RESUMEN
The innate immune system is the first line of host defense against infection by pathogenic microorganisms, among which macrophages are important innate immune cells. Macrophages are widely distributed throughout the body and recognize and eliminate viruses through pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs). In the present chapter, we provide detailed protocols for vesicular stomatitis virus (VSV) amplification, VSV titer detection, isolation of mouse primary peritoneal macrophages, in vitro and in vivo VSV infection, detection of interferon-beta (IFN-ß) expression, and lung injury. These protocols provide efficient and typical methods to evaluate virus-induced innate immunity in vitro and in vivo.
Asunto(s)
Inmunidad Innata , Interferón beta , Macrófagos Peritoneales , Vesiculovirus , Animales , Ratones , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/virología , Macrófagos Peritoneales/metabolismo , Interferón beta/inmunología , Interferón beta/metabolismo , Interferón beta/genética , Vesiculovirus/inmunología , Vesiculovirus/genética , Estomatitis Vesicular/inmunología , Estomatitis Vesicular/virología , Virus de la Estomatitis Vesicular Indiana/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Receptores de Reconocimiento de Patrones/inmunologíaAsunto(s)
Epidemiología , Urgencias Médicas , Encefalitis , Rhabdoviridae , Mpox , Orthobunyavirus , Vesiculovirus , LepraRESUMEN
Filoviruses, like the Marburg (MARV) and Ebola (EBOV) viruses, have caused outbreaks associated with significant hemorrhagic morbidity and high fatality rates. Vaccines offer one of the best countermeasures for fatal infection, but to date only the EBOV vaccine has received FDA licensure. Given the limited cross protection between the EBOV vaccine and Marburg hemorrhagic fever (MHF), we analyzed the protective efficacy of a similar vaccine, rVSV-MARV, in the lethal cynomolgus macaque model. NHPs vaccinated with a single dose (as little as 1.6 × 107 pfu) of rVSV-MARV seroconverted to MARV G-protein prior to challenge on day 42. Vaccinemia was measured in all vaccinated primates, self-resolved by day 14 post vaccination. Importantly, all vaccinated NHPs survived lethal MARV challenge, and showed no significant alterations in key markers of morbid disease, including clinical signs, and certain hematological and clinical chemistry parameters. Further, apart from one primate (from which tissues were not collected and no causal link was established), no pathology associated with Marburg disease was observed in vaccinated animals. Taken together, rVSV-MARV is a safe and efficacious vaccine against MHF in cynomolgus macaques.
Asunto(s)
Macaca fascicularis , Enfermedad del Virus de Marburg , Marburgvirus , Vesiculovirus , Vacunas Virales , Animales , Enfermedad del Virus de Marburg/prevención & control , Enfermedad del Virus de Marburg/inmunología , Enfermedad del Virus de Marburg/virología , Marburgvirus/inmunología , Marburgvirus/genética , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Vesiculovirus/genética , Vesiculovirus/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/administración & dosificación , Modelos Animales de Enfermedad , Vacunación , Masculino , Femenino , Eficacia de las Vacunas , Vectores Genéticos , Inmunogenicidad VacunalRESUMEN
Marburg virus (MARV), a filovirus, was first identified in 1967 in Marburg, Germany, and Belgrade, former Yugoslavia. Since then, MARV has caused sporadic outbreaks of human disease with high case fatality rates in parts of Africa, with the largest outbreak occurring in 2004/05 in Angola. From 2021 to 2023, MARV outbreaks occurred in Guinea, Ghana, New Guinea, and Tanzania, emphasizing the expansion of its endemic area into new geographical regions. There are currently no approved vaccines or therapeutics targeting MARV, but several vaccine candidates have shown promise in preclinical studies. We compared three vaccine platforms simultaneously by vaccinating hamsters with either a single dose of an adenovirus-based (ChAdOx-1 MARV) vaccine, an alphavirus replicon-based RNA (LION-MARV) vaccine, or a recombinant vesicular stomatitis virus-based (VSV-MARV) vaccine, all expressing the MARV glycoprotein as the antigen. Lethal challenge with hamster-adapted MARV 4 weeks after vaccination resulted in uniform protection of the VSV-MARV and LION-MARV groups and 83% of the ChAdOx-1 MARV group. Assessment of the antigen-specific humoral response and its functionality revealed vaccine-platform-dependent differences, particularly in the Fc effector functions.
Asunto(s)
Enfermedad del Virus de Marburg , Marburgvirus , Vacunas Virales , Animales , Cricetinae , Vacunas Virales/inmunología , Marburgvirus/inmunología , Enfermedad del Virus de Marburg/prevención & control , Enfermedad del Virus de Marburg/inmunología , Modelos Animales de Enfermedad , Adenoviridae/genética , Adenoviridae/inmunología , Vesiculovirus/inmunología , Vesiculovirus/genética , Anticuerpos Antivirales/inmunología , Vacunación/métodosRESUMEN
One major limitation of effective vaccine delivery is its dependency on a robust cold chain infrastructure. While Vesicular stomatitis virus (VSV) has been demonstrated to be an effective viral vaccine vector for diseases including Ebola, its -70 °C storage requirement is a significant limitation for accessing disadvantaged locations and populations. Previous work has shown thermal stabilization of viral vaccines with a combination of pullulan and trehalose (PT) dried films. To improve the thermal stability of VSV, we optimized PT formulation concentrations and components, as well as drying methodology with enhanced vacuum drying. When formulated in PT films, VSV can be stored for 32 weeks at 4 °C with less than 2 log PFU loss, at 25 °C with 2.5 log PFU loss, and at 37 °C with 3.1 log PFU loss. These results demonstrate a significant advancement in VSV thermal stabilization, decreasing the cold chain requirements for VSV vectored vaccines.
Asunto(s)
Glucanos , Trehalosa , Trehalosa/química , Glucanos/química , Vacio , Vectores Genéticos , Desecación/métodos , Vacunas Virales/química , Vesiculovirus/genética , Animales , TemperaturaRESUMEN
Host cells have evolved an intricate regulatory network to fine tune the type-I interferon responses. However, the full picture of this regulatory network remains to be depicted. In this study, we found that knock out of zinc-finger CCHC-type containing protein 8 (ZCCHC8) impairs the replication of influenza A virus (IAV), Sendai virus (Sev), Japanese encephalitis virus (JEV), and vesicular stomatitis virus (VSV). Further investigation unveiled that ZCCHC8 suppresses the type-I interferon responses by targeting the interferon regulatory factor 3 (IRF3) signaling pathway. Mechanistically, ZCCHC8 associates with phosphorylated IRF3 and disrupts the interaction of IRF3 with the co-activator CREB-binding protein (CBP). Additionally, the direct binding of ZCCHC8 with the IFN-stimulated response element (ISRE) impairs the ISRE-binding of IRF3. Our study contributes to the comprehensive understanding for the negative regulatory network of the type-I interferon responses and provides valuable insights for the control of multiple viruses from a host-centric perspective.IMPORTANCEThe innate immune responses serve as the initial line of defense against invading pathogens and harmful substances. Negative regulation of the innate immune responses plays an essential role in avoiding auto-immune diseases and over-activated immune responses. Hence, the comprehensive understanding of the negative regulation network for innate immune responses could provide novel therapeutic insights for the control of viral infections and immune dysfunction. In this study, we report that ZCCHC8 negatively regulates the type-I interferon responses. We illustrate that ZCCHC8 impedes the IRF3-CBP association by interacting with phosphorylated IRF3 and competes with IRF3 for binding to ISRE. Our study demonstrates the role of ZCCHC8 in the replication of multiple RNA viruses and contributes to a deeper understanding of the negative regulation system for the type-I interferon responses.
Asunto(s)
Proteína de Unión a CREB , Inmunidad Innata , Factor 3 Regulador del Interferón , Interferón Tipo I , Virus Sendai , Transducción de Señal , Replicación Viral , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Humanos , Células HEK293 , Virus Sendai/fisiología , Virus Sendai/genética , Proteína de Unión a CREB/metabolismo , Proteína de Unión a CREB/genética , Virus ARN/fisiología , Virus ARN/inmunología , Virus ARN/genética , Animales , Células A549 , Virus de la Influenza A/fisiología , Virus de la Influenza A/inmunología , Fosforilación , Interacciones Huésped-Patógeno , Vesiculovirus/fisiología , Virus de la Encefalitis Japonesa (Especie)/fisiología , Virus de la Encefalitis Japonesa (Especie)/inmunologíaRESUMEN
Viral infection is frequently assayed by ongoing expression of viral genes. These assays fail to identify cells that have been exposed to the virus but limit or inhibit viral replication. To address this limitation, we used a dual-labeling vesicular stomatitis virus (DL-VSV), which has a deletion of the viral glycoprotein gene, to allow evaluation of primary infection outcomes. This virus encodes Cre, which can stably mark any cell with even a minimal level of viral gene expression. Additionally, the virus encodes GFP, which distinguishes cells with higher levels of viral gene expression, typically due to genome replication. Stereotactic injections of DL-VSV into the murine brain showed that different cell types had very different responses to the virus. Almost all neurons hosted high levels of viral gene expression, while glial cells varied in their responses. Astrocytes (Sox9+) were predominantly productively infected, while oligodendrocytes (Sox10+) were largely abortively infected. Microglial cells (Iba1+) were primarily uninfected. Furthermore, we monitored the early innate immune response to viral infection and identified unique patterns of interferon (IFN) induction. Shortly after infection, microglia were the main producers of IFNb, whereas later, oligodendrocytes were the main producers. IFNb+ cells were primarily abortively infected regardless of cell type. Last, we investigated whether IFN signaling had any impact on the outcome of primary infection and did not observe significant changes, suggesting that intrinsic factors are likely responsible for determining the outcome of primary infection.
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Astrocitos , Animales , Ratones , Astrocitos/virología , Astrocitos/metabolismo , Replicación Viral , Microglía/virología , Microglía/metabolismo , Microglía/inmunología , Neuronas/virología , Neuronas/metabolismo , Factor de Transcripción SOX9/metabolismo , Factor de Transcripción SOX9/genética , Vesiculovirus/fisiología , Vesiculovirus/inmunología , Vesiculovirus/genética , Oligodendroglía/virología , Oligodendroglía/metabolismo , Estomatitis Vesicular/virología , Estomatitis Vesicular/inmunología , Inmunidad Innata , Ratones Endogámicos C57BL , Encéfalo/virología , Encéfalo/metabolismo , Encéfalo/inmunología , Neuroglía/virología , Neuroglía/metabolismoRESUMEN
Targeted antineoplastic immunotherapies have achieved remarkable clinical outcomes. However, resistance to these therapies due to target absence or antigen shedding limits their efficacy and excludes tumours from candidacy. To address this limitation, here we engineer an oncolytic rhabdovirus, vesicular stomatitis virus (VSVΔ51), to express a truncated targeted antigen, which allows for HER2-targeting with trastuzumab. The truncated HER2 (HER2T) lacks signaling capabilities and is efficiently expressed on infected cell surfaces. VSVΔ51-mediated HER2T expression simulates HER2-positive status in tumours, enabling effective treatment with the antibody-drug conjugate trastuzumab emtansine in vitro, ex vivo, and in vivo. Additionally, we combine VSVΔ51-HER2T with an oncolytic vaccinia virus expressing a HER2-targeted T-cell engager. This dual-virus therapeutic strategy demonstrates potent curative efficacy in vivo in female mice using CD3+ infiltrate for anti-tumour immunity. Our findings showcase the ability to tailor the tumour microenvironment using oncolytic viruses, thereby enhancing compatibility with "off-the-shelf" targeted therapies.
Asunto(s)
Inmunoterapia , Viroterapia Oncolítica , Virus Oncolíticos , Receptor ErbB-2 , Linfocitos T , Trastuzumab , Virus Vaccinia , Animales , Femenino , Humanos , Inmunoterapia/métodos , Ratones , Receptor ErbB-2/metabolismo , Receptor ErbB-2/inmunología , Receptor ErbB-2/genética , Viroterapia Oncolítica/métodos , Virus Oncolíticos/genética , Virus Oncolíticos/inmunología , Linfocitos T/inmunología , Línea Celular Tumoral , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Trastuzumab/uso terapéutico , Trastuzumab/farmacología , Microambiente Tumoral/inmunología , Vesiculovirus/genética , Vesiculovirus/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Ratones Endogámicos BALB CRESUMEN
Rhabdoviral vectors can induce lysis of cancer cells. While studied almost exclusively at 37 °C, viruses are subject to a range of temperatures in vivo, including temperatures ≤31 °C. Despite potential implications, the effect of temperatures <37 °C on the performance of rhabdoviral vectors is unknown. We investigated the effect of low anatomical temperatures on two rhabdoviruses, vesicular stomatitis virus (VSV) and Maraba virus (MG1). Using a metabolic resazurin assay, VSV- and MG1-mediated oncolysis was characterized in a panel of cell lines at 28, 31, 34 and 37 °C. The oncolytic ability of both viruses was hindered at 31 and 28 °C. Cold adaptation of both viruses was attempted as a mitigation strategy. Viruses were serially passaged at decreasing temperatures in an attempt to induce mutations. Unfortunately, the cold-adaptation strategies failed to potentiate the oncolytic activity of the viruses at temperatures <37 °C. Interestingly, we discovered that viral replication was unaffected at low temperatures despite the abrogation of oncolytic activity. In contrast, the proliferation of cancer cells was reduced at low temperatures. Equivalent oncolytic effects could be achieved if cells at low temperatures were treated with viruses for longer times. This suggests that rhabdovirus-mediated oncolysis could be compromised at low temperatures in vivo where therapeutic windows are limited.
Asunto(s)
Frío , Virus Oncolíticos , Rhabdoviridae , Replicación Viral , Humanos , Rhabdoviridae/fisiología , Rhabdoviridae/genética , Animales , Virus Oncolíticos/fisiología , Virus Oncolíticos/genética , Vesiculovirus/fisiología , Vesiculovirus/genética , Viroterapia Oncolítica/métodos , Línea Celular , Vectores Genéticos/genética , Línea Celular Tumoral , TemperaturaRESUMEN
Histone deacetylates family proteins have been studied for their function in regulating viral replication by deacetylating non-histone proteins. RIG-I (Retinoic acid-inducible gene I) is a critical protein in RNA virus-induced innate antiviral signaling pathways. Our previous research showed that HDAC8 (histone deacetylase 8) involved in innate antiviral immune response, but the underlying mechanism during virus infection is still unclear. In this study, we showed that HDAC8 was involved in the regulation of vesicular stomatitis virus (VSV) replication. Over-expression of HDAC8 inhibited while knockdown promoted VSV replication. Further exploration demonstrated that HDAC8 interacted with and deacetylated RIG-I, which eventually lead to enhance innate antiviral immune response. Collectively, our data clearly demonstrated that HDAC8 inhibited VSV replication by promoting RIG-I mediated interferon production and downstream signaling pathway.
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Proteína 58 DEAD Box , Histona Desacetilasas , Inmunidad Innata , Receptores Inmunológicos , Transducción de Señal , Vesiculovirus , Replicación Viral , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Humanos , Histona Desacetilasas/metabolismo , Vesiculovirus/inmunología , Receptores Inmunológicos/metabolismo , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Acetilación , Células HEK293 , Interferones/metabolismo , Interferones/inmunología , Línea Celular , Interacciones Huésped-Patógeno/inmunología , Animales , Virus de la Estomatitis Vesicular Indiana/inmunologíaRESUMEN
Non-segmented negative-strand (NNS) RNA viruses, such as rabies, Nipah and Ebola, produce 5'-capped and 3'-polyadenylated mRNAs resembling higher eukaryotic mRNAs. Here, we developed a transcription elongation-coupled pre-mRNA capping system for vesicular stomatitis virus (VSV, a prototypic NNS RNA virus). Using this system, we demonstrate that the single-polypeptide RNA-dependent RNA polymerase (RdRp) large protein (L) catalyzes all pre-mRNA modifications co-transcriptionally in the following order: (i) 5'-capping (polyribonucleotidylation of GDP) to form a GpppA cap core structure, (ii) 2'-O-methylation of GpppA into GpppAm, (iii) guanine-N7-methylation of GpppAm into m7GpppAm (cap 1), (iv) 3'-polyadenylation to yield a poly(A) tail. The GDP polyribonucleotidyltransferase (PRNTase) domain of L generated capped pre-mRNAs of 18 nucleotides or longer via the formation of covalent enzyme-pre-mRNA intermediates. The single methyltransferase domain of L sequentially methylated the cap structure only when pre-mRNAs of 40 nucleotides or longer were associated with elongation complexes. These results suggest that the formation of pre-mRNA closed loop structures in elongation complexes via the RdRp and PRNTase domains followed by the RdRp and MTase domains on the same polypeptide is required for the cap 1 formation during transcription. Taken together, our findings indicate that NNS RNA virus L acts as an all-in-one viral mRNA assembly machinery.
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Caperuzas de ARN , ARN Mensajero , ARN Viral , Proteínas Virales , ARN Mensajero/metabolismo , ARN Mensajero/genética , Caperuzas de ARN/metabolismo , Caperuzas de ARN/genética , ARN Viral/metabolismo , ARN Viral/genética , ARN Viral/química , Proteínas Virales/metabolismo , Proteínas Virales/genética , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/genética , Metilación , Vesiculovirus/genética , Precursores del ARN/metabolismo , Precursores del ARN/genética , Transcripción Genética , Procesamiento Postranscripcional del ARN , Animales , Poliadenilación , Elongación de la Transcripción GenéticaRESUMEN
The interferon (IFN) system protects mammals from diseases caused by virus infections. IFN synthesis is induced by pattern recognition receptor signaling pathways activated by virus infection. IFN is secreted from the infected cells and acts upon neighboring cells by binding cell surface receptors and triggering induction of hundreds of IFN-stimulated genes and proteins, many of which block different steps of virus replication. The IFN-induced tetratricopeptide repeat proteins (IFIT) are a family of RNA-binding proteins. We and others have previously reported that IFIT2 protects mice from many neurotropic RNA viruses; indeed, Ifit2-/- mice are very susceptible to intranasal or subcutaneous infections with vesicular stomatitis virus (VSV). Here, using a newly generated conditional knockout mouse, we report that ablation of Ifit2 expression only in neuronal cells was sufficient to render mice susceptible to neuropathogenesis caused by intranasal, but not subcutaneous, infection of VSV. Another genetically modified mouse line, expressing a mutant IFIT2 that cannot bind RNA, was as susceptible to VSV infection as Ifit2-/- mice. These results demonstrated that IFIT2 RNA-binding activity is essential for protecting mice against neurological diseases caused by intranasal infection of VSV.IMPORTANCEInterferon's (IFN's) antiviral effects are mediated by the proteins encoded by the interferon-stimulated genes. IFN-stimulated genes (IFIT2) is one such protein, which inhibits replication of many RNA viruses in the mouse brain and the resultant neuropathology. Our study sheds light on how IFIT2 works. By ablating Ifit2 expression only in neuronal cells, using a newly generated conditional knockout mouse line, we showed that Ifit2 induction in the neurons of the infected mouse was necessary for antiviral function of interferon. IFIT2 has no known enzyme activity; instead, it functions by binding to cellular or viral proteins or RNAs. We engineered a new mouse line that expressed a mutant IFIT2 that cannot bind RNA. These mice were very susceptible to infection with vesicular stomatitis virus indicating that the RNA-binding property of IFIT2 was essential for its antiviral function in vivo.
Asunto(s)
Ratones Noqueados , Neuronas , Proteínas de Unión al ARN , Estomatitis Vesicular , Animales , Ratones , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Neuronas/virología , Neuronas/metabolismo , Estomatitis Vesicular/virología , Estomatitis Vesicular/inmunología , Estomatitis Vesicular/genética , Replicación Viral , Vesiculovirus/inmunología , Vesiculovirus/genética , Ratones Endogámicos C57BL , Virus de la Estomatitis Vesicular Indiana/inmunología , Virus de la Estomatitis Vesicular Indiana/genética , Proteínas Reguladoras de la ApoptosisRESUMEN
The rapidly developing field of oncolytic virus (OV) therapy necessitates the development of new and improved analytical approaches for the characterization of the virus during production and development. Accurate monitoring and absolute quantification of viral proteins are crucial for OV product characterization and can facilitate the understanding of infection, immunogenicity, and development stages of viral replication. Targeted mass spectrometry methods like multiple reaction monitoring (MRM) offer a robust way to directly detect and quantify specific targeted proteins represented by surrogate peptides. We have leveraged the power of MRM by combining ultra-high performance liquid chromatography (UPLC) with a Sciex 6500 triple-stage quadrupole mass spectrometer to develop an assay that accurately and absolutely quantifies the structural proteins of a pseudotyped vesicular stomatitis virus (VSV) intended for use as a new biotherapeutic (designated hereafter as VSV-GP to differentiate it from native VSV). The new UPLC-MRM method provides absolute quantification with the use of heavy-labeled reference standard surrogate peptides. When added in known exact amounts to standards and samples, the reference standards normalize and account for any small perturbations during sample preparation and/or instrument performance, resulting in accurate and precise quantification. Because of the multiplexed nature of MRM, all targeted proteins are quantified at the same time. The optimized assay has been enhanced to quantify the ratios of the processed GP1 and GP2 proteins while simultaneously measuring any remaining or unprocessed form of the envelope protein GP complex (GPC; full-length GPC). IMPORTANCE: The development of oncolytic viral therapy has gained considerable momentum in recent years. Vesicular stomatitis virus glycoprotein (VSV-GP) is a new biotherapeutic emerging in the oncolytic viral therapy platform. Novel analytical assays that can accurately and precisely quantify the viral proteins are a necessity for the successful development of viral vector as a biotherapeutic. We developed an ultra-high performance liquid chromatography multiple reaction monitoring-based assay to quantify the absolute concentrations of the different structural proteins of VSV-GP. The complete processing of GP complex (GPC) is a prerequisite for the infectivity of the virus. The assay extends the potential for quantifying full-length GPC, which provides an understanding of the processing of GPC (along with the quantification of GP1 and GP2 separately). We used this assay in tracking GPC processing in HEK-293-F production cell lines infected with VSV-GP.
Asunto(s)
Vesiculovirus , Proteínas Virales , Humanos , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Virus Oncolíticos/genética , Virus de la Estomatitis Vesicular Indiana/genética , Vesiculovirus/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Nucleoprotein (N) is well known for its function in the encapsidation of the genomic RNAs of negative-strand RNA viruses, which leads to the formation of ribonucleoproteins that serve as templates for viral transcription and replication. However, the function of the N protein in other aspects during viral infection is far from clear. In this study, the N protein of snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus, was proved to be ubiquitinated mainly via K63-linked ubiquitination. We identified nine host E3 ubiquitin ligases that interacted with SHVV N, among which seven E3 ubiquitin ligases facilitated ubiquitination of the N protein. Further investigation revealed that only two E3 ubiquitin ligases, Siah E3 ubiquitin protein ligase 2 (Siah2) and leucine-rich repeat and sterile alpha motif containing 1 (LRSAM1), mediated K63-linked ubiquitination of the N protein. SHVV infection upregulated the expression of Siah2 and LRSAM1, which maintained the stability of SHVV N. Besides, overexpression of Siah2 or LRSAM1 promoted SHVV replication, while knockdown of Siah2 or LRSAM1 inhibited SHVV replication. Deletion of the ligase domain of Siah2 or LRSAM1 did not affect their interactions with SHVV N but reduced the K63-linked ubiquitination of SHVV N and SHVV replication. In summary, Siah2 and LRSAM1 mediate K63-linked ubiquitination of SHVV N to facilitate SHVV replication, which provides novel insights into the role of the N proteins of negative-strand RNA viruses. IMPORTANCE: Ubiquitination of viral protein plays an important role in viral replication. However, the ubiquitination of the nucleoprotein (N) of negative-strand RNA viruses has rarely been investigated. This study aimed at investigating the ubiquitination of the N protein of a fish rhabdovirus SHVV (snakehead vesiculovirus), identifying the related host E3 ubiquitin ligases, and determining the role of SHVV N ubiquitination and host E3 ubiquitin ligases in viral replication. We found that SHVV N was ubiquitinated mainly via K63-linked ubiquitination, which was mediated by host E3 ubiquitin ligases Siah2 (Siah E3 ubiquitin protein ligase 2) and LRSAM1 (leucine-rich repeat and sterile alpha motif containing 1). The data suggested that Siah2 and LRSAM1 were hijacked by SHVV to ubiquitinate the N protein for viral replication, which exhibited novel anti-SHVV targets for drug design.
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Nucleoproteínas , Ubiquitina-Proteína Ligasas , Ubiquitinación , Vesiculovirus , Replicación Viral , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Animales , Nucleoproteínas/metabolismo , Nucleoproteínas/genética , Vesiculovirus/fisiología , Vesiculovirus/metabolismo , Vesiculovirus/genética , Humanos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Células HEK293 , Proteínas Virales/metabolismo , Proteínas Virales/genética , Línea Celular , Infecciones por Rhabdoviridae/virología , Infecciones por Rhabdoviridae/metabolismo , Enfermedades de los Peces/virología , Enfermedades de los Peces/metabolismoRESUMEN
Although patients benefit from immune checkpoint inhibition (ICI) therapy in a broad variety of tumors, resistance may arise from immune suppressive tumor microenvironments (TME), which is particularly true of hepatocellular carcinoma (HCC). Since oncolytic viruses (OV) can generate a highly immune-infiltrated, inflammatory TME, OVs could potentially restore ICI responsiveness via recruitment, priming, and activation of anti-tumor T cells. Here we find that on the contrary, an oncolytic vesicular stomatitis virus, expressing interferon-ß (VSV-IFNß), antagonizes the effect of anti-PD-L1 therapy in a partially anti-PD-L1-responsive model of HCC. Cytometry by Time of Flight shows that VSV-IFNß expands dominant anti-viral effector CD8 T cells with concomitant relative disappearance of anti-tumor T cell populations, which are the target of anti-PD-L1. However, by expressing a range of HCC tumor antigens within VSV, combination OV and anti-PD-L1 therapeutic benefit could be restored. Our data provide a cautionary message for the use of highly immunogenic viruses as tumor-specific immune-therapeutics by showing that dominant anti-viral T cell responses can inhibit sub-dominant anti-tumor T cell responses. However, through encoding tumor antigens within the virus, oncolytic virotherapy can generate anti-tumor T cell populations upon which immune checkpoint blockade can effectively work.
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Antígenos de Neoplasias , Antígeno B7-H1 , Linfocitos T CD8-positivos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Viroterapia Oncolítica , Virus Oncolíticos , Microambiente Tumoral , Virus Oncolíticos/genética , Virus Oncolíticos/inmunología , Animales , Viroterapia Oncolítica/métodos , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/inmunología , Microambiente Tumoral/inmunología , Ratones , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Humanos , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/inmunología , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Interferón beta/metabolismo , Interferón beta/inmunología , Ratones Endogámicos C57BL , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , Linfocitos T/inmunología , Femenino , Vesiculovirus/inmunología , Vesiculovirus/genéticaRESUMEN
Viral glycoproteins mediate entry into host cells, thereby dictating host range and pathogenesis. In addition, they constitute the principal target of neutralizing antibody responses, making them important antigens in vaccine development. Recombinant vesicular stomatitis virus (VSV) encoding foreign glycoproteins can provide a convenient and safe surrogate system to interrogate the function, evolution, and antigenicity of viral glycoproteins from viruses that are difficult to manipulate or those requiring high biosafety level containment. However, the production of recombinant VSV can be technically challenging. In this work, we present an efficient and robust plasmid-based system for the production of recombinant VSV encoding foreign glycoproteins. We validate the system using glycoproteins from different viral families, including arenaviruses, coronaviruses, and hantaviruses, as well as highlight their utility for studying the effects of mutations on viral fitness. Overall, the methods described herein can facilitate the study of both native and recombinant VSV encoding foreign glycoproteins and can serve as the basis for the production of VSV-based vaccines.
Asunto(s)
Glicoproteínas , Plásmidos , Plásmidos/genética , Glicoproteínas/genética , Glicoproteínas/inmunología , Animales , Humanos , Vesiculovirus/genética , Proteínas Virales/genética , Proteínas Virales/inmunología , Células HEK293RESUMEN
Herpes B virus (BV) is a zoonotic virus and belongs to the genus Simplexvius, the same genus as human herpes simplex virus (HSV). BV typically establishes asymptomatic infection in its natural hosts, macaque monkeys. However, in humans, BV infection causes serious neurological diseases and death. As such, BV research can only be conducted in a high containment level facility (i.e., biosafety level [BSL] 4), and the mechanisms of BV entry have not been fully elucidated. In this study, we generated a pseudotyped vesicular stomatitis virus (VSV) expressing BV glycoproteins using G-complemented VSV∆G system, which we named VSV/BVpv. We found that four BV glycoproteins (i.e., gB, gD, gH, and gL) were required for the production of a high-titer VSV/BVpv. Moreover, VSV/BVpv cell entry was dependent on the binding of gD to its cellular receptor nectin-1. Pretreatment of Vero cells with endosomal acidification inhibitors did not affect the VSV/BVpv infection. The result indicated that VSV/BVpv entry occurred by direct fusion with the plasma membrane of Vero cells and suggested that the entry pathway was similar to that of native HSV. Furthermore, we developed a VSV/BVpv-based chemiluminescence reduction neutralization test (CRNT), which detected the neutralization antibodies against BV in macaque plasma samples with high sensitivity and specificity. Crucially, the VSV/BVpv generated in this study can be used under BSL-2 condition to study the initial entry process through gD-nectin-1 interaction and the direct fusion of BV with the plasma membrane of Vero cells.IMPORTANCEHerpes B virus (BV) is a highly pathogenic zoonotic virus against humans. BV belongs to the genus Simplexvius, the same genus as human herpes simplex virus (HSV). By contrast to HSV, cell entry mechanisms of BV are not fully understood. The research procedures to manipulate infectious BV should be conducted in biosafety level (BSL)-4 facilities. As pseudotyped viruses provide a safe viral entry model because of their inability to produce infectious progeny virus, we tried to generate a pseudotyped vesicular stomatitis virus bearing BV glycoproteins (VSV/BVpv) by modification of expression constructs of BV glycoproteins, and successfully obtained VSV/BVpv with a high titer. This study has provided novel information for constructing VSV/BVpv and its usefulness to study BV infection.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Internalización del Virus , Animales , Anticuerpos Neutralizantes/inmunología , Chlorocebus aethiops , Células Vero , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Humanos , Pruebas de Neutralización , Vesiculovirus/genética , Vesiculovirus/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Glicoproteínas/genética , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Proteínas Virales/metabolismoRESUMEN
BACKGROUND: Metastatic breast cancer is a leading cause of cancer death in woman. Current treatment options are often associated with adverse side effects and poor outcomes, demonstrating the need for effective new treatments. Immunotherapies can provide durable outcomes in many cancers; however, limited success has been achieved in metastatic triple negative breast cancer. We tested whether combining different immunotherapies can target metastatic triple negative breast cancer in pre-clinical models. METHODS: Using primary and metastatic 4T1 triple negative mammary carcinoma models, we examined the therapeutic effects of oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express reovirus-derived fusion associated small transmembrane proteins p14 (VSV-p14) or p15 (VSV-p15). These viruses were delivered alone or in combination with natural killer T (NKT) cell activation therapy mediated by adoptive transfer of α-galactosylceramide-loaded dendritic cells. RESULTS: Treatment of primary 4T1 tumors with VSV-p14 or VSV-p15 alone increased immunogenic tumor cell death, attenuated tumor growth, and enhanced immune cell infiltration and activation compared to control oncolytic virus (VSV-GFP) treatments and untreated mice. When combined with NKT cell activation therapy, oncolytic VSV-p14 and VSV-p15 reduced metastatic lung burden to undetectable levels in all mice and generated immune memory as evidenced by enhanced in vitro recall responses (tumor killing and cytokine production) and impaired tumor growth upon rechallenge. CONCLUSION: Combining NKT cell immunotherapy with enhanced oncolytic virotherapy increased anti-tumor immune targeting of lung metastasis and presents a promising treatment strategy for metastatic breast cancer.