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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.
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The highly prevalent herpes simplex virus type 1 (HSV-1) causes a range of diseases, including cold sores, blinding keratitis, and life-threatening encephalitis. HSV-1 initially replicates in epithelial cells, enters the peripheral nervous system via neurites, and establishes lifelong infection in the neuronal cell bodies. Neurites are highly dynamic structures that grow or retract in response to attractive or repulsive cues, respectively. Here, we show that infection with HSV-1, but not with a mutant virus lacking glycoprotein G (gG), reduced the repulsive effect of epithelial cells on neurite outgrowth and facilitated HSV-1 invasion of neurons. HSV-1 gG was required and sufficient to induce neurite outgrowth by modifying the protein composition of extracellular vesicles, increasing the amount of neurotrophic and neuroprotective proteins, including galectin-1. Antibodies directed against galectin-1 neutralized the capacity of extracellular vesicles released from HSV-1-infected cells to promote neurite outgrowth. Our study provides new insights into the neurotropism of HSV-1 and identifies a viral protein that modifies the protein composition of extracellular vesicles to stimulate neurite outgrowth and invasion of the nervous system.IMPORTANCEHerpes simplex virus type 1 (HSV-1) must infect neurites (or nerve endings) to establish a chronic infection in neurons. Neurites are highly dynamic structures that retract or grow in the presence of repulsive or attractive proteins. Some of these proteins are released by epithelial cells in extracellular vesicles and act upon interaction with their receptor present on neurites. We show here that HSV-1 infection of epithelial cells modulated their effect on neurites, increasing neurite growth. Mechanistically, HSV-1 glycoprotein G (gG) modifies the protein composition of extracellular vesicles released by epithelial cells, increasing the amount of attractive proteins that enhance neurite outgrowth and facilitate neuronal infection. These results could inform of therapeutic strategies to block HSV-1 induction of neurite outgrowth and, thereby, neuronal infection.
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Enfermedades Transmisibles , Vesículas Extracelulares , Herpes Simple , Herpesvirus Humano 1 , Humanos , Herpesvirus Humano 1/fisiología , Galectina 1/metabolismo , Vesículas Extracelulares/metabolismo , Proyección Neuronal , Glicoproteínas/metabolismoRESUMEN
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.
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Alphaherpesvirinae , Herpes Simple , Humanos , Carioferinas , alfa Carioferinas/genética , Poro Nuclear , Proteínas de la CápsideRESUMEN
The evolutionarily conserved, structural HSV-1 tegument protein pUL36 is essential for both virus entry and assembly. While its N-terminal deubiquitinase (DUB) activity is dispensable for infection in cell culture, it is required for efficient virus spread in vivo, as it acts as a potent viral immune evasin. Interferon (IFN) induces the expression of hundreds of antiviral factors, including many ubiquitin modulators, which HSV-1 needs to neutralize to efficiently initiate a productive infection. Herein, we discover two functions of the conserved pUL36 DUB during lytic replication in cell culture in an understudied but equally important scenario of HSV-1 infection in IFN-treated cells. Our data indicate that the pUL36 DUB contributes to overcoming the IFN-mediated suppression of productive infection in both the early and late phases of HSV-1 infection. We show that incoming tegument-derived pUL36 DUB activity contributes to the IFN resistance of HSV-1 in IFN-primed cells to efficiently initiate lytic virus replication. Subsequently, the de novo expressed DUB augmented the efficiency of virus replication and increased the output of infectious virus. Notably, the DUB defect was only apparent when IFN was applied prior to infection. Our data indicate that IFN-induced defense mechanisms exist and that they work to both neutralize infectivity early on and slow the progression of HSV-1 replication in the late stages of infection. Also, our data indicate that pUL36 DUB activity contributes to the disarming of these host responses. IMPORTANCE HSV-1 is a ubiquitous human pathogen that is responsible for common cold sores and may also cause life-threatening disease. pUL36 is an essential, conserved herpesvirus protein with N-terminal deubiquitinating (DUB) activity. The DUB is dispensable for HSV-1 replication in cell culture but represents an important viral immune evasin in vivo. IFN plays a pivotal role in HSV-1 infection and suppresses viral replication both in vitro and in vivo. Here, we show that DUB activity contributes to overcoming IFN-induced cellular resistance in order to more efficiently initiate lytic replication and produce infectious virions. As such, DUB activity in the incoming virions increases their infectivity, while the de novo synthesized DUB augments productive infection. Thus, the HSV-1 DUB antagonizes the activity of IFN-inducible effector proteins to facilitate productive infection at multiple levels. Our findings underscore the importance of using more challenging cell culture systems to fully understand virus protein functions.
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Enzimas Desubicuitinizantes , Herpes Simple , Herpesvirus Humano 1 , Proteínas Virales , Humanos , Enzimas Desubicuitinizantes/metabolismo , Herpesvirus Humano 1/fisiología , Proteínas Virales/metabolismo , Replicación Viral , InterferonesRESUMEN
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.
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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
Host proteins sense viral products and induce defence mechanisms, particularly in immune cells. Using cell-free assays and quantitative mass spectrometry, we determined the interactome of capsid-host protein complexes of herpes simplex virus and identified the large dynamin-like GTPase myxovirus resistance protein B (MxB) as an interferon-inducible protein interacting with capsids. Electron microscopy analyses showed that cytosols containing MxB had the remarkable capability to disassemble the icosahedral capsids of herpes simplex viruses and varicella zoster virus into flat sheets of connected triangular faces. In contrast, capsids remained intact in cytosols with MxB mutants unable to hydrolyse GTP or to dimerize. Our data suggest that MxB senses herpesviral capsids, mediates their disassembly, and thereby restricts the efficiency of nuclear targeting of incoming capsids and/or the assembly of progeny capsids. The resulting premature release of viral genomes from capsids may enhance the activation of DNA sensors, and thereby amplify the innate immune responses.
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Cápside , Herpesviridae , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , GTP Fosfohidrolasas/metabolismo , Interferones/metabolismo , SimplexvirusRESUMEN
Herpes simplex virus capsids are assembled and packaged in the nucleus and move by diffusion through the nucleoplasm to the nuclear envelope for egress. Analyzing their motion provides conclusions not only on capsid transport but also on the properties of the nuclear environment during infection. We utilized live-cell imaging and single-particle tracking to characterize capsid motion relative to the host chromatin. The data indicate that as the chromatin was marginalized toward the nuclear envelope it presented a restrictive barrier to the capsids. However, later in infection this barrier became more permissive and the probability of capsids to enter the chromatin increased. Thus, although chromatin marginalization initially restricted capsid transport to the nuclear envelope, a structural reorganization of the chromatin counteracted that to promote capsid transport later. Analyses of capsid motion revealed that it was subdiffusive, and that the diffusion coefficients were lower in the chromatin than in regions lacking chromatin. In addition, the diffusion coefficient in both regions increased during infection. Throughout the infection, the capsids were never enriched at the nuclear envelope, which suggests that instead of nuclear export the transport through the chromatin is the rate-limiting step for the nuclear egress of capsids. This provides motivation for further studies by validating the importance of intranuclear transport to the life cycle of HSV-1.
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Transporte Biológico Activo/fisiología , Cápside/metabolismo , Cromatina/metabolismo , Membrana Nuclear/metabolismo , Simplexvirus/metabolismo , Animales , Chlorocebus aethiops , Herpes Simple , Células Vero , Replicación Viral/fisiologíaRESUMEN
Herpesviruses cause severe diseases particularly in immunocompromised patients. Both genome packaging and release from the capsid require a unique portal channel occupying one of the 12 capsid vertices. Here, we report the 2.6 Å crystal structure of the pentameric pORF19 of the γ-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) resembling the portal cap that seals this portal channel. We also present the structure of its ß-herpesviral ortholog, revealing a striking structural similarity to its α- and γ-herpesviral counterparts despite apparent differences in capsid association. We demonstrate pORF19 pentamer formation in solution and provide insights into how pentamerization is triggered in infected cells. Mutagenesis in its lateral interfaces blocked pORF19 pentamerization and severely affected KSHV capsid assembly and production of infectious progeny. Our results pave the way to better understand the role of pORF19 in capsid assembly and identify a potential novel drug target for the treatment of herpesvirus-induced diseases.
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Herpesvirus Humano 8/fisiología , Sistemas de Lectura Abierta/genética , Multimerización de Proteína , Proteínas Virales/metabolismo , Ensamble de Virus/fisiología , Animales , Cápside/química , Secuencia Conservada , Cristalografía por Rayos X , Empaquetamiento del ADN , ADN Viral/genética , Drosophila , Células HEK293 , Herpesvirus Humano 8/ultraestructura , Humanos , Modelos Moleculares , Mutagénesis/genética , Proteínas Mutantes/metabolismo , Proteínas Virales/químicaRESUMEN
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.
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Proteínas de la Cápside , Cápside , Proteínas de la Cápside/genética , Núcleo Celular , Citosol , SimplexvirusRESUMEN
BACKGROUND: The high susceptibility of AD patients to microbial skin infections has been attributed to a deficient antimicrobial peptide (AMP) expression, which is contradicted by a growing amount of recent studies clearly demonstrating that AMP expression is not impaired in lesional skin of AD patients. The reasons for the high susceptibility of AD patients to microbial infections are still unknown. METHODS: The influence of self-DNA on the antimicrobial activity of RNase 7, LL-37, and hBD2 has been investigated using antibacterial and antiviral assays. The amount of self-DNA on skin has been analyzed by skin rinsings and subsequent quantification using dsDNA assays. DNA source was identified by qPCR. RESULTS: Complex formation of the AMPs with self-DNA significantly impaired their antibacterial activity against Staphylococcus aureus and their antiviral activity against HSV-1. The inhibition of the antibacterial activity was dependent on the DNA concentration but not on the length of the DNA molecules. Of note, we detected significant higher amounts of cell-free self-DNA in skin rinses taken from lesional AD skin compared to skin rinses from non-lesional skin and from normal skin of healthy donors. Consequently, rinse solution from AD lesional skin prevented antibacterial activity of LL-37. CONCLUSION: Our study indicates that extracellular self-DNA is released in considerable amounts in AD skin lesions and AMP-self-DNA-complex formation leads to a significant loss of antibacterial and antiviral activity in atopic dermatitis. Studies on strategies to reduce the amount of extracellular DNA in AD are needed to identify possible methods relevant in clinical settings.
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Dermatitis Atópica , Péptidos Catiónicos Antimicrobianos , ADN , Dermatitis Atópica/tratamiento farmacológico , Humanos , Proteínas Citotóxicas Formadoras de Poros , PielRESUMEN
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.
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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
Although infection with the human enteropathogen Giardia lamblia causes self-limited diarrhea in adults, infant populations in endemic areas experience persistent pathogen carriage in the absence of diarrhea. The persistence of this protozoan parasite in infants has been associated with reduced weight gain and linear growth (height-for-age). The mechanisms that support persistent infection and determine the different disease outcomes in the infant host are incompletely understood. Using a neonatal mouse model of persistent G. lamblia infection, we demonstrate that G. lamblia induced bile secretion and used the bile constituent phosphatidylcholine as a substrate for parasite growth. In addition, we show that G. lamblia infection altered the enteric microbiota composition, leading to enhanced bile acid deconjugation and increased expression of fibroblast growth factor 15. This resulted in elevated energy expenditure and dysregulated lipid metabolism with reduced adipose tissue, body weight gain, and growth in the infected mice. Our results indicate that this enteropathogen's modulation of bile acid metabolism and lipid metabolism in the neonatal mouse host led to an altered body composition, suggesting how G. lamblia infection could contribute to growth restriction in infants in endemic areas.
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Microbioma Gastrointestinal , Giardiasis , Animales , Bilis , Giardia , Homeostasis , RatonesRESUMEN
Infections of the brain with herpes simplex virus type 1 (HSV-1) cause life-threatening Herpes simplex encephalitis (HSE) characterized by viral replication in neurons and neuro-inflammation including an infiltration of peripheral immune cells. HSV-1 reprograms host cells to foster its own replication and for immune evasion, but eventually the immune responses clear the infection in most patients. However, many survivors suffer from long-term neuronal damage and cannot regenerate all brain functions. HSV-1 influences the physiology of neurons, astrocytes, oligodendrocytes and microglia, and significantly changes their protein expression and secretion pattern. To characterize temporal changes upon HSV-1 infection in detail, we inoculated mixed primary cultures of the murine brain cortex, and performed quantitative mass spectrometry analyses of the cell-associated proteome and the secretome. We identified 28 differentially regulated host proteins influencing inflammasome formation and intracellular vesicle trafficking during endocytosis and secretion. The NIMA-related kinase 7 (NEK7), a critical component of the inflammasome, and ArfGap1, a regulator of endocytosis, were significantly up-regulated upon HSV-1 infection. In the secretome, we identified 71 proteins including guidance cues regulating axonal regeneration, such as semaphorin6D, which were enriched in the conditioned media of HSV-1 infected cells. Modulation of inflammasome activity and intracellular membrane traffic are critical for HSV-1 cell entry, virus assembly, and intracellular spread. Our proteome analysis provides first clues on host factors that might dampen the inflammasome response and modulate intracellular vesicle transport to promote HSV infection of the brain. Furthermore, our secretome analysis revealed a set of proteins involved in neuroregeneration that might foster neuronal repair processes to restore brain functions after clearance of an HSV-1 infection.
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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.
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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.
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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
Herpes simplex virus (HSV) is the main cause of viral encephalitis in the Western world, and the type I interferon (IFN) system is important for antiviral control in the brain. Here, we have compared Ifnb induction in mixed murine brain cell cultures by a panel of HSV1 mutants, each devoid of one mechanism to counteract the IFN-stimulating cGAS-STING pathway. We found that a mutant lacking the deubiquitinase (DUB) activity of the VP1-2 protein induced particularly strong expression of Ifnb and IFN-stimulated genes. HSV1 ΔDUB also induced elevated IFN expression in murine and human microglia and exhibited reduced viral replication in the brain. This was associated with increased ubiquitination of STING and elevated phosphorylation of STING, TBK1, and IRF3. VP1-2 associated directly with STING, leading to its deubiquitination. Recruitment of VP1-2 to STING was dependent on K150 of STING, which was ubiquitinated by TRIM32. Thus, the DUB activity of HSV1 VP1-2 is a major viral immune-evasion mechanism in the brain.
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Encéfalo/virología , Enzimas Desubicuitinizantes/metabolismo , Herpesvirus Humano 1/metabolismo , Interferón Tipo I/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Virales/metabolismo , Animales , Encéfalo/patología , Células Cultivadas , Citoplasma/metabolismo , ADN Viral/metabolismo , Células HEK293 , Humanos , Lisina/metabolismo , Ratones Endogámicos C57BL , Microglía/metabolismo , Mutación/genética , Nucleotidiltransferasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Ubiquitina/metabolismo , Ubiquitinación , Replicación Viral/fisiologíaRESUMEN
Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1-/- mice results in replication of HSV-1 and Asah1-/- mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol.
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Ceramidasa Ácida/metabolismo , Herpes Simple/enzimología , Herpes Simple/prevención & control , Herpesvirus Humano 1/fisiología , Macrófagos/enzimología , Cuerpos Multivesiculares/virología , Ceramidasa Ácida/genética , Animales , Femenino , Herpes Simple/virología , Humanos , Macrófagos/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Replicación ViralRESUMEN
RNase 7 is one of the major antimicrobial peptides (AMPs) secreted by keratinocytes. The AMPs human beta defensin 2 and LL-37 promote the toll-like receptor 9-mediated activation of human plasmacytoid dendritic cells (pDCs) by human self-DNA; however, whether keratinocytes respond in a similar way has not yet been addressed. Keratinocytes express several receptors for the detection of cytosolic DNA. Here, we investigated the activation of keratinocytes by RNase 7 in combination with human DNA. The stimulation of keratinocytes with RNase 7 and human DNA induced a strong increase in the production of IP-10. Of note, the stimulation of keratinocytes with human beta defensin 2 and LL-37 in combination with DNA failed to induce the production of IP-10. The production of IP-10 was mediated by the induction of the type I interferon IFN-ß and was significantly downregulated by blocking of the interferon-α/ß receptor and inhibition of stimulator of IFN genes. In addition, the pretreatment of keratinocytes with RNase 7 and DNA significantly reduced the herpes simplex virus-1 infection of human keratinocytes. This study demonstrates that RNase 7 functions as an alarmin by converting self-DNA into a danger signal that directly activates an antiviral immune response in human keratinocytes without the involvement of plasmacytoid dendritic cells.
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ADN/metabolismo , Herpes Simple/inmunología , Inmunidad Innata , Queratinocitos/inmunología , Ribonucleasas/metabolismo , Alarminas/metabolismo , Células Cultivadas , Quimiocina CXCL10/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/inmunología , Interacciones Microbiota-Huesped/inmunología , Humanos , Queratinocitos/metabolismo , Cultivo Primario de CélulasRESUMEN
BACKGROUND: Herpes simplex virus-1 (HSV-1) infections of the central nervous system (CNS) can result in HSV-1 encephalitis (HSE) which is characterized by severe brain damage and long-term disabilities. Different cell types including neurons and astrocytes become infected in the course of an HSE which leads to an activation of glial cells. Activated glial cells change their neurotrophic factor profile and modulate inflammation and repair. The superfamily of fibroblast growth factors (FGFs) is one of the largest family of neurotrophic factors comprising 22 ligands. FGFs induce pro-survival signaling in neurons and an anti-inflammatory answer in glial cells thereby providing a coordinated tissue response which favors repair over inflammation. Here, we hypothesize that FGF expression is altered in HSV-1-infected CNS cells. METHOD: We employed primary murine cortical cultures comprising a mixed cell population of astrocytes, neurons, microglia, and oligodendrocytes. Astrocyte reactivity was morphometrically monitored by an automated image analysis algorithm as well as by analyses of A1/A2 marker expression. Altered FGF expression was detected by quantitative real-time PCR and its paracrine FGF activity. In addition, HSV-1 mutants were employed to characterize viral factors important for FGF responses of infected host cells. RESULTS: Astrocytes in HSV-1-infected cortical cultures were transiently activated and became hypertrophic and expressed both A1- and A2-markers. Consistently, a number of FGFs were transiently upregulated inducing paracrine neurotrophic signaling in neighboring cells. Most prominently, FGF-4, FGF-8, FGF-9, and FGF-15 became upregulated in a switch-on like mechanism. This effect was specific for CNS cells and for a fully functional HSV-1. Moreover, the viral protein ICP0 critically mediated the FGF switch-on mechanism. CONCLUSIONS: HSV-1 uses the viral protein ICP0 for the induction of FGF-expression in CNS cells. Thus, we propose that HSV-1 triggers FGF activity in the CNS for a modulation of tissue response upon infection.
Asunto(s)
Corteza Cerebral/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Herpesvirus Humano 1/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Comunicación Paracrina/fisiología , Proteínas Virales/metabolismo , Animales , Animales Recién Nacidos , Corteza Cerebral/virología , Chlorocebus aethiops , Técnicas de Cocultivo , Cricetinae , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Células VeroRESUMEN
During lytic herpes simplex virus 1 (HSV-1) infection, the expansion of the viral replication compartments leads to an enrichment of the host chromatin in the peripheral nucleoplasm. We have shown previously that HSV-1 infection induces the formation of channels through the compacted peripheral chromatin. Here, we used three-dimensional confocal and expansion microscopy, soft X-ray tomography, electron microscopy, and random walk simulations to analyze the kinetics of host chromatin redistribution and capsid localization relative to their egress site at the nuclear envelope. Our data demonstrated a gradual increase in chromatin marginalization, and the kinetics of chromatin smoothening around the viral replication compartments correlated with their expansion. We also observed a gradual transfer of capsids to the nuclear envelope. Later in the infection, random walk modeling indicated a gradually faster transport of capsids to the nuclear envelope that correlated with an increase in the interchromatin channels in the nuclear periphery. Our study reveals a stepwise and time-dependent mechanism of herpesvirus nuclear egress, in which progeny viral capsids approach the egress sites at the nuclear envelope via interchromatin spaces.