modelos Animales , Mutagénesis , Plásmidos/genética , Dominios y Motivos de Interacción de Proteínas/genética , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Regulación hacia Arriba , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Varicella-zoster virus (VZV) is the alphaherpesvirus that causes chicken pox (varicella) and shingles (zoster). The two VZV glycoproteins gE and gI form a heterodimer that mediates efficient cell-to-cell spread. Deletion of gI yields a small-plaque-phenotype virus, ΔgI virus, which is avirulent in human skin using the xenograft model of VZV pathogenesis. In the present study, 10 mutant viruses were generated to determine which residues were required for the typical function of gI. Three phosphorylation sites in the cytoplasmic domain of gI were not required for VZV virulence in vivo. Two deletion mutants mapped a gE binding region in gI to residues 105 to 125. A glycosylation site, N116, in this region did not affect virulence. Substitution of four cysteine residues highly conserved in the Alphaherpesvirinae established that C95 is required for gE/gI heterodimer formation. The C95A and Δ105-125 (with residues 105 to 125 deleted) viruses had small-plaque phenotypes with reduced replication kinetics in vitro similar to those of the ΔgI virus. The Δ105-125 virus was avirulent for human skin in vivo. In contrast, the C95A mutant replicated in vivo but with significantly reduced kinetics compared to those of the wild-type virus. In addition to abolished gE/gI heterodimer formation, gI from the C95A or the Δ105-125 mutant was not recognized by monoclonal antibodies that detect the canonical conformation of gI, demonstrating structural disruption of gI in these viruses. This alteration prevented gI incorporation into virus particles. Thus, residues C95 and 105 to 125 are critical for gI structure required for gE/gI heterodimer formation, virion incorporation, and ultimately, effective viral spread in human skin.