RESUMEN
There is a realistic expectation that the global effort in vaccination will bring the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) under control. Nonetheless, uncertainties remain about the type of long-term association that the virus will establish with the human population and, in particular, whether coronavirus disease 2019 (COVID-19) will become an endemic disease. Although the trajectory is difficult to predict, the conditions, concepts and variables that influence this transition can be anticipated. Persistence of SARS-CoV-2 as an endemic virus, perhaps with seasonal epidemic peaks, may be fuelled by pockets of susceptible individuals and waning immunity after infection or vaccination, changes in the virus through antigenic drift that diminish protection and re-entries from zoonotic reservoirs. Here we review relevant observations from previous epidemics and discuss the potential evolution of SARS-CoV-2 as it adapts during persistent transmission in the presence of a level of population immunity. Lack of effective surveillance or adequate response could enable the emergence of new epidemic or pandemic patterns from an endemic infection of SARS-CoV-2. There are key pieces of data that are urgently needed in order to make good decisions; we outline these and propose a way forward.
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COVID-19/epidemiología , COVID-19/virología , Pandemias , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Animales , COVID-19/inmunología , COVID-19/transmisión , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/provisión & distribución , Evolución Molecular , Humanos , Evasión Inmune , Programas de Inmunización , Gripe Humana/inmunología , Gripe Humana/prevención & control , Gripe Humana/virología , SARS-CoV-2/inmunología , Factores de TiempoRESUMEN
Antibody-dependent enhancement (ADE) of disease is a general concern for the development of vaccines and antibody therapies because the mechanisms that underlie antibody protection against any virus have a theoretical potential to amplify the infection or trigger harmful immunopathology. This possibility requires careful consideration at this critical point in the pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we review observations relevant to the risks of ADE of disease, and their potential implications for SARS-CoV-2 infection. At present, there are no known clinical findings, immunological assays or biomarkers that can differentiate any severe viral infection from immune-enhanced disease, whether by measuring antibodies, T cells or intrinsic host responses. In vitro systems and animal models do not predict the risk of ADE of disease, in part because protective and potentially detrimental antibody-mediated mechanisms are the same and designing animal models depends on understanding how antiviral host responses may become harmful in humans. The implications of our lack of knowledge are twofold. First, comprehensive studies are urgently needed to define clinical correlates of protective immunity against SARS-CoV-2. Second, because ADE of disease cannot be reliably predicted after either vaccination or treatment with antibodies-regardless of what virus is the causative agent-it will be essential to depend on careful analysis of safety in humans as immune interventions for COVID-19 move forward.
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Anticuerpos Antivirales/efectos adversos , Anticuerpos Antivirales/inmunología , Acrecentamiento Dependiente de Anticuerpo/inmunología , Betacoronavirus/inmunología , Betacoronavirus/patogenicidad , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Neumonía Viral/inmunología , Neumonía Viral/virología , Animales , Anticuerpos Neutralizantes/efectos adversos , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , COVID-19 , Vacunas contra la COVID-19 , Infecciones por Coronavirus/prevención & control , Virus del Dengue/inmunología , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Macaca mulatta , Ratones , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Orthomyxoviridae/inmunología , Pandemias , Ratas , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , SARS-CoV-2 , Vacunas Virales/efectos adversos , Vacunas Virales/inmunologíaRESUMEN
Host-pathogen interactions involve complex inside-out and outside-in signal transmission through critical cellular networks that dictate disease outcomes. The phosphoinositide 3-kinase (PI3K)/Akt pathway is a pivotal junction that regulates several cell functions, and phospho-Akt (pAkt) is often found to be constitutively active in cancer cells, similar to phospho-STAT3. In this chapter, we discuss the regulation of PI3K/Akt pathway in VZV infected cells and of other pathways including p53 which, unlike pAkt and pSTAT3, directs cells towards apoptosis. The fine spatio-temporal balance of activation of pro- and anti-apoptotic factors during VZV infection likely provides an optimum environment for the virus to replicate and cause disease in the human host.
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Herpesvirus Humano 3 , Fosfatidilinositol 3-Quinasas , Humanos , Herpesvirus Humano 3/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Interacciones Huésped-PatógenoRESUMEN
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.
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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
While the varicella vaccine was created with approaches established for other live attenuated viral vaccines, novel methods to probe virus-host interactions have been used to explore the genetics, pathogenesis, and immunogenicity of the vaccine compared to wild-type varicella-zoster virus (VZV). As summarized here, a mechanism-based understanding of the safety and efficacy of the varicella vaccine has been achieved through these investigations.
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Vacuna contra el Herpes Zóster , Herpes Zóster , Vacunas Virales , Humanos , Vacuna contra la Varicela/genética , Herpes Zóster/prevención & control , Herpesvirus Humano 3/genética , Vacunas Atenuadas/genética , Antígenos ViralesRESUMEN
Cell-cell fusion (abbreviated as cell fusion) is a characteristic pathology of medically important viruses, including varicella-zoster virus (VZV), the causative agent of chickenpox and shingles. Cell fusion is mediated by a complex of VZV glycoproteins, gB and gH-gL, and must be tightly regulated to enable skin pathogenesis based on studies with gB and gH hyperfusogenic VZV mutants. Although the function of gB and gH-gL in the regulation of cell fusion has been explored, whether host factors are directly involved in this regulation process is unknown. Here, we discovered host factors that modulated VZV gB/gH-gL mediated cell fusion via high-throughput screening of bioactive compounds with known cellular targets. Two structurally related non-antibiotic macrolides, tacrolimus and pimecrolimus, both significantly increased VZV gB/gH-gL mediated cell fusion. These compounds form a drug-protein complex with FKBP1A, which binds to calcineurin and specifically inhibits calcineurin phosphatase activity. Inhibition of calcineurin phosphatase activity also enhanced both herpes simplex virus-1 fusion complex and syncytin-1 mediated cell fusion, indicating a broad role of calcineurin in modulating this process. To characterize the role of calcineurin phosphatase activity in VZV gB/gH-gL mediated fusion, a series of biochemical, biological and infectivity assays was performed. Pimecrolimus-induced, enhanced cell fusion was significantly reduced by shRNA knockdown of FKBP1A, further supporting the role of calcineurin phosphatase activity in fusion regulation. Importantly, inhibition of calcineurin phosphatase activity during VZV infection caused exaggerated syncytia formation and suppressed virus propagation, which was consistent with the previously reported phenotypes of gB and gH hyperfusogenic VZV mutants. Seven host cell proteins that remained uniquely phosphorylated when calcineurin phosphatase activity was inhibited were identified as potential downstream factors involved in fusion regulation. These findings demonstrate that calcineurin is a critical host cell factor pivotal in the regulation of VZV induced cell fusion, which is essential for VZV pathogenesis.
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Calcineurina/metabolismo , Varicela/virología , Herpes Zóster/virología , Herpesvirus Humano 3/fisiología , Glicoproteínas de Membrana/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/metabolismo , Fusión Celular , Línea Celular , Glicoproteínas/metabolismo , Herpesvirus Humano 3/genética , Humanos , MutaciónRESUMEN
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.
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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
The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.
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Modelos Moleculares , Conformación Proteica , Proteínas/química , Programas Informáticos , Secuencia de Aminoácidos , Biología Computacional , Microscopía por Crioelectrón , Cristalografía por Rayos X , Análisis de Secuencia de ProteínaRESUMEN
During all stages of infection, herpes simplex virus 1 (HSV-1) expresses viral microRNAs (miRNAs). There are at least 20 confirmed HSV-1 miRNAs, yet the roles of individual miRNAs in the context of viral infection remain largely uncharacterized. We constructed a recombinant virus lacking the sequences for miR-H1-5p and miR-H6-3p (17dmiR-H1/H6). The seed sequences for these miRNAs are antisense to each other and are transcribed from divergent noncoding RNAs in the latency-associated transcript (LAT) promoter region. Comparing phenotypes exhibited by the recombinant virus lacking these miRNAs to the wild type (17syn+), we found that during acute infection in cell culture, 17dmiR-H1/H6 exhibited a modest increase in viral yields. Analysis of pathogenesis in the mouse following footpad infection revealed a slight increase in virulence for 17dmiR-H1/H6 but no significant difference in the establishment or maintenance of latency. Strikingly, explant of latently infected dorsal root ganglia revealed a decreased and delayed reactivation phenotype. Further, 17dmiR-H1/H6 was severely impaired in epinephrine-induced reactivation in the rabbit ocular model. Finally, we demonstrated that deletion of miR-H1/H6 increased the accumulation of the LAT as well as several of the LAT region miRNAs. These results suggest that miR-H1/H6 plays an important role in facilitating efficient reactivation from latency.IMPORTANCE While HSV antivirals reduce the severity and duration of clinical disease in some individuals, there is no vaccine or cure. Therefore, understanding the mechanisms regulating latency and reactivation as a potential to elucidate targets for better therapeutics is important. There are at least 20 confirmed HSV-1 miRNAs, yet the roles of individual miRNAs in the context of viral infection remain largely uncharacterized. The present study focuses on two of the miRNAs (miR-H1/H6) that are encoded within the latency-associated transcript (LAT) region, a portion of the genome that has been associated with efficient reactivation. Here, we demonstrate that the deletion of the seed sequences of these miRNAs results in a severe reduction in reactivation of HSV-1 in the mouse and rabbit models. These results suggest a linkage between these miRNAs and reactivation.
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Ganglios Espinales/metabolismo , Herpes Simple/metabolismo , Herpesvirus Humano 1/fisiología , MicroARNs/metabolismo , ARN Viral/metabolismo , Activación Viral , Latencia del Virus , Animales , Ganglios Espinales/virología , Células HEK293 , Herpes Simple/genética , Humanos , Ratones , MicroARNs/genética , ARN Viral/genética , ConejosRESUMEN
Varicella-zoster virus (VZV) is the causative agent of chicken pox (varicella) and shingles (zoster). Although considered benign diseases, both varicella and zoster can cause complications. Zoster is painful and can lead to post herpetic neuralgia. VZV has also been linked to stroke, related to giant cell arteritis in some cases. Vaccines are available but the attenuated vaccine is not recommended in immunocompromised individuals and the efficacy of the glycoprotein E (gE) based subunit vaccine has not been evaluated for the prevention of varicella. A hallmark of VZV pathology is the formation of multinucleated cells termed polykaryocytes in skin lesions. This cell-cell fusion (abbreviated as cell fusion) is mediated by the VZV glycoproteins gB, gH and gL, which constitute the fusion complex of VZV, also needed for virion entry. Expression of gB, gH and gL during VZV infection and trafficking to the cell surface enables cell fusion. Recent evidence supports the concept that cellular processes are required for regulating cell fusion induced by gB/gH-gL. Mutations within the carboxyl domains of either gB or gH have profound effects on fusion regulation and dramatically restrict the ability of VZV to replicate in human skin. This loss of regulation modifies the transcriptome of VZV infected cells. Furthermore, cellular proteins have significant effects on the regulation of gB/gH-gL-mediated cell fusion and the replication of VZV, exemplified by the cellular phosphatase, calcineurin. This review provides the current state-of-the-art knowledge about the molecular controls of cell fusion-dependent pathogenesis caused by VZV.
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Herpesvirus Humano 3/inmunología , Interacciones Huésped-Patógeno , Infección por el Virus de la Varicela-Zóster/virología , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Animales , Fusión Celular , Vacuna contra la Varicela , Dimerización , Regulación Viral de la Expresión Génica , Herpesvirus Humano 3/genética , Humanos , Inmunoglobulina E/química , Glicoproteínas de Membrana/metabolismo , Ratones , Mutagénesis , Mutación , Sistemas de Lectura Abierta , Conformación Proteica , Infección por el Virus de la Varicela-Zóster/inmunología , Infección por el Virus de la Varicela-Zóster/prevención & control , Proteínas Virales/metabolismo , Virión/metabolismoRESUMEN
BACKGROUND: Recipients of autologous haemopoietic stem-cell transplants (auto-HSCT) have an increased risk of herpes zoster and herpes zoster-related complications. The aim of this study was to establish the efficacy and safety of an inactivated varicella zoster vaccine for the prevention of herpes zoster after auto-HSCT. METHODS: In this randomised, double-blind, placebo-controlled phase 3 trial, participants were recruited from 135 medical centres (ie, stem-cell transplant centres and hospitals) in North America, South America, Europe, and Asia. Patients were eligible if they were aged 18 years or older, scheduled to receive an auto-HSCT within 60 days of enrolment, and had a history of varicella infection or were seropositive for antibodies to varicella zoster virus, or both. Exclusion criteria included a history of herpes zoster within the previous year of enrolment, and intended antiviral prophylaxis for longer than 6 months after transplantation. Participants were randomly assigned according to a central randomisation schedule generated by the trial statistician, to receive either the inactivated-virus vaccine from one of three consistency lots, a high-antigen lot, or placebo, stratified by age (<50 vs ≥50 years) and intended duration of antiviral prophylaxis after transplantation (≤3 months vs >3 to ≤6 months). Participants, investigators, trial staff, and the funder's clinical and laboratory personnel were masked to group assignment. Participants were given four doses of inactivated vaccine or placebo, with the first dose 5-60 days before auto-HSCT, and the second, third, and fourth doses at about 30, 60, and 90 days after transplantation. The primary efficacy endpoint was the incidence of herpes zoster, confirmed by PCR or adjudication by a masked clinical committee, or both, assessed in all participants randomly assigned to the vaccine consistency lot group or placebo group who received at least one dose of vaccine and had auto-HSCT. Safety was assessed in all randomised participants who received at least one dose of vaccine and had follow-up data. A prespecified vaccine efficacy success criterion required the lower bound of the 95% CI be higher than 25% for the relative reduction of the hazard ratio of herpes zoster infection in participants given the vaccine from one of the consistency lots compared with those given placebo. This trial is registered on ClinicalTrials.gov (NCT01229267) and EudraCT (2010-020150-34). FINDINGS: Between Dec 7, 2010, and April 25, 2013, 560 participants were randomly assigned to the vaccine consistency lot group, 106 to the high-antigen lot group, and 564 to the placebo group. 249 (44%) of patients in the vaccine consistency lot group, 35 (33%) in the high-antigen lot group, and 220 (39%) in the placebo group discontinued before study end, mostly because of death or withdrawal. 51 participants were excluded from the primary efficacy endpoint analyses because they did not undergo auto-HSCT or were not vaccinated, or both (22 [4%] in the vaccine consistency lot group, and 29 [5%] in the placebo group). Mean follow-up for efficacy was 2·4 years (SD 1·3) in the vaccine consistency lot group and 2·3 years (SD 1·3) in the placebo group. 42 (8%) of 538 participants in the vaccine consistency lot group (32·9 per 1000 person-years) and 113 (21%) of 535 in the placebo group (91·9 per 1000 person-years) had a confirmed case of herpes zoster. The estimated vaccine efficacy was 63·8% (95% CI 48·4-74·6), meeting the pre-specified success criterion. For the combined vaccine groups versus the placebo group, the proportion of patients with serious adverse events (216 [33%] of 657 vs 181 [33%] of 554; risk difference 0·2%, 95% CI -5·1 to 5·5) and serious vaccine-related adverse events (five [1%] vs five [1%]; risk difference 0·1%, -1·4 to 1·1) were similar. Vaccine-related injection-site adverse events occurred more frequently in participants given vaccine than those given placebo (191 [29%] vs 36 [7%]; risk difference 22·6%, 95% CI 18·5-26·6; p<0·0001). INTERPRETATION: This study shows for the first time in a large phase 3 trial that early vaccination of auto-HSCT recipients during the peri-transplant period can be effective for the prevention of an opportunistic infection like herpes zoster and that the vaccine is well tolerated. FUNDING: Merck & Co., Inc.
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Trasplante de Células Madre Hematopoyéticas , Vacuna contra el Herpes Zóster , Herpes Zóster/prevención & control , Adulto , Anciano , Método Doble Ciego , Femenino , Humanos , Leucemia/terapia , Linfoma/terapia , Masculino , Persona de Mediana Edad , Mieloma Múltiple/terapia , Trasplante Autólogo , Vacunas de Productos Inactivados , Adulto JovenRESUMEN
Both type I and type II interferons (IFNs) have been implicated in the host defense against varicella-zoster virus (VZV), a common human herpesvirus that causes varicella and zoster. The purpose of this study was to compare their contributions to the control of VZV replication, to identify the signaling pathways that are critical for mediating their antiviral activity, and to define the mechanisms by which the virus counteracts their effects. Gamma interferon (IFN-γ) was much more potent than IFN-α in blocking VZV infection, which was associated with a differential induction of the interferon regulatory factor (IRF) proteins IRF1 and IRF9, respectively. These observations account for the clinical experience that while the formation of VZV skin lesions is initially controlled by local immunity, adaptive virus-specific T cell responses are required to prevent life-threatening VZV infections.IMPORTANCE While both type I and type II IFNs are involved in the control of herpesvirus infections in the human host, to our knowledge, their relative contributions to the restriction of viral replication and spread have not been assessed. We report that IFN-γ has more potent activity than IFN-α against VZV. Findings from this comparative analysis show that the IFN-α-IRF9 axis functions as a first line of defense to delay the onset of viral replication and spread, whereas the IFN-γ-IRF1 axis has the capacity to block the infectious process. Our findings underscore the importance of IRFs in IFN regulation of herpesvirus infection and account for the clinical experience of the initial control of VZV skin infection attributable to IFN-α production, together with the requirement for induction of adaptive IFN-γ-producing VZV-specific T cells to resolve the infection.
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Herpesvirus Humano 3/inmunología , Factor 1 Regulador del Interferón/inmunología , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/inmunología , Interferón-alfa/inmunología , Interferón gamma/inmunología , Infección por el Virus de la Varicela-Zóster/inmunología , Línea Celular Tumoral , Células HEK293 , Humanos , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT2/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Infección por el Virus de la Varicela-Zóster/virología , Replicación Viral/inmunologíaRESUMEN
Varicella-zoster virus (VZV) is the skin-tropic human alphaherpesvirus responsible for both varicella-zoster and herpes zoster. Varicella-zoster and herpes zoster skin lesions have similar morphologies, but herpes zoster occurs disproportionally in older individuals and is often associated with a more extensive local rash and severe zoster-related neuralgia. We hypothesized that skin aging could also influence the outcome of the anterograde axonal transport of VZV to skin. We utilized human skin xenografts maintained in immunodeficient (SCID) mice to study VZV-induced skin pathology in vivo in fetal and adult skin xenografts. Here we found that VZV replication is enhanced in skin from older compared to younger adults, correlating with clinical observations. In addition to measures of VZV infection, we examined the expression of type I interferon (IFN) pathway components in adult skin and investigated elements of the cutaneous proliferative and inflammatory response to VZV infection in vivo Our results demonstrated that VZV infection of adult skin triggers intrinsic IFN-mediated responses such as we have described in VZV-infected fetal skin xenografts, including MxA as well as promyelocytic leukemia protein (PML), in skin cells surrounding lesions. Further, we observed that VZV elicited altered cell signaling and proliferative and inflammatory responses that are involved in wound healing, driven by follicular stem cells. These cellular changes are consistent with VZV-induced activation of STAT3 and suggest that VZV exploits the wound healing process to ensure efficient delivery of the virus to keratinocytes. Adult skin xenografts offer an approach to further investigate VZV-induced skin pathologies in vivoIMPORTANCE Varicella-zoster virus (VZV) is the agent responsible for both varicella-zoster and herpes zoster. Herpes zoster occurs disproportionally in older individuals and is often associated with a more extensive local rash and severe zoster-related neuralgia. To examine the effect of skin aging on VZV skin lesions, we utilized fetal and adult human skin xenografts maintained in immunodeficient (SCID) mice. We measured VZV-induced skin pathology, examined the expression of type I interferon (IFN) pathway components in adult skin, and investigated elements of the cutaneous proliferative and inflammatory response to VZV infection in vivo Our results demonstrate that characteristics of aging skin are preserved in xenografts; that VZV replication is enhanced in skin from older compared to younger adults, correlating with clinical observations; and that VZV infection elicits altered cell signaling and inflammatory responses. Adult skin xenografts offer an approach to further investigate VZV-induced skin pathologies in vivo.
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Envejecimiento/patología , Herpesvirus Humano 3/crecimiento & desarrollo , Factor de Transcripción STAT3/metabolismo , Infección por el Virus de la Varicela-Zóster/patología , Replicación Viral/fisiología , Adulto , Factores de Edad , Anciano , Animales , Modelos Animales de Enfermedad , Activación Enzimática , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Proteína de la Leucemia Promielocítica/metabolismo , Piel/virología , Enfermedades de la Piel/patología , Enfermedades de la Piel/virología , Trasplante de Piel , Trasplante Heterólogo , Infección por el Virus de la Varicela-Zóster/virología , Cicatrización de Heridas/fisiologíaRESUMEN
The highly conserved herpesvirus glycoprotein complex gB/gH-gL mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multinucleated cells, or syncytia, during the infection of human tissues, but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. gBcyt regulation is necessary for VZV pathogenesis, as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt-regulated fusion was investigated by comparing melanoma cells infected with wild-type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytium formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization, and rapid displacement of nuclei to dense central structures compared to pOka using live-cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using whole transcriptome sequencing (RNA-seq) to identify viral and cellular responses induced when gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and the genes for glycoproteins gC, gE, and gI, was significantly reduced at 36 h postinfection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate gBcyt in the regulation of gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection. IMPORTANCE: The highly infectious, human-restricted pathogen varicella-zoster virus (VZV) causes chickenpox and shingles. Postherpetic neuralgia (PHN) is a common complication of shingles that manifests as prolonged excruciating pain, which has proven difficult to treat. The formation of fused multinucleated cells in ganglia might be associated with this condition. An effective vaccine against VZV is available but not recommended for immunocompromised individuals, highlighting the need for new therapies. This study investigated the viral and cellular responses to hyperfusion, a condition where the usual constraints of cell membranes are overcome and cells form multinucleated cells. This process hinders VZV and is regulated by a viral glycoprotein, gB. A combination of live-cell imaging and next-generation genomics revealed an alteration in viral and cellular responses during hyperfusion that was caused by the loss of gB regulation. These studies reveal mechanisms central to VZV pathogenesis, potentially leading to improved therapies.
Asunto(s)
Herpesvirus Humano 3/genética , Interacciones Huésped-Patógeno , Melanocitos/virología , Transcriptoma , Proteínas del Envoltorio Viral/genética , Proteínas ras/genética , Sustitución de Aminoácidos , Fusión Celular , Línea Celular Tumoral , Regulación de la Expresión Génica , Ontología de Genes , Genes Reporteros , Células Gigantes/inmunología , Células Gigantes/ultraestructura , Células Gigantes/virología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Herpesvirus Humano 3/crecimiento & desarrollo , Herpesvirus Humano 3/inmunología , Humanos , Melanocitos/inmunología , Melanocitos/ultraestructura , Anotación de Secuencia Molecular , Mutación , Dominios Proteicos , Análisis de Secuencia de ARN , Transducción de Señal , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Internalización del Virus , Proteínas ras/inmunologíaRESUMEN
The conserved glycoproteins gB and gH-gL are essential for herpesvirus entry and cell-cell fusion induced syncytium formation, a characteristic of varicella-zoster virus (VZV) pathology in skin and sensory ganglia. VZV syncytium formation, which has been implicated in the painful condition of postherpetic neuralgia, is regulated by the cytoplasmic domains of gB (gBcyt) via an immunoreceptor tyrosine-based inhibition motif (ITIM) and gH (gHcyt). A lysine cluster (K894, K897, K898, and K900) in the VZV gBcyt was identified by sequence alignment to be conserved among alphaherpesviruses, suggesting a functional role. Alanine and arginine substitutions were used to determine if the positive charge and susceptibility to posttranslational modifications of these lysines contributed to gB/gH-gL cell-cell fusion. Critically, the positive charge of the lysine residues was necessary for fusion regulation, as alanine substitutions induced a 440% increase in fusion compared to that of the wild-type gBcyt while arginine substitutions had wild-type-like fusion levels in an in vitro gB/gH-gL cell fusion assay. Consistent with these results, the alanine substitutions in the viral genome caused exaggerated syncytium formation, reduced VZV titers (-1.5 log10), and smaller plaques than with the parental Oka (pOka) strain. In contrast, arginine substitutions resulted in syncytia with only 2-fold more nuclei, a -0.5-log10 reduction in titers, and pOka-like plaques. VZV mutants with both an ITIM mutation and either alanine or arginine substitutions had reduced titers and small plaques but differed in syncytium morphology. Thus, effective VZV propagation is dependent on cell-cell fusion regulation by the conserved gBcyt lysine cluster, in addition to the gBcyt ITIM and the gHcyt. IMPORTANCE: Varicella-zoster virus (VZV) is a ubiquitous pathogen that causes chickenpox and shingles. Individuals afflicted with shingles risk developing the painful condition of postherpetic neuralgia (PHN), which has been difficult to treat because the underlying cause is not well understood. Additional therapies are needed, as the current vaccine is not recommended for immunocompromised individuals and its efficacy decreases with the age of the recipient. VZV is known to induce the formation of multinuclear cells in neuronal tissue, which has been proposed to be a factor contributing to PHN. This study examines the role of a lysine cluster in the cytoplasmic domain of the VZV fusion protein, gB, in the formation of VZV induced multinuclear cells and in virus replication kinetics and spread. The findings further elucidate how VZV self-regulates multinuclear cell formation and may provide insight into the development of new PHN therapies.
Asunto(s)
Células Epiteliales/virología , Herpesvirus Humano 3/crecimiento & desarrollo , Lisina/química , Glicoproteínas de Membrana/química , Procesamiento Proteico-Postraduccional , Proteínas del Envoltorio Viral/química , Proteínas Virales/química , Alanina/química , Alanina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Arginina/química , Arginina/metabolismo , Células CHO , Fusión Celular , Línea Celular Tumoral , Secuencia Conservada , Cricetulus , Células Epiteliales/inmunología , Expresión Génica , Células Gigantes/ultraestructura , Células Gigantes/virología , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/inmunología , Interacciones Huésped-Patógeno , Humanos , Lisina/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Mutación , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Electricidad Estática , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Internalización del VirusRESUMEN
Autophagy is a process by which misfolded and damaged proteins are sequestered into autophagosomes, before degradation in and recycling from lysosomes. We have extensively studied the role of autophagy in varicella-zoster virus (VZV) infection, and have observed that vesicular cells are filled with >100 autophagosomes that are easily detectable after immunolabeling for the LC3 protein. To confirm our hypothesis that increased autophagosome formation was not secondary to a block, we examined all conditions of VZV infection as well as carrying out two assessments of autophagic flux. We first investigated autophagy in human skin xenografts in the severe combined immunodeficiency (SCID) mouse model of VZV pathogenesis, and observed that autophagosomes were abundant in infected human skin tissues. We next investigated autophagy following infection with sonically prepared cell-free virus in cultured cells. Under these conditions, autophagy was detected in a majority of infected cells, but was much less than that seen after an infected-cell inoculum. In other words, inoculation with lower-titered cell-free virus did not reflect the level of stress to the VZV-infected cell that was seen after inoculation of human skin in the SCID mouse model or monolayers with higher-titered infected cells. Finally, we investigated VZV-induced autophagic flux by two different methods (radiolabeling proteins and a dual-colored LC3 plasmid); both showed no evidence of a block in autophagy. Overall, therefore, autophagy within a VZV-infected cell was remarkably different from autophagy within an HSV-infected cell, whose genome contains two modifiers of autophagy, ICP34.5 and US11, not present in VZV.
Asunto(s)
Autofagia , Herpes Simple/patología , Herpes Simple/virología , Herpes Zóster/patología , Herpes Zóster/virología , Herpesvirus Humano 3/fisiología , Simplexvirus/fisiología , Animales , Línea Celular , Sistema Libre de Células , Modelos Animales de Enfermedad , Fibroblastos/patología , Fibroblastos/virología , Proteínas Fluorescentes Verdes/metabolismo , Xenoinjertos , Humanos , Ratones SCID , Proteínas Asociadas a Microtúbulos/metabolismo , Fagosomas/metabolismo , Plásmidos/metabolismo , Piel/patología , Piel/virologíaRESUMEN
Varicella-zoster virus (VZV), of the family Alphaherpesvirinae, causes varicella in children and young adults, potentially leading to herpes zoster later in life on reactivation from latency. The conserved herpesvirus glycoprotein gB and the heterodimer gHgL mediate virion envelope fusion with cell membranes during virus entry. Naturally occurring neutralizing antibodies against herpesviruses target these entry proteins. To determine the molecular basis for VZV neutralization, crystal structures of gHgL were determined in complex with fragments of antigen binding (Fabs) from two human monoclonal antibodies, IgG-94 and IgG-RC, isolated from seropositive subjects. These structures reveal that the antibodies target the same site, composed of residues from both gH and gL, distinct from two other neutralizing epitopes identified by negative-stain electron microscopy and mutational analysis. Inhibition of gB/gHgL-mediated membrane fusion and structural comparisons with herpesvirus homologs suggest that the IgG-RC/94 epitope is in proximity to the site on VZV gHgL that activates gB. Immunization studies proved that the anti-gHgL IgG-RC/94 epitope is a critical target for antibodies that neutralize VZV. Thus, the gHgL/Fab structures delineate a site of herpesvirus vulnerability targeted by natural immunity.
Asunto(s)
Anticuerpos Neutralizantes/química , Glicoproteínas/química , Herpesvirus Humano 3/inmunología , Proteínas del Envoltorio Viral/química , Animales , Anticuerpos Monoclonales/química , Anticuerpos Antivirales/inmunología , Cristalografía por Rayos X , Epítopos/química , Humanos , Fragmentos de Inmunoglobulinas/química , Ratones , Modelos Moleculares , Pruebas de Neutralización , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
Studies of varicella-zoster virus (VZV) tropism for T cells support their role in viral transport to the skin during primary infection. Multiparametric single-cell mass cytometry demonstrates that, instead of preferentially infecting skin-homing T cells, VZV alters cell signaling and remodels surface proteins to enhance T cell skin trafficking. Viral proteins dispensable in skin, such as that encoded by open reading frame 66, are necessary in T cells. Interference with VZV T cell tropism may offer novel strategies for drug and vaccine design.
Asunto(s)
Varicela/virología , Herpes Zóster/virología , Herpesvirus Humano 3/inmunología , Piel/virología , Linfocitos T/virología , Exantema/virología , Vacuna contra el Herpes Zóster/inmunología , Herpesvirus Humano 3/genética , Humanos , Sistemas de Lectura Abierta/genética , Transducción de Señal , Piel/patología , Linfocitos T/inmunología , TropismoRESUMEN
UNLABELLED: Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella and herpes zoster. Membrane fusion is essential for VZV entry and the distinctive syncytium formation in VZV-infected skin and neuronal tissue. Herpesvirus fusion is mediated by a complex of glycoproteins gB and gH-gL, which are necessary and sufficient for VZV to induce membrane fusion. However, the cellular requirements of fusion are poorly understood. Integrins have been implicated to facilitate entry of several human herpesviruses, but their role in VZV entry has not yet been explored. To determine the involvement of integrins in VZV fusion, a quantitative cell-cell fusion assay was developed using a VZV-permissive melanoma cell line. The cells constitutively expressed a reporter protein and short hairpin RNAs (shRNAs) to knock down the expression of integrin subunits shown to be expressed in these cells by RNA sequencing. The αV integrin subunit was identified as mediating VZV gB/gH-gL fusion, as its knockdown by shRNAs reduced fusion levels to 60% of that of control cells. A comparable reduction in fusion levels was observed when an anti-αV antibody specific to its extracellular domain was tested in the fusion assay, confirming that the domain was important for VZV fusion. In addition, reduced spread was observed in αV knockdown cells infected with the VZV pOka strain relative to that of the control cells. This was demonstrated by reductions in plaque size, replication kinetics, and virion entry in the αV subunit knockdown cells. Thus, the αV integrin subunit is important for VZV gB/gH-gL fusion and infection. IMPORTANCE: Varicella-zoster virus (VZV) is a highly infectious pathogen that causes chickenpox and shingles. A common complication of shingles is the excruciating condition called postherpetic neuralgia, which has proven difficult to treat. While a vaccine is now available, it is not recommended for immunocompromised individuals and its efficacy decreases with the recipient's age. These limitations highlight the need for new therapies. This study examines the role of integrins in membrane fusion mediated by VZV glycoproteins gB and gH-gL, a required process for VZV infection. This knowledge will further the understanding of VZV entry and provide insight into the development of better therapies.
Asunto(s)
Herpesvirus Humano 3/fisiología , Interacciones Huésped-Patógeno , Integrina alfaV/metabolismo , Internalización del Virus , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Humanos , Integrina alfaV/genética , Ensayo de Placa ViralRESUMEN
UNLABELLED: Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella upon primary infection and zoster upon reactivation from latency in sensory ganglion neurons. The replication of herpesviruses requires manipulation of cell signaling pathways. Notably, CREB, a factor involved in the regulation of several cellular processes, is activated upon infection of T cells with VZV. Here, we report that VZV infection also induced CREB phosphorylation in fibroblasts and that XX-650-23, a newly identified inhibitor of the phosphorylated-CREB (pCREB) interaction with p300/CBP, restricted cell-cell spread of VZV in vitro CREB phosphorylation did not require the viral open reading frame 47 (ORF47) and ORF66 kinases encoded by VZV. Evaluating the biological relevance of these observations during VZV infection of human skin xenografts in the SCID mouse model of VZV pathogenesis showed both that pCREB was upregulated in infected skin and that treatment with XX-650-23 reduced infectious-virus production and limited lesion formation compared to treatment with a vehicle control. Thus, processes of CREB activation and p300/CBP binding are important for VZV skin infection and may be targeted for antiviral drug development. IMPORTANCE: Varicella-zoster virus (VZV) is a common pathogen that causes chicken pox and shingles. As with all herpesviruses, the infection is acquired for life, and the virus can periodically reactivate from latency. Although VZV infection is usually benign with few or no deleterious consequences, infection can be life threatening in immunocompromised patients. Otherwise healthy elderly individuals who develop zoster as a consequence of viral reactivation are at risk for postherpetic neuralgia (PHN), a painful and long-lasting complication. Current vaccines use a live attenuated virus that is usually safe but cannot be given to many immunodeficient patients and retains the capacity to establish latency and reactivate, causing zoster. Antiviral drugs are effective against severe VZV infections but have little impact on PHN. A better understanding of virus-host cell interactions is relevant for developing improved therapies to safely interfere with cellular processes that are crucial for VZV pathogenesis.