RESUMO
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.
Assuntos
Herpes Genital/metabolismo , Herpesvirus Humano 2/metabolismo , Fator de Crescimento Neural/metabolismo , Neuritos , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Herpesvirus Humano 2/patogenicidade , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neuritos/metabolismo , Neuritos/virologia , Células VeroRESUMO
Herpes Simplex Virus type 2 (HSV-2) is a neurotropic human pathogen. Upon de novo infection, the viral infected cell protein 0 (ICP0) is immediately expressed and interacts with various cellular components during the viral replication cycle. ICP0 is a multifunctional regulatory protein that has been shown to be important for both efficient viral replication and virus reactivation from latency. In particular, as previously demonstrated in transfected tissue culture models, ICP0 interacts with the cellular E3 ubiquitin ligase SIAH-1, which targets ICP0 for proteasomal degradation. However, the consequence of this virus-host interaction during the establishment of HSV-2 infection in vivo has not yet been elucidated. Here we confirmed that ICP0 of HSV-2 interacts with SIAH-1 via two conserved PxAxVxP amino acid binding motifs. We also demonstrate in vitro that a SIAH-1 binding-deficient HSV-2 strain, constructed by homologous recombination technology, exhibits an attenuated growth curve and impaired DNA and protein synthesis. This attenuated phenotype was also confirmed in an in vivo ocular infection mouse model. Specifically, viral load of the SIAH-1 binding-deficient HSV-2 mutant was significantly reduced in the trigeminal ganglia and brain stem at day 5 and 7 post infection. Our findings indicate that the interplay between ICP0 and SIAH-1 is important for efficient HSV-2 replication in vivo, thereby affecting viral dissemination kinetics in newly infected organisms, and possibly revealing novel targets for antiviral therapy.
Assuntos
Herpesvirus Humano 2/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Animais , Sítios de Ligação/genética , Tronco Encefálico/metabolismo , Tronco Encefálico/virologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Modelos Animais de Doenças , Olho/metabolismo , Olho/virologia , Infecções Oculares Virais/genética , Infecções Oculares Virais/metabolismo , Feminino , Herpes Simples/genética , Herpes Simples/metabolismo , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas Imediatamente Precoces/genética , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Gânglio Trigeminal/metabolismo , Gânglio Trigeminal/virologia , Ubiquitina-Proteína Ligases/genética , Proteínas Virais/genética , Replicação Viral/genéticaRESUMO
Therapy or prophylaxis of herpes simplex virus type 2 (HSV-2) infections with the nucleoside analog aciclovir (ACV) can lead to the emergence of drug-resistant HSV-2 strains, particularly in immunocompromised patients. In this context, multiple amino acid (aa) changes can accumulate in the ACV-converting viral thymidine kinase (TK) which hampers sequence-based diagnostics significantly. In this study, the so far unknown or still doubted relevance of several individual aa changes for drug resistance in HSV-2 was clarified. For this purpose, ten recombinant fluorescent HSV-2 strains differing in the respective aa within their TK were constructed using the bacterial artificial chromosome (BAC) pHSV2(MS)Lox. Similar TK expression levels and similar replication behavior patterns were demonstrated for the mutants as compared to the unmodified BAC-derived HSV-2 strain. Subsequently, the resulting strains were tested for their susceptibility to ACV as well as penciclovir (PCV) in parallel to a modified cytopathic effect (CPE) inhibition assay and by determining the relative fluorescence intensity (quantified using units, RFU) as a measure for the viral replication capacity. While aa changes Y53N and R221H conferred ACV resistance with cross-resistance to PCV, the aa changes G25A, G39E, T131M, Y133F, G150D, A157T, R248W, and L342W maintained a susceptible phenotype against both antivirals. The CPE inhibition assay and the measurement of relative fluorescence intensity yielded comparable results for the phenotypic testing of recombinant viruses. The latter test showed some technical advantages. In conclusion, the significance of single aa changes in HSV-2 TK on ACV/PCV resistance was clarified by the construction and phenotypic testing of recombinant viral strains. This was facilitated by the fluorescence based method.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Herpes Simples/virologia , Herpesvirus Humano 2/efeitos dos fármacos , Herpesvirus Humano 2/enzimologia , Timidina Quinase/genética , Proteínas Virais/genética , Aciclovir/análogos & derivados , Aciclovir/farmacologia , Guanina , Herpesvirus Humano 2/genética , Humanos , Mutação , Timidina Quinase/metabolismo , Proteínas Virais/metabolismoRESUMO
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.
Assuntos
Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 1/patogenicidade , Neurônios/virologia , Animais , Transporte Axonal , Axônios/ultraestrutura , Axônios/virologia , Capsídeo/fisiologia , Capsídeo/ultraestrutura , Células Cultivadas , Chlorocebus aethiops , Gânglios Espinais/virologia , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Movimento/fisiologia , Mutação , Neurônios/ultraestrutura , Células Vero , Proteínas Virais/genética , Proteínas Virais/fisiologia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/fisiologiaRESUMO
BACKGROUND: Chronic infections with herpes simplex virus (HSV) type 1 are highly prevalent in populations worldwide and cause recurrent oral lesions in up to 40% of infected subjects. OBJECTIVE: We investigated the antiviral activity of a defined Spirulina platensis microalga extract and of purified calcium spirulan (Ca-SP), a sulfated polysaccharide contained therein. METHODS: The inhibitory effects of HSV-1 were assessed by using a plaque reduction assay and quantitative PCR in a susceptible mammalian epithelial cell line and confirmed in human keratinocytes. Time-of-addition and attachment experiments and fluorescence detection of the HSV-1 tegument protein VP16 were used to analyze the mechanism of HSV-1 inhibition. Effects of Ca-SP on Kaposi sarcoma-associated herpesvirus/human herpes virus 8 replication and uptake of the ORF45 tegument protein were tested in human retinal pigment epithelial cells. In an observational trial the prophylactic effects of topically applied Ca-SP were compared with those of systemic and topical nucleoside analogues in 198 volunteers with recurrent herpes labialis receiving permanent lip makeup. RESULTS: Ca-SP inhibited HSV-1 infection in vitro with a potency at least comparable to that of acyclovir by blocking viral attachment and penetration into host cells. Ca-SP also inhibited entry of Kaposi sarcoma-associated herpesvirus/human herpes virus 8. In the clinical model of herpes exacerbation, the prophylactic effect of a Ca-SP and microalgae extract containing cream was superior to that of acyclovir cream. CONCLUSION: These data indicate a potential clinical use of Ca-SP containing Spirulina species extract for the prophylactic treatment of herpes labialis and suggest possible activity of Ca-SP against infections caused by other herpesviruses.
Assuntos
Antivirais/farmacologia , Antivirais/uso terapêutico , Herpes Labial/prevenção & controle , Polissacarídeos/farmacologia , Polissacarídeos/uso terapêutico , Spirulina , Adulto , Idoso , Animais , Linhagem Celular , Chlorocebus aethiops , Cosméticos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Feminino , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 8/efeitos dos fármacos , Herpesvirus Humano 8/patogenicidade , Herpesvirus Humano 8/fisiologia , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/virologia , Pessoa de Meia-Idade , Células Vero , Ligação Viral/efeitos dos fármacos , Adulto JovemRESUMO
Hypusine modification of the eukaryotic initiation factor 5A (eIF-5A) is emerging as a crucial regulator in cancer, infections, and inflammation. Although its contribution in translational regulation of proline repeat-rich proteins has been sufficiently demonstrated, its biological role in higher eukaryotes remains poorly understood. To establish the hypusine modification system as a novel platform for therapeutic strategies, we aimed to investigate its functional relevance in mammals by generating and using a range of new knock-out mouse models for the hypusine-modifying enzymes deoxyhypusine synthase and deoxyhypusine hydroxylase as well as for the cancer-related isoform eIF-5A2. We discovered that homozygous depletion of deoxyhypusine synthase and/or deoxyhypusine hydroxylase causes lethality in adult mice with different penetrance compared with haploinsufficiency. Network-based bioinformatic analysis of proline repeat-rich proteins, which are putative eIF-5A targets, revealed that these proteins are organized in highly connected protein-protein interaction networks. Hypusine-dependent translational control of essential proteins (hubs) and protein complexes inside these networks might explain the lethal phenotype observed after deletion of hypusine-modifying enzymes. Remarkably, our results also demonstrate that the cancer-associated isoform eIF-5A2 is dispensable for normal development and viability. Together, our results provide the first genetic evidence that the hypusine modification in eIF-5A is crucial for homeostasis in mammals. Moreover, these findings highlight functional diversity of the hypusine system compared with lower eukaryotes and indicate eIF-5A2 as a valuable and safe target for therapeutic intervention in cancer.
Assuntos
Lisina/análogos & derivados , Oxigenases de Função Mista/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Fatores de Iniciação de Peptídeos/metabolismo , Animais , Homeostase/genética , Humanos , Lisina/genética , Lisina/metabolismo , Camundongos , Camundongos Knockout , Oxigenases de Função Mista/metabolismo , Neoplasias/genética , Neoplasias/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Fatores de Iniciação de Peptídeos/genética , Biossíntese de Proteínas , Mapas de Interação de Proteínas , Processamento de Proteína Pós-TraducionalRESUMO
Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral mutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17+)Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary envelopment.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Herpesvirus Humano 1/fisiologia , Proteínas do Nucleocapsídeo/metabolismo , Montagem de Vírus/fisiologia , Animais , Chlorocebus aethiops , Células HeLa , Humanos , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Células VeroRESUMO
Bacterial artificial chromosomes (BACs) are suitable vectors not only to maintain the large genomes of herpesviruses in Escherichia coli but also to enable the traceless introduction of any mutation using modern tools of bacterial genetics. To clone a herpes simplex virus genome, a BAC replication origin is first introduced into the viral genome by homologous recombination in eukaryotic host cells. As part of their nuclear replication cycle, genomes of herpesviruses circularize and these replication intermediates are then used to transform bacteria. After cloning, the integrity of the recombinant viral genomes is confirmed by restriction length polymorphism analysis and sequencing. The BACs may then be used to design virus mutants. Upon transfection into eukaryotic cells new herpesvirus strains harboring the desired mutations can be recovered and used for experiments in cultured cells as well as in animal infection models.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Genoma Viral , Biologia Molecular/métodos , Simplexvirus/genética , Células Cultivadas , Clonagem Molecular , Replicação do DNA , Escherichia coli/genética , Células Eucarióticas , HumanosRESUMO
Herpes simplex virus type 1 (HSV-1) has properties that can be exploited for the development of gene therapy vectors. The neurotropism of HSV enables delivery of therapeutic genes to the nervous system. Using a bacterial artificial chromosome (BAC), we constructed an HSV-1(17(+))-based replicative vector deleted of the neurovirulence gene γ134.5, and expressing leukemia inhibitory factor (LIF) as a transgene for treatment of experimental autoimmune encephalomyelitis (EAE). EAE is an inducible T-cell mediated autoimmune disease of the central nervous system (CNS) and is used as an animal model for multiple sclerosis. Demyelination and inflammation are hallmarks of both diseases. LIF is a cytokine that has the potential to limit demyelination and oligodendrocyte loss in CNS autoimmune diseases and to affect the T-cell mediated autoimmune response. In this study SJL/J mice, induced for EAE, were treated with a HSV-LIF vector intracranially and the subsequent changes in disease parameters and immune responses during the acute disease were investigated. Replicating HSV-LIF and its DNA were detected in the CNS during the acute infection, and the vector spread to the spinal cord but was non-virulent. The HSV-LIF significantly ameliorated the EAE and contributed to a higher number of oligodendrocytes in the brains when compared to untreated mice. The HSV-LIF therapy also induced favorable changes in the expression of immunoregulatory cytokines and T-cell population markers in the CNS during the acute disease. These data suggest that BAC-derived HSV vectors are suitable for gene therapy of CNS disease and can be used to test the therapeutic potential of immunomodulatory factors for treatment of EAE.
Assuntos
Encefalomielite Autoimune Experimental/terapia , Fatores Imunológicos/biossíntese , Fator Inibidor de Leucemia/biossíntese , Simplexvirus/genética , Animais , Autoimunidade , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/virologia , Chlorocebus aethiops , Citocinas/genética , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Feminino , Terapia Genética , Vetores Genéticos , Fatores Imunológicos/genética , Imunomodulação , Fator Inibidor de Leucemia/genética , Camundongos , Bainha de Mielina/patologia , Oligodendroglia/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/virologia , Células Vero , Replicação ViralRESUMO
To analyze the subcellular trafficking of herpesvirus capsids, the small capsid protein has been labeled with different fluorescent proteins. Here, we analyzed the infectivity of several HSV1(17(+)) strains in which the N-terminal region of the non-essential small capsid protein VP26 had been tagged at different positions. While some variants replicated with similar kinetics as their parental wild type strain, others were not infectious at all. Improper tagging resulted in the aggregation of VP26 in the nucleus, prevented efficient nuclear egress of viral capsids, and thus virion formation. Correlative fluorescence and electron microscopy showed that these aggregates had sequestered several other viral proteins, but often did not contain viral capsids. The propensity for aggregate formation was influenced by the type of the fluorescent protein domain, the position of the inserted tag, the cell type, and the progression of infection. Among the tags that we have tested, mRFPVP26 had the lowest tendency to induce nuclear aggregates, and showed the least reduction in replication when compared to wild type. Our data suggest that bona fide monomeric fluorescent protein tags have less impact on proper assembly of HSV1 capsids and nuclear capsid egress than tags that tend to dimerize. Small chemical compounds capable of inducing aggregate formation of VP26 may lead to new antiviral drugs against HSV infections.
Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Núcleo Celular/virologia , Proteínas Recombinantes de Fusão/metabolismo , Simplexvirus/fisiologia , Liberação de Vírus/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Capsídeo/ultraestrutura , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Humanos , Corpos de Inclusão Intranuclear/metabolismo , Corpos de Inclusão Intranuclear/ultraestrutura , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Hypusine modification of eukaryotic initiation factor 5A (eIF-5A) represents a unique and highly specific post-translational modification with regulatory functions in cancer, diabetes, and infectious diseases. However, the specific cellular pathways that are influenced by the hypusine modification remain largely unknown. To globally characterize eIF-5A and hypusine-dependent pathways, we used an approach that combines large-scale bioreactor cell culture with tandem affinity purification and mass spectrometry: "bioreactor-TAP-MS/MS." By applying this approach systematically to all four components of the hypusine modification system (eIF-5A1, eIF-5A2, DHS, and DOHH), we identified 248 interacting proteins as components of the cellular hypusine network, with diverse functions including regulation of translation, mRNA processing, DNA replication, and cell cycle regulation. Network analysis of this data set enabled us to provide a comprehensive overview of the protein-protein interaction landscape of the hypusine modification system. In addition, we validated the interaction of eIF-5A with some of the newly identified associated proteins in more detail. Our analysis has revealed numerous novel interactions, and thus provides a valuable resource for understanding how this crucial homeostatic signaling pathway affects different cellular functions.
Assuntos
Lisina/análogos & derivados , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Animais , Biologia Computacional , Proteínas de Ligação a DNA/metabolismo , Humanos , Lisina/metabolismo , Espectrometria de Massas , Camundongos , Oxigenases de Função Mista/metabolismo , Corpos Multivesiculares/metabolismo , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Nucleofosmina , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Fragmentos de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Transporte Proteico , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Proteínas Ribossômicas/metabolismo , Frações Subcelulares/metabolismo , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Many enveloped viruses, including herpes viruses, hepatitis B virus (HBV), and hepatitis C virus (HCV), and human immunodeficiency virus (HIV), are among the most important human pathogens and are often responsible for coinfections involving ≥2 types of viruses. However, therapies that are effective against multiple virus classes are rare. Here we present a new class of synthetic anti-lipopolysaccharide peptides (SALPs) that bind to heparan sulfate moieties on the cell surface and inhibit infection with a variety of enveloped viruses. We demonstrate that SALPs inhibit entry of human immunodeficiency virus type 1 (HIV-1), herpes simplex virus (HSV) 1 and 2, HBV, and HCV to their respective host cells. Despite their high antiviral efficiency, SALPs were well tolerated, and neither toxicity nor measurable inhibitor-induced adverse effects were observed. Since these broad-spectrum antiviral peptides target a host cell rather than a viral component, they may also be useful for suppression of viruses that are resistant to antiviral drugs.
Assuntos
Antivirais/farmacologia , Peptídeos/farmacologia , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Vírus/efeitos dos fármacos , Antivirais/toxicidade , Linhagem Celular , Sobrevivência Celular , Heparitina Sulfato/metabolismo , Humanos , Lipopolissacarídeos/metabolismo , Peptídeos/toxicidade , Ligação ProteicaRESUMO
The largest tegument protein of herpes simplex virus type 1 (HSV1), pUL36, is a multivalent cross-linker between the viral capsids and the tegument and associated membrane proteins during assembly that upon subsequent cell entry releases the incoming capsids from the outer tegument and viral envelope. Here we show that pUL36 was recruited to cytosolic progeny capsids that later colocalized with membrane proteins of herpes simplex virus type 1 (HSV1) and the trans-Golgi network. During cell entry, pUL36 dissociated from viral membrane proteins but remained associated with cytosolic capsids until arrival at the nucleus. HSV1 UL36 mutants lacking C-terminal portions of increasing size expressed truncated pUL36 but could not form plaques. Cytosolic capsids of mutants lacking the C-terminal 735 of the 3,164 amino acid residues accumulated in the cytosol but did not recruit pUL36 or associate with membranes. In contrast, pUL36 lacking only the 167 C-terminal residues bound to cytosolic capsids and subsequently colocalized with viral and host membrane proteins. Progeny virions fused with neighboring cells, but incoming capsids did not retain pUL36, nor could they target the nucleus or initiate HSV1 gene expression. Our data suggest that residues 2430 to 2893 of HSV1 pUL36, containing one binding site for the capsid protein pUL25, are sufficient to recruit pUL36 onto cytosolic capsids during assembly for secondary envelopment, whereas the 167 residues of the very C terminus with the second pUL25 binding site are crucial to maintain pUL36 on incoming capsids during cell entry. Capsids lacking pUL36 are targeted neither to membranes for virus assembly nor to nuclear pores for genome uncoating.
Assuntos
Proteínas do Capsídeo/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Montagem de Vírus , Internalização do Vírus , Motivos de Aminoácidos , Animais , Sítios de Ligação , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Linhagem Celular , Herpesvirus Humano 1/química , Herpesvirus Humano 1/genética , Humanos , Ligação Proteica , Proteínas Virais/genética , Rede trans-Golgi/virologiaRESUMO
Herpes simplex virus (HSV) immediate-early protein ICP0 is a transcriptional activator with E3 ubiquitin ligase activity that induces the degradation of ND10 proteins, including the promyelocytic leukemia protein (PML) and Sp100. Moreover, ICP0 has a role in the derepression of viral genomes and in the modulation of the host interferon response to virus infection. Here, we report that ICP0 interacts with SIAH-1, a cellular E3 ubiquitin ligase that is involved in multiple cellular pathways and is itself capable of mediating PML degradation. This novel virus-host interaction profoundly stabilized SIAH-1 and recruited this cellular E3 ligase into ICP0-containing nuclear bodies. Moreover, SIAH-1 mediated the polyubiquitination of HSV ICP0 in vitro and in vivo. After infection of SIAH-1 knockdown cells with HSV, higher levels of ICP0 were produced, ICP0 was less ubiquitinated, and the half-life of this multifunctional viral regulatory protein was increased. These results indicate an inhibitory role of SIAH-1 during lytic infection by targeting ICP0 for proteasomal degradation.
Assuntos
Proteínas Imediatamente Precoces/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Bases , Linhagem Celular , Primers do DNA , Interações Hospedeiro-Patógeno , Humanos , Hidrólise , Reação em Cadeia da Polimerase , Ligação Proteica , UbiquitinaçãoRESUMO
Thirteen different glycoproteins are incorporated into mature herpes simplex virus type 1 (HSV-1) virions. Five of them play important roles during entry, while others intervene during egress of the virus. Although HSV-1 gM is not essential in cell culture, its deletion reduces viral yields and promotes syncytium formation. Furthermore, gM is conserved among herpesviruses, is essential for several of them, and can redirect the gD and gH/gL viral glycoproteins from the cell surface to the trans-Golgi network, where gM presumably modulates final capsid envelopment. Late in infection, gM reaches the nuclear envelope and decorates perinuclear virions. This process seemingly requires U(L)31 and U(L)34 and occurs when several markers of the trans-Golgi network have relocalized to the nucleus. However, the precise mechanism of gM nuclear targeting is unclear. We now report that gM is quickly and specifically targeted to nuclear membranes in a virus-dependent manner. This occurs prior to the HSV-1-induced reorganization of the trans-Golgi network and before gM enters the secretory pathway. The presence of a high-mannose glycosylation pattern on gM further corroborated these findings. While gM was targeted to the inner nuclear membrane early in infection, its partners gD, gH, gN, VP22, U(L)31, and U(L)34 did not colocalize with gM. These data suggest that nuclear gM fulfills an early nuclear function that is independent of its known interaction partners and its function in viral egress.
Assuntos
Herpesvirus Humano 1/metabolismo , Glicoproteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Sequência de Bases , Núcleo Celular/metabolismo , Células Cultivadas , Retículo Endoplasmático/metabolismo , Humanos , Dados de Sequência Molecular , Rede trans-Golgi/metabolismoRESUMO
Dendritic cells (DC), which can be subdivided into different phenotypic and functional subsets, play a pivotal role in the generation of cytotoxic T cell immunity against viral infections. Understanding the modes of Ag acquisition, processing and presentation by DC is essential for the design of effective antiviral vaccines. We aimed to assess the contribution of direct vs cross-presentation for the induction of HSV1-specific CD8(+) T lymphocyte responses in mice. Using HSV1 strains expressing fluorescence proteins, we provide evidence for the ability of HSV1 to induce viral transcription. Using HSV1-wild-type as well as gB- or gH-deficient mutants to either directly inoculate DC or to infect target cells, which then were given to DC, we show that DC acquired viral Ag via phagocytosis of target cells and via direct inoculation of virus being released from target cells. Our study corroborates the function of the CD8(+) DC specialized in Ag cross-presentation and confirms this specific feature for Ags that these DC acquire directly from HSV1. However, although infection of cross-presenting DC amplified T cell responses, it was not a requirement for presentation of viral Ags, both in vitro and in vivo. Finally, we provide evidence that direct presentation did not contribute to the Ag presentation capacity of CD8(+) DC after phagocytosis of infected target cells. We conclude that cross-presentation is of major importance for the induction of CTL immunity in mice.
Assuntos
Apresentação de Antígeno/imunologia , Apresentação Cruzada/imunologia , Testes Imunológicos de Citotoxicidade , Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Antígenos CD8/imunologia , Antígenos CD8/metabolismo , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Herpes Simples/prevenção & controle , Herpes Simples/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fagocitose/imunologia , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/virologiaRESUMO
To analyze the assembly of herpes simplex virus type 1 (HSV1) by triple-label fluorescence microscopy, we generated a bacterial artificial chromosome (BAC) and inserted eukaryotic Cre recombinase, as well as beta-galactosidase expression cassettes. When the BAC pHSV1(17(+))blueLox was transfected back into eukaryotic cells, the Cre recombinase excised the BAC sequences, which had been flanked with loxP sites, from the viral genome, leading to HSV1(17(+))blueLox. We then tagged the capsid protein VP26 and the envelope protein glycoprotein D (gD) with fluorescent protein domains to obtain HSV1(17(+))blueLox-GFPVP26-gDRFP and -RFPVP26-gDGFP. All HSV1 BACs had variations in the a-sequences and lost the oriL but were fully infectious. The tagged proteins behaved as their corresponding wild type, and were incorporated into virions. Fluorescent gD first accumulated in cytoplasmic membranes but was later also detected in the endoplasmic reticulum and the plasma membrane. Initially, cytoplasmic capsids did not colocalize with viral glycoproteins, indicating that they were naked, cytosolic capsids. As the infection progressed, they were enveloped and colocalized with the viral membrane proteins. We then analyzed the subcellular distribution of capsids, envelope proteins, and nuclear pores during a synchronous infection. Although the nuclear pore network had changed in ca. 20% of the cells, an HSV1-induced reorganization of the nuclear pore architecture was not required for efficient nuclear egress of capsids. Our data are consistent with an HSV1 assembly model involving primary envelopment of nuclear capsids at the inner nuclear membrane and primary fusion to transfer capsids into the cytosol, followed by their secondary envelopment on cytoplasmic membranes.
Assuntos
Capsídeo/metabolismo , Núcleo Celular/metabolismo , Herpesvirus Humano 1/metabolismo , Animais , Southern Blotting , Linhagem Celular , Núcleo Celular/virologia , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos , Clonagem Molecular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Fluorescência , Herpesvirus Humano 1/crescimento & desenvolvimento , Transporte ProteicoRESUMO
After viral fusion, capsids of the neurotropic herpes simplex virus are transported along microtubules (MT) to the nuclear pores for viral genome uncoating, nuclear transcription and replication. After assembly and egress from the nucleus, cytosolic capsids are transported to host membranes for secondary envelopment or to the axon terminal for further viral spread. Using GFP-tagged capsids, Cy3-labelled MT and cytosol, we have reconstituted viral capsid transport in vitro. In the presence of ATP, capsids moved along MT up to 30 microm. Blocking the function of dynactin, a cofactor of dynein and kinesin-2, inhibited the transport. Removing outer tegument proteins from the capsids increased in vitro motility. In contrast, capsids isolated from infected nuclei that were devoid of inner as well as outer tegument proteins showed little interaction with dynein and its cofactor dynactin. Our data suggest that the inner tegument of alphaherpesviruses contains viral receptors for MT motors.
Assuntos
Herpesvirus Humano 1/fisiologia , Microtúbulos/virologia , Trifosfato de Adenosina/fisiologia , Animais , Capsídeo/fisiologia , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Núcleo Celular/virologia , Cricetinae , Citosol/virologia , Complexo Dinactina , Dineínas/fisiologia , Feminino , Proteínas de Fluorescência Verde/metabolismo , Técnicas In Vitro , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Motores Moleculares/fisiologia , Movimento , Oócitos/virologia , Proteínas Recombinantes de Fusão/metabolismo , Xenopus laevisRESUMO
Incoming viral particles move from the cell surface to sites of viral transcription and replication. By contrast, during assembly and egress, subviral nucleoprotein complexes and virions travel back to the plasma membrane. Because diffusion of large molecules is severely restricted in the cytoplasm, viruses use ATP-hydrolyzing molecular motors of the host for propelling along the microtubules, which are the intracellular highways. Recent studies have revealed that, besides travelling inside endocytic or exocytic vesicles, viral proteins interact directly with dynein or kinesin motors. Understanding the molecular mechanisms of cytoplasmic viral transport will aid in the construction of viral vectors for human gene therapy and the search for new antiviral targets.