RESUMEN
The nucleoplasm, the cytosol, the inside of virions, and again the cytosol comprise the world in which the capsids of alphaherpesviruses encounter viral and host proteins that support or limit them in performing their tasks. Here, we review the fascinating conundrum of how specific protein-protein interactions late in alphaherpesvirus infection orchestrate capsid nuclear assembly, nuclear egress, and cytoplasmic envelopment, but target incoming capsids to the nuclear pores in naive cells to inject the viral genomes into the nucleoplasm for viral transcription and replication. Multiple capsid interactions with viral and host proteins have been characterized using viral mutants and assays that reconstitute key stages of the infection cycle. Keratinocytes, fibroblasts, mucosal epithelial cells, neurons, and immune cells employ cell type-specific intrinsic and cytokine-induced resistance mechanisms to restrict several stages of the viral infection cycle. However, concomitantly, alphaherpesviruses have evolved countermeasures to ensure efficient capsid function during infection.
RESUMEN
BACKGROUND: The antimicrobial ribonuclease RNase 7 is abundantly expressed in the epidermis of lesional skin of atopic dermatitis (AD). Host RNase inhibitor (RI) binds to RNase 7 and blocks its ribonuclease activity. This study aimed to evaluate the impact of RNase 7-RI interactions on AD. METHODS: Cultured human primary keratinocytes, with siRNA-mediated downregulation of RNase 7 and RI, were stimulated with the synthetic RNA polyinosinic-polycytidylic acid (poly I:C). Induction of proinflammatory mediators was analyzed by real-time PCR and ELISA. RI expression in AD non-lesional and lesional skin biopsies and healthy controls was analyzed by real-time PCR and immunostaining. RI protein release in vivo on the AD skin surface was determined by western blot. Antimicrobial and ribonuclease assays were used to investigate the functional role of RI. RESULTS: RNase 7 inhibited the RNA-induced expression of proinflammatory mediators in keratinocytes. Accordingly, downregulation of RNase 7 in keratinocytes enhanced RNA-mediated induction of proinflammatory mediators, whereas downregulation of RI had the opposite effect. RI was released by damaged keratinocytes and epidermis. In vivo expression and release of RI on the skin surface were enhanced in lesional AD skin. Rinsing solution from the surface of lesional AD skin blocked the ribonuclease activity of RNase 7. The anti-Staphylococcus aureus activity of RNase 7 was abrogated by RI. CONCLUSIONS: Our data suggest a novel role of RI as a trigger factor of inflammation in AD by blocking the ribonuclease and antimicrobial activity of RNase 7, thereby enhancing RNA-mediated inflammation and S. aureus growth.
Asunto(s)
Dermatitis Atópica , Queratinocitos , Ribonucleasas , Staphylococcus aureus , Humanos , Dermatitis Atópica/metabolismo , Ribonucleasas/metabolismo , Queratinocitos/metabolismo , Inflamación/metabolismo , Células CultivadasRESUMEN
Microtubule transport and nuclear import are functionally connected, and the nuclear pore complex (NPC) can interact with microtubule motors. For several alphaherpesvirus proteins, nuclear localization signals (NLSs) and their interactions with specific importin-α proteins have been characterized. Here, we review recent insights on the roles of microtubule motors, capsid-associated NLSs, and importin-α proteins for capsid transport, capsid docking to NPCs, and genome release into the nucleoplasm, as well as the role of importins for nuclear viral transcription, replication, capsid assembly, genome packaging, and nuclear capsid egress. Moreover, importin-α proteins exert antiviral effects by promoting the nuclear import of transcription factors inducing the expression of interferons (IFN), cytokines, and IFN-stimulated genes, and the IFN-inducible MxB restricts capsid docking to NPCs.
Asunto(s)
Alphaherpesvirinae , Herpes Simple , Humanos , Carioferinas , alfa Carioferinas/genética , Poro Nuclear , Proteínas de la CápsideRESUMEN
Upon recognition of aberrantly located DNA, the innate immune sensor cyclic GMP-AMP synthase (cGAS) activates stimulator of IFN genes (STING)/IFN regulatory factor (IRF)3-driven antiviral responses. In this study, we characterized the ability of a specific variant of the human cGAS-encoding gene MB21D1, rs610913, to alter cGAS-mediated DNA sensing and viral infection. rs610913 is a frequent G>T polymorphism resulting in a P261H exchange in the cGAS protein. Data from the International Collaboration for the Genomics of HIV suggested that rs610913 nominally associates with HIV-1 acquisition in vivo. Molecular modeling of cGAS(P261H) hinted toward the possibility for an additional binding site for a potential cellular cofactor in cGAS dimers. However, cGAS(wild-type [WT]) or cGAS(P261H)-reconstituted THP-1 cGAS knockout cells shared steady-state expression of IFN-stimulated genes, as opposed to cells expressing the enzymatically inactive cGAS(G212A/S213A). Accordingly, cGAS(WT) and cGAS(P261H) cells were less susceptible to lentiviral transduction and infection with HIV-1, HSV-1, and Chikungunya virus as compared with cGAS knockout or cGAS(G212A/S213A) cells. Upon DNA challenge, innate immune activation appeared to be mildly reduced upon expression of cGAS(P261H) compared with cGAS(WT). Finally, DNA challenge of PBMCs from donors homozygously expressing rs610913 provoked a trend toward a slightly reduced type I IFN response as compared with PBMCs from GG donors. Taken together, the steady-state activity of cGAS maintains a baseline antiviral state rendering cells more refractory to IFN-stimulated gene-sensitive viral infections. rs610913 failed to grossly differ phenotypically from the WT gene, suggesting that cGAS(P261H) and WT cGAS share a similar ability to sense viral infections in vivo.
Asunto(s)
Inmunidad Innata , Virosis , Humanos , ADN Viral/inmunología , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Nucleotidiltransferasas/metabolismo , Transducción de Señal , Virosis/genética , Virosis/inmunología , Virosis/prevención & controlRESUMEN
Starting a herpesviral infection is a steeplechase across membranes, cytosol, and nuclear envelopes and against antiviral defence mechanisms. Here, we highlight recent insights on capsid stabilization at the portals during assembly, early capsid-host interactions ensuring nuclear targeting of incoming capsids, and genome uncoating. After fusion with a host membrane, incoming capsids recruit microtubule motors for traveling to the centrosome, and by unknown mechanisms get forward towards the nucleus. The interaction of capsid-associated tegument proteins with nucleoporins orients the capsid portal towards the nuclear pore, and presumably after removal of the portal caps the genomes that have been packaged under pressure can be injected into the nucleoplasm for transcription and replication. Some cell types disarm the incoming capsids or silence the incoming genomes to reduce the likelihood of infection.
Asunto(s)
Proteínas de la Cápside , Cápside , Proteínas de la Cápside/genética , Núcleo Celular , Citosol , SimplexvirusRESUMEN
Interferon-induced transmembrane (IFITM) proteins restrict membrane fusion and virion internalization of several enveloped viruses. The role of IFITM proteins during alphaviral infection of human cells and viral counteraction strategies are insufficiently understood. Here, we characterized the impact of human IFITMs on the entry and spread of chikungunya virus and Mayaro virus and provide first evidence for a CHIKV-mediated antagonism of IFITMs. IFITM1, 2, and 3 restricted infection at the level of alphavirus glycoprotein-mediated entry, both in the context of direct infection and cell-to-cell transmission. Relocalization of normally endosomal IFITM3 to the plasma membrane resulted in loss of antiviral activity. rs12252-C, a naturally occurring variant of IFITM3 that may associate with severe influenza in humans, restricted CHIKV, MAYV, and influenza A virus infection as efficiently as wild-type IFITM3 Antivirally active IFITM variants displayed reduced cell surface levels in CHIKV-infected cells involving a posttranscriptional process mediated by one or several nonstructural protein(s) of CHIKV. Finally, IFITM3-imposed reduction of specific infectivity of nascent particles provides a rationale for the necessity of a virus-encoded counteraction strategy against this restriction factor.
Asunto(s)
Infecciones por Alphavirus/prevención & control , Fiebre Chikungunya/prevención & control , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/metabolismo , Alphavirus/patogenicidad , Infecciones por Alphavirus/metabolismo , Infecciones por Alphavirus/virología , Línea Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Fiebre Chikungunya/metabolismo , Fiebre Chikungunya/virología , Virus Chikungunya/patogenicidad , Endosomas/metabolismo , Humanos , Proteínas de la Membrana/fisiología , Proteínas de Unión al ARN/fisiología , Internalización del VirusRESUMEN
The DNA sensor cGAS catalyzes the production of the cyclic dinucleotide cGAMP, resulting in type I interferon responses. We addressed the functionality of cGAS-mediated DNA sensing in human and murine T cells. Activated primary CD4+ T cells expressed cGAS and responded to plasmid DNA by upregulation of ISGs and release of bioactive interferon. In mouse T cells, cGAS KO ablated sensing of plasmid DNA, and TREX1 KO enabled cells to sense short immunostimulatory DNA. Expression of IFIT1 and MX2 was downregulated and upregulated in cGAS KO and TREX1 KO T cell lines, respectively, compared to parental cells. Despite their intact cGAS sensing pathway, human CD4+ T cells failed to mount a reverse transcriptase (RT) inhibitor-sensitive immune response following HIV-1 infection. In contrast, infection of human T cells with HSV-1 that is functionally deficient for the cGAS antagonist pUL41 (HSV-1ΔUL41N) resulted in a cGAS-dependent type I interferon response. In accordance with our results in primary CD4+ T cells, plasmid challenge or HSV-1ΔUL41N inoculation of T cell lines provoked an entirely cGAS-dependent type I interferon response, including IRF3 phosphorylation and expression of ISGs. In contrast, no RT-dependent interferon response was detected following transduction of T cell lines with VSV-G-pseudotyped lentiviral or gammaretroviral particles. Together, T cells are capable to raise a cGAS-dependent cell-intrinsic response to both plasmid DNA challenge or inoculation with HSV-1ΔUL41N. However, HIV-1 infection does not appear to trigger cGAS-mediated sensing of viral DNA in T cells, possibly by revealing viral DNA of insufficient quantity, length, and/or accessibility to cGAS.
Asunto(s)
Linfocitos T CD4-Positivos/virología , VIH-1/fisiología , Interferón Tipo I/metabolismo , Nucleotidiltransferasas/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , ADN Viral/fisiología , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Factor 3 Regulador del Interferón/metabolismo , Ratones , Nucleotidiltransferasas/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Especificidad de la Especie , Replicación ViralRESUMEN
During primary infection, herpes simplex virus 2 (HSV-2) replicates in epithelial cells and enters neurites to infect neurons of the peripheral nervous system. Growth factors and attractive and repulsive directional cues influence neurite outgrowth and neuronal survival. We hypothesized that HSV-2 modulates the activity of such cues to increase neurite outgrowth. To test this hypothesis, we exposed sensory neurons to nerve growth factor (NGF) and mock- or HSV-2-infected HEK-293T cells, since they express repellents of neurite outgrowth. We show that HEK-293T cells secrete factors that inhibit neurite outgrowth, while infection with HSV-2 strains MS and 333 reduces this repelling phenotype, increasing neurite numbers. The HSV-2-mediated restoration of neurite outgrowth required the activity of NGF. In the absence of infection, however, NGF did not overcome the repulsion mediated by HEK-293T cells. We previously showed that recombinant, soluble glycoprotein G of HSV-2 (rSgG2) binds and enhances NGF activity, increasing neurite outgrowth. However, the effect of gG2 during infection has not been investigated. Therefore, we addressed whether gG2 contributes to overcoming neurite outgrowth repulsion. To do so, we generated viruses lacking gG2 expression and complemented them by exogenous expression of gG2. Overall, our results suggest that HSV-2 infection of nonneuronal cells reduces their repelling effect on neurite outgrowth in an NGF-dependent manner. gG2 contributed to this phenotype, but it was not the only factor. The enhanced neurite outgrowth may facilitate HSV-2 spread from epithelial cells into neurons expressing NGF receptors and increase HSV-2-mediated pathogenesis.IMPORTANCE Herpes simplex virus 2 (HSV-2) is a prevalent human pathogen that establishes lifelong latency in neurons of the peripheral nervous system. Colonization of neurons is required for HSV-2 persistence and pathogenesis. The viral and cellular factors required for efficient infection of neurons are not fully understood. We show here that nonneuronal cells repel neurite outgrowth of sensory neurons, while HSV-2 infection overcomes this inhibition and, rather, stimulates neurite outgrowth. HSV-2 glycoprotein G and nerve growth factor contribute to this phenotype, which may attract neurites to sites of infection and facilitate virus spread to neurons. Understanding the mechanisms that modulate neurite outgrowth and facilitate HSV-2 infection of neurons might foster the development of therapeutics to reduce HSV-2 colonization of the nervous system and provide insights on neurite outgrowth and regeneration.
Asunto(s)
Herpes Genital/metabolismo , Herpesvirus Humano 2/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Neuritas , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Herpesvirus Humano 2/patogenicidad , Humanos , Ratones , Ratones Endogámicos BALB C , Neuritas/metabolismo , Neuritas/virología , Células VeroRESUMEN
Filoviruses infect a wide range of cell types with the exception of lymphocytes. The intracellular proteins cathepsin B and L, two-pore channel 1 and 2, and bona fide receptor Niemannâ»Pick Disease C1 (NPC1) are essential for the endosomal phase of cell entry. However, earlier steps of filoviral infection remain poorly characterized. Numerous plasma membrane proteins have been implicated in attachment but it is still unclear which ones are sufficient for productive entry. To define a minimal set of host factors required for filoviral glycoprotein-driven cell entry, we screened twelve cell lines and identified the nonlymphocytic cell line SH-SY5Y to be specifically resistant to filovirus infection. Heterokaryons of SH-SY5Y cells fused to susceptible cells were susceptible to filoviruses, indicating that SH-SY5Y cells do not express a restriction factor but lack an enabling factor critical for filovirus entry. However, all tested cell lines expressed functional intracellular factors. Global gene expression profiling of known cell surface entry factors and protein expression levels of analyzed attachment factors did not reveal any correlation between susceptibility and expression of a specific host factor. Using binding assays with recombinant filovirus glycoprotein, we identified cell attachment as the step impaired in filovirus entry in SH-SY5Y cells. Individual overexpression of attachment factors T-cell immunoglobulin and mucin domain 1 (TIM-1), Axl, Mer, or dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) rendered SH-SY5Y cells susceptible to filovirus glycoprotein-driven transduction. Our study reveals that a lack of attachment factors limits filovirus entry and provides direct experimental support for a model of filoviral cell attachment where host factor usage at the cell surface is highly promiscuous.
Asunto(s)
Membrana Celular/virología , Filoviridae/fisiología , Interacciones Huésped-Patógeno , Receptores Virales/genética , Proteínas Virales/genética , Internalización del Virus , Células A549 , Proteínas Portadoras/genética , Línea Celular , Ebolavirus/genética , Ebolavirus/fisiología , Filoviridae/genética , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Glicoproteínas de Membrana/genéticaRESUMEN
Dendritic cells (DCs) are crucial for the induction of potent antiviral immune responses. In contrast to immature DCs (iDCs), mature DCs (mDCs) are not permissive for infection with herpes simplex virus type 1 (HSV-1). Here, we demonstrate that HSV-1 infection of iDCs and mDCs induces autophagy, which promotes the degradation of lamin A/C, B1, and B2 in iDCs only. This in turn facilitates the nuclear egress of progeny viral capsids and thus the formation of new infectious particles. In contrast, lamin protein levels remain stable in HSV-1-infected mDCs due to an inefficient autophagic flux. Elevated protein levels of KIF1B and KIF2A in mDCs inhibited lamin degradation, likely by hampering autophagosome-lysosome fusion. Therefore, in mDCs, fewer progeny capsids were released from the nuclei into the cytosol, and fewer infectious virions were assembled. We hypothesize that inhibition of autophagic lamin degradation in mDCs represents a very powerful cellular counterstrike to inhibit the production of progeny virus and thus viral spread.
Asunto(s)
Cápside/metabolismo , Núcleo Celular , Citosol , Células Dendríticas , Herpesvirus Humano 1/metabolismo , Liberación del Virus/fisiología , Núcleo Celular/metabolismo , Núcleo Celular/virología , Citosol/metabolismo , Citosol/virología , Células Dendríticas/metabolismo , Células Dendríticas/virología , Herpesvirus Humano 1/genética , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Laminas/genética , Laminas/metabolismo , ProteolisisRESUMEN
Given the endemic seroprevalence of herpes simplex viruses (HSV), its associated human diseases, and the emergence of acyclovir-resistant strains, there is a continuous need for better antiviral therapies. Towards this aim, identifying mechanistic details of how HSV-1 manipulates infected cells, how it modulates the immune responses, and how it causes diseases are essential. Measuring titers and growth kinetics of clinical isolates and viral mutants are important for a thorough characterization of viral phenotypes in vitro and in vivo. We provide protocols for the preparation as well as titration of HSV-1 stocks, and explain how to perform single-step growth curves to characterize the functions of viral proteins or host factors during infection. In particular, we describe methods to prepare and characterize high-titer HSV-1 stocks with low genome to titer ratios that are required for infection studies in cell culture and animal experiments.
RESUMEN
To enter host cells, herpes simplex virus 1 (HSV-1) initially attaches to cell surface glycosaminoglycans, followed by the requisite binding to one of several cellular receptors, leading to viral internalization. Although virus-receptor interactions have been studied in various cell lines, the contributions of individual receptors to uptake into target tissues such as mucosa, skin, and cornea are not well understood. We demonstrated that nectin-1 acts as a major receptor for HSV-1 entry into murine epidermis, while herpesvirus entry mediator (HVEM) can serve as an alternative receptor. Recently, the macrophage receptor with collagenous structure (MARCO) has been described to mediate adsorption of HSV-1 to epithelial cells. Here, we investigated the impact of MARCO on the entry process of HSV-1 into the two major cell types of skin, keratinocytes in the epidermis and fibroblasts in the underlying dermis. Using ex vivo infection of murine epidermis, we showed that HSV-1 entered basal keratinocytes of MARCO-/- epidermis as efficiently as those of control epidermis. In addition, entry into dermal fibroblasts was not impaired in the absence of MARCO. When we treated epidermis, primary keratinocytes, or fibroblasts with poly(I), a ligand for class A scavenger receptors, HSV-1 entry was strongly reduced. As we also observed reducing effects of poly(I) in the absence of both MARCO and scavenger receptor A1, we concluded that the inhibitory effects of poly(I) on HSV-1 infection are not directly linked to class A scavenger receptors. Overall, our results support that HSV-1 entry into skin cells is independent of MARCO.IMPORTANCE During entry into its host cells, the human pathogen herpes simplex virus (HSV) interacts with various cellular receptors. Initially, receptor interaction can mediate cellular adsorption, followed by receptor binding that triggers viral internalization. The intriguing question is which receptors are responsible for the various steps during entry into the natural target tissues of HSV? Previously, we demonstrated the role of nectin-1 as a major receptor and that of HVEM as an alternative receptor for HSV-1 to invade murine epidermis. As MARCO has been described to promote infection in skin, we explored the predicted role of MARCO as a receptor that mediates adsorption to epithelial cells. Our infection studies of murine skin cells indicate that the absence of MARCO does not interfere with the efficiency of HSV-1 entry and that the inhibitory effect on viral adsorption by poly(I), a ligand of MARCO, is independent of MARCO.
Asunto(s)
Dermis/metabolismo , Epidermis/metabolismo , Fibroblastos/metabolismo , Herpesvirus Humano 1/metabolismo , Receptores Inmunológicos/metabolismo , Internalización del Virus , Animales , Dermis/virología , Epidermis/virología , Fibroblastos/virología , Herpesvirus Humano 1/genética , Humanos , Queratinocitos/metabolismo , Queratinocitos/virología , Ratones , Ratones Noqueados , Receptores Inmunológicos/genéticaRESUMEN
Herpesviruses are large DNA viruses which depend on many nuclear functions, and therefore on host transport factors to ensure specific nuclear import of viral and host components. While some import cargoes bind directly to certain transport factors, most recruit importin ß1 via importin α. We identified importin α1 in a small targeted siRNA screen to be important for herpes simplex virus (HSV-1) gene expression. Production of infectious virions was delayed in the absence of importin α1, but not in cells lacking importin α3 or importin α4. While nuclear targeting of the incoming capsids, of the HSV-1 transcription activator VP16, and of the viral genomes were not affected, the nuclear import of the HSV-1 proteins ICP4 and ICP0, required for efficient viral transcription, and of ICP8 and pUL42, necessary for DNA replication, were reduced. Furthermore, quantitative electron microscopy showed that fibroblasts lacking importin α1 contained overall fewer nuclear capsids, but an increased proportion of mature nuclear capsids indicating that capsid formation and capsid egress into the cytoplasm were impaired. In neurons, importin α1 was also not required for nuclear targeting of incoming capsids, but for nuclear import of ICP4 and for the formation of nuclear capsid assembly compartments. Our data suggest that importin α1 is specifically required for the nuclear localization of several important HSV1 proteins, capsid assembly, and capsid egress into the cytoplasm, and may become rate limiting in situ upon infection at low multiplicity or in terminally differentiated cells such as neurons.
Asunto(s)
Proteínas de la Cápside/metabolismo , Núcleo Celular/metabolismo , Fibroblastos/virología , Herpesvirus Humano 1/fisiología , Neuronas/virología , Ensamble de Virus/genética , alfa Carioferinas/fisiología , Transporte Activo de Núcleo Celular/genética , Animales , Cápside/metabolismo , Línea Celular , Núcleo Celular/virología , Cricetinae , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Herpesvirus Humano 1/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , alfa Carioferinas/genéticaRESUMEN
Upon reactivation from latency and during lytic infections in neurons, alphaherpesviruses assemble cytosolic capsids, capsids associated with enveloping membranes, and transport vesicles harboring fully enveloped capsids. It is debated whether capsid envelopment of herpes simplex virus (HSV) is completed in the soma prior to axonal targeting or later, and whether the mechanisms are the same in neurons derived from embryos or from adult hosts. We used HSV mutants impaired in capsid envelopment to test whether the inner tegument proteins pUL36 or pUL37 necessary for microtubule-mediated capsid transport were sufficient for axonal capsid targeting in neurons derived from the dorsal root ganglia of adult mice. Such neurons were infected with HSV1-ΔUL20 whose capsids recruited pUL36 and pUL37, with HSV1-ΔUL37 whose capsids associate only with pUL36, or with HSV1-ΔUL36 that assembles capsids lacking both proteins. While capsids of HSV1-ΔUL20 were actively transported along microtubules in epithelial cells and in the somata of neurons, those of HSV1-ΔUL36 and -ΔUL37 could only diffuse in the cytoplasm. Employing a novel image analysis algorithm to quantify capsid targeting to axons, we show that only a few capsids of HSV1-ΔUL20 entered axons, while vesicles transporting gD utilized axonal transport efficiently and independently of pUL36, pUL37, or pUL20. Our data indicate that capsid motility in the somata of neurons mediated by pUL36 and pUL37 does not suffice for targeting capsids to axons, and suggest that capsid envelopment needs to be completed in the soma prior to targeting of herpes simplex virus to the axons, and to spreading from neurons to neighboring cells.
Asunto(s)
Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/patogenicidad , Neuronas/virología , Animales , Transporte Axonal , Axones/ultraestructura , Axones/virología , Cápside/fisiología , Cápside/ultraestructura , Células Cultivadas , Chlorocebus aethiops , Ganglios Espinales/virología , Herpes Simple/virología , Herpesvirus Humano 1/genética , Interacciones Huésped-Patógeno , Humanos , Ratones , Microscopía Electrónica de Transmisión , Movimiento/fisiología , Mutación , Neuronas/ultraestructura , Células Vero , Proteínas Virales/genética , Proteínas Virales/fisiología , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/fisiologíaRESUMEN
The MHC class I presentation is responsible for the presentation of viral proteins to CD8+ T lymphocytes and mainly depends on the classical antigen processing pathway. Recently, a second pathway involving autophagy has been implicated in this process. Here, we show an increase in the capacity of murine dendritic cells (DCs) to present viral antigens on MHC class I after infection with a mutant herpes simplex virus 1 (HSV-1-Δ34.5), lacking infected cell protein 34.5 (ICP34.5), when compared to its parental HSV-1 strain. The ICP34.5 protein counteracts host cell translational arrest and suppresses macroautophagy, and the lack of this protein resulted in a low viral protein abundance, which was processed and presented in an efficient way. Our study demonstrates an important role of autophagy in processing endogenous viral proteins in HSV-1-infected DCs.
Asunto(s)
Autofagia/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/fisiología , Activación de Linfocitos , Animales , Presentación de Antígeno , Antígenos Virales/inmunología , Linfocitos T CD8-positivos/fisiología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Células Dendríticas/virología , Herpesvirus Humano 1/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Ratones , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Hepatitis delta virus (HDV) is a minimalistic satellite virus of hepatitis B virus (HBV). HBV/HDV co-infection, i.e. "hepatitis D", is the most severe form of viral hepatitis. No effective therapy for HDV infection is available partly due to the fact that HDV is a highly host-dependent virus devoid of any potentially drugable enzyme encoded in its small genome. In this study we present a semi-automated method to evaluate HDV infection and replication under the influence of different drugs. We utilized a Huh-7/hNTCP cell culture based system in a 96-well plate format, an automated microscope and image acquisition as well as analysis with the CellProfiler software to quantify the impact of these drugs on HDV infection. For validation, three groups of potential anti-HDV agents were evaluated: To target ribozyme activity of HDV RNA, we screened ribozyme inhibitors but only observed marked toxicity. Testing innate antiviral mediators showed that interferons alpha-2a and beta-1a had a specific inhibitory effect on HDV infection. Finally, we screened a library of 160 human kinase inhibitors covering all parts of the human kinome. Overall, only inhibitors targeting the tyrosine kinase-like group had significant average anti-HDV activity. Looking at individual substances, kenpaullone, a GSK-3ß and Cdk inhibitor, had the highest selective index of 3.44. Thus, we provide a potentially useful tool to screen for substances with anti-HDV activity and novel insights into interactions between HDV replication and the human kinome.
Asunto(s)
Antivirales/farmacología , Anticuerpos Antihepatitis/inmunología , Hepatitis D/tratamiento farmacológico , Virus de la Hepatitis Delta/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Interacciones Huésped-Patógeno/efectos de los fármacos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Antivirales/aislamiento & purificación , Automatización , Benzazepinas/farmacología , Coinfección/tratamiento farmacológico , Hepatitis B/virología , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis D/virología , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Humanos , Indoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , ARN Catalítico/antagonistas & inhibidores , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
The human protein DDX3X is a DEAD box ATP-dependent RNA helicase that regulates transcription, mRNA maturation, and mRNA export and translation. DDX3X concomitantly modulates the replication of several RNA viruses and promotes innate immunity. We previously showed that herpes simplex virus 1 (HSV-1), a human DNA virus, incorporates DDX3X into its mature particles and that DDX3X is required for optimal HSV-1 infectivity. Here, we show that viral gene expression, replication, and propagation depend on optimal DDX3X protein levels. Surprisingly, DDX3X from incoming viral particles was not required for the early stages of the HSV-1 infection, but, rather, the protein controlled the assembly of new viral particles. This was independent of the previously reported ability of DDX3X to stimulate interferon type I production. Instead, both the lack and overexpression of DDX3X disturbed viral gene transcription and thus subsequent genome replication. This suggests that in addition to its effect on RNA viruses, DDX3X impacts DNA viruses such as HSV-1 by an interferon-independent pathway.IMPORTANCE Viruses interact with a variety of cellular proteins to complete their life cycle. Among them is DDX3X, an RNA helicase that participates in most aspects of RNA biology, including transcription, splicing, nuclear export, and translation. Several RNA viruses and a limited number of DNA viruses are known to manipulate DDX3X for their own benefit. In contrast, DDX3X is also known to promote interferon production to limit viral propagation. Here, we show that DDX3X, which we previously identified in mature HSV-1 virions, stimulates HSV-1 gene expression and, consequently, virion assembly by a process that is independent of its ability to promote the interferon pathway.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Regulación Viral de la Expresión Génica , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno , Ensamble de Virus , Animales , Línea Celular , Herpesvirus Humano 1/genética , Humanos , Replicación ViralRESUMEN
UNLABELLED: Herpes simplex virus (HSV) replicates in the skin and mucous membranes, and initiates lytic or latent infections in sensory neurons. Assembly of progeny virions depends on the essential large tegument protein pUL36 of 3,164 amino acid residues that links the capsids to the tegument proteins pUL37 and VP16. Of the 32 tryptophans of HSV-1-pUL36, the tryptophan-acidic motifs (1766)WD(1767) and (1862)WE(1863) are conserved in all HSV-1 and HSV-2 isolates. Here, we characterized the role of these motifs in the HSV life cycle since the rare tryptophans often have unique roles in protein function due to their large hydrophobic surface. The infectivity of the mutants HSV-1(17(+))Lox-pUL36-WD/AA-WE/AA and HSV-1(17(+))Lox-CheVP26-pUL36-WD/AA-WE/AA, in which the capsid has been tagged with the fluorescent protein Cherry, was significantly reduced. Quantitative electron microscopy shows that there were a larger number of cytosolic capsids and fewer enveloped virions compared to their respective parental strains, indicating a severe impairment in secondary capsid envelopment. The capsids of the mutant viruses accumulated in the perinuclear region around the microtubule-organizing center and were not dispersed to the cell periphery but still acquired the inner tegument proteins pUL36 and pUL37. Furthermore, cytoplasmic capsids colocalized with tegument protein VP16 and, to some extent, with tegument protein VP22 but not with the envelope glycoprotein gD. These results indicate that the unique conserved tryptophan-acidic motifs in the central region of pUL36 are required for efficient targeting of progeny capsids to the membranes of secondary capsid envelopment and for efficient virion assembly. IMPORTANCE: Herpesvirus infections give rise to severe animal and human diseases, especially in young, immunocompromised, and elderly individuals. The structural hallmark of herpesvirus virions is the tegument, which contains evolutionarily conserved proteins that are essential for several stages of the herpesvirus life cycle. Here we characterized two conserved tryptophan-acidic motifs in the central region of the large tegument protein pUL36 of herpes simplex virus. When we mutated these motifs, secondary envelopment of cytosolic capsids and the production of infectious particles were severely impaired. Our data suggest that pUL36 and its homologs in other herpesviruses, and in particular such tryptophan-acidic motifs, could provide attractive targets for the development of novel drugs to prevent herpesvirus assembly and spread.
Asunto(s)
Cápside/metabolismo , Herpesvirus Humano 1/fisiología , Triptófano/química , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/metabolismo , Ensamble de Virus , Secuencias de Aminoácidos , Cápside/ultraestructura , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Línea Celular , Citoplasma/virología , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Herpesvirus Humano 1/química , Herpesvirus Humano 1/genética , Humanos , Estadios del Ciclo de Vida , Microscopía Electrónica , Mutación , Unión Proteica , Dominios Proteicos , Triptófano/metabolismo , Proteínas Estructurales Virales/genéticaRESUMEN
BACKGROUND: The sodium-taurocholate cotransporting polypeptide (NTCP) is both a key bile acid (BA) transporter mediating uptake of BA into hepatocytes and an essential receptor for hepatitis B virus (HBV) and hepatitis D virus (HDV). In this study we aimed to characterize to what extent and through what mechanism BA affect HDV cell entry. METHODS: HuH-7 cells stably expressing NTCP (HuH-7/NTCP) and primary human hepatocytes (PHH) were infected with in vitro generated HDV particles. Infectivity in the absence or presence of compounds was assessed using immunofluorescence staining for HDV antigen, standard 50% tissue culture infectious dose (TCID50) assays and quantitative PCR. RESULTS: Addition of primary conjugated and unconjugated BA resulted in a dose dependent reduction in the number of infected cells while secondary, tertiary and synthetic BA had a lesser effect. This effect was observed both in HuH-7/NTCP and in PHH. Other replication cycle steps such as replication and particle assembly and release were unaffected. Moreover, inhibitory BA competed with a fragment from the large HBV envelope protein for binding to NTCP-expressing cells. Conversely, the sodium/BA-cotransporter function of NTCP seemed not to be required for HDV infection since infection was similar in the presence or absence of a sodium gradient across the plasma membrane. When chenodeoxycolic acid (15 mg per kg body weight) was administered to three chronically HDV infected individuals over a period of up to 16 days there was no change in serum HDV RNA. CONCLUSIONS: Primary BA inhibit NTCP-mediated HDV entry into hepatocytes suggesting that modulation of the BA pool may affect HDV infection of hepatocytes.
Asunto(s)
Ácidos y Sales Biliares/farmacología , Carcinoma Hepatocelular/patología , Regulación Neoplásica de la Expresión Génica , Virus de la Hepatitis Delta/fisiología , Neoplasias Hepáticas/patología , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismo , Internalización del Virus/efectos de los fármacos , Adulto , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Ácido Quenodesoxicólico/farmacología , Coinfección/virología , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Femenino , Hepatitis B Crónica/virología , Hepatitis D Crónica/virología , Virus de la Hepatitis Delta/efectos de los fármacos , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virología , Masculino , Persona de Mediana Edad , Sodio/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
UNLABELLED: Herpes simplex virus 1 (HSV-1) is an alphaherpesvirus that has been reported to infect some epithelial cell types by fusion at the plasma membrane but others by endocytosis. To determine the molecular mechanisms of productive HSV-1 cell entry, we perturbed key endocytosis host factors using specific inhibitors, RNA interference (RNAi), or overexpression of dominant negative proteins and investigated their effects on HSV-1 infection in the permissive epithelial cell lines Vero, HeLa, HEp-2, and PtK2. HSV-1 internalization required neither endosomal acidification nor clathrin- or caveolin-mediated endocytosis. In contrast, HSV-1 gene expression and internalization were significantly reduced after treatment with 5-(N-ethyl-N-isopropyl)amiloride (EIPA). EIPA blocks the activity of Na(+)/H(+) exchangers, which are plasma membrane proteins implicated in all forms of macropinocytosis. HSV-1 internalization furthermore required the function of p21-activated kinases that contribute to macropinosome formation. However, in contrast to some forms of macropinocytosis, HSV-1 did not enlist the activities of protein kinase C (PKC), tyrosine kinases, C-terminal binding protein 1, or dynamin to activate its internalization. These data suggest that HSV-1 depends on Na(+)/H(+) exchangers and p21-activated kinases either for macropinocytosis or for local actin rearrangements required for fusion at the plasma membrane or subsequent passage through the actin cortex underneath the plasma membrane. IMPORTANCE: After initial replication in epithelial cells, herpes simplex viruses (HSVs) establish latent infections in neurons innervating these regions. Upon primary infection and reactivation from latency, HSVs cause many human skin and neurological diseases, particularly in immunocompromised hosts, despite the availability of effective antiviral drugs. Many viruses use macropinocytosis for virus internalization, and many host factors mediating this entry route have been identified, although the specific perturbation profiles vary for different host and viral cargo. In addition to an established entry pathway via acidic endosomes, we show here that HSV-1 internalization depended on sodium-proton exchangers at the plasma membrane and p21-activated kinases. These results suggest that HSV-1 requires a reorganization of the cortical actin cytoskeleton, either for productive cell entry via pH-independent fusion from macropinosomes or for fusion at the plasma membrane, and subsequent cytosolic passage to microtubules that mediate capsid transport to the nucleus for genome uncoating and replication.