RESUMEN
The Ebola virus (EBOV) is a member of the Orthoebolavirus genus, Filoviridae family, which causes severe hemorrhagic diseases in humans and non-human primates (NHPs), with a case fatality rate of up to 90%. The development of countermeasures against EBOV has been hindered by the lack of ideal animal models, as EBOV requires handling in biosafety level (BSL)-4 facilities. Therefore, accessible and convenient animal models are urgently needed to promote prophylactic and therapeutic approaches against EBOV. In this study, a recombinant vesicular stomatitis virus expressing Ebola virus glycoprotein (VSV-EBOV/GP) was constructed and applied as a surrogate virus, establishing a lethal infection in hamsters. Following infection with VSV-EBOV/GP, 3-week-old female Syrian hamsters exhibited disease signs such as weight loss, multi-organ failure, severe uveitis, high viral loads, and developed severe systemic diseases similar to those observed in human EBOV patients. All animals succumbed at 2-3 days post-infection (dpi). Histopathological changes indicated that VSV-EBOV/GP targeted liver cells, suggesting that the tissue tropism of VSV-EBOV/GP was comparable to wild-type EBOV (WT EBOV). Notably, the pathogenicity of the VSV-EBOV/GP was found to be species-specific, age-related, gender-associated, and challenge route-dependent. Subsequently, equine anti-EBOV immunoglobulins and a subunit vaccine were validated using this model. Overall, this surrogate model represents a safe, effective, and economical tool for rapid preclinical evaluation of medical countermeasures against EBOV under BSL-2 conditions, which would accelerate technological advances and breakthroughs in confronting Ebola virus disease.
Asunto(s)
Modelos Animales de Enfermedad , Ebolavirus , Fiebre Hemorrágica Ebola , Mesocricetus , Animales , Fiebre Hemorrágica Ebola/virología , Fiebre Hemorrágica Ebola/patología , Ebolavirus/genética , Ebolavirus/patogenicidad , Femenino , Humanos , Vesiculovirus/genética , Vesiculovirus/patogenicidad , Anticuerpos Antivirales/sangre , Cricetinae , Carga Viral , Glicoproteínas/genética , Glicoproteínas/inmunologíaRESUMEN
Asp-Glu-Ala-Asp (DEAD) box helicase 3 X-linked (DDX3X) plays important regulatory roles in the replication of many viruses. However, the role of DDX3X in rhabdovirus replication has seldomly been investigated. In this study, snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus, was used to study the role of DDX3X in rhabdovirus replication. DDX3X was identified as an interacting partner of SHVV phosphoprotein (P). The expression level of DDX3X was increased at an early stage of SHVV infection and then decreased to a normal level at a later infection stage. Overexpression of DDX3X promoted, while knockdown of DDX3X using specific small interfering RNAs (siRNAs) suppressed, SHVV replication, indicating that DDX3X was a proviral factor for SHVV replication. The N-terminal and core domains of DDX3X (DDX3X-N and DDX3X-Core) were determined to be the regions responsible for its interaction with SHVV P. Overexpression of DDX3X-Core suppressed SHVV replication by competitively disrupting the interaction between full-length DDX3X and SHVV P, suggesting that full-length DDX3X-P interaction was required for SHVV replication. Mechanistically, DDX3X-mediated promotion of SHVV replication was due not to inhibition of interferon expression but to maintenance of the stability of SHVV P to avoid autophagy-lysosome-dependent degradation. Collectively, our data suggest that DDX3X is hijacked by SHVV P to ensure effective replication of SHVV, which suggests an important anti-SHVV target. This study will help elucidate the role of DDX3X in regulating the replication of rhabdoviruses. IMPORTANCE Growing evidence has suggested that DDX3X plays important roles in virus replication. In one respect, DDX3X inhibits the replication of viruses, including hepatitis B virus, influenza A virus, Newcastle disease virus, duck Tembusu virus, and red-spotted grouper nervous necrosis virus. In another respect, DDX3X is required for the replication of viruses, including hepatitis C virus, Japanese encephalitis virus, West Nile virus, murine norovirus, herpes simplex virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because DDX3X has rarely been investigated in rhabdovirus replication, this study aimed at investigating the role of DDX3X in rhabdovirus replication by using the fish rhabdovirus SHVV as a model. We found that DDX3X was required for SHVV replication, with the mechanism that DDX3X interacts with and maintains the stability of SHVV phosphoprotein. Our data provide novel insights into the role of DDX3X in virus replication and will facilitate the design of antiviral drugs against rhabdovirus infection.
Asunto(s)
ARN Helicasas DEAD-box , Perciformes , Fosfoproteínas , Vesiculovirus , Replicación Viral , Animales , ARN Helicasas DEAD-box/genética , Peces , Perciformes/virología , ARN Interferente Pequeño , Vesiculovirus/patogenicidad , Vesiculovirus/fisiología , Proteínas ViralesRESUMEN
Chandipura virus (CHPV), belonging to the genus Vesiculovirus of the family Rhabdoviridae, has been identified as one of the causes of pediatric encephalitis in India. Currently, neither vaccines nor therapeutic drugs are available against this agent. Considering that the disease progresses very fast with a high mortality rate, working towards the development of potential therapeutics against it will have a public health impact. Although the use of viral inhibitors as antiviral agents is the most common way to curb virus replication, the mutation-prone nature of viruses results in the development of resistance to antiviral agents. The recent development of proteomic platforms for analysis of purified viral agents has allowed certain upregulated host proteins that are involved in the morphogenesis and replication of viruses to be identified. Thus, the alternative approach of inhibition of host proteins involved in the regulation of virus replication could be explored for their therapeutic effectiveness. In the current study, we have evaluated the effect of inhibition of cyclophilin A (CypA), an immunophilin with peptidyl-prolyl cis/trans-isomerase activity, on the replication of CHPV. Treatment with cyclosporin A, used in vitro for the inhibition of CypA, resulted in a 3-log reduction in CHPV titer and an undetectable level of CypA in comparison to an untreated control. An in silico analysis of the interaction of the CHPV nucleoprotein with the human CypA protein showed stable interaction in molecular docking and molecular dynamics simulations. Overall, the results of this study suggest a possible role of CypA in facilitating CHPV replication, thus making it one of the potential host factors to be explored in future antiviral studies.
Asunto(s)
Ciclofilina A/metabolismo , Interacciones Huésped-Patógeno/fisiología , Infecciones por Rhabdoviridae/virología , Vesiculovirus/patogenicidad , Ciclofilina A/antagonistas & inhibidores , Ciclofilina A/química , Ciclosporina/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Vesiculovirus/efectos de los fármacos , Vesiculovirus/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
The antiviral innate immunity is the first line of host defense against viral infection. Mitochondrial antiviral signaling protein (MAVS, also named Cardif/IPS-1/VISA) is a critical protein in RNA virus-induced antiviral signaling pathways. Our previous research suggested that E3 ubiquitin-protein ligases RING-finger protein (RNF90) negatively regulate cellular antiviral responses by targeting STING for degradation, though its role in RNA virus infection remains unknown. This study demonstrated that RNF90 negatively regulated RNA virus-triggered antiviral innate immune responses in RNF90-silenced PMA-THP1 cells, RNF90-deficient cells (including HaCaTs, MEFs, and BMDMs), and RNF90-deficient mice. However, RNF90 regulated RNA virus-triggered antiviral innate immune responses independent of STING. RNF90 promoted K48-linked ubiquitination of MAVS and its proteasome-dependent degradation, leading to the inhibition of innate immune responses. Altogether, our findings suggested a novel function and mechanism of RNF90 in antiviral innate immunity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Inmunidad Innata , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Estomatitis Vesicular/metabolismo , Vesiculovirus/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Chlorocebus aethiops , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Células HEK293 , Células HaCaT , Interacciones Huésped-Patógeno , Humanos , Ratones Noqueados , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Transducción de Señal , Células THP-1 , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/inmunología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/inmunología , Ubiquitinación , Células Vero , Estomatitis Vesicular/genética , Estomatitis Vesicular/inmunología , Estomatitis Vesicular/virología , Vesiculovirus/patogenicidadRESUMEN
Viral encephalitis is the most common cause of encephalitis. It is responsible for high morbidity rates, permanent neurological sequelae, and even high mortality rates. The host immune response plays a critical role in preventing or clearing invading pathogens, especially when effective antiviral treatment is lacking. However, due to blockade of the blood-brain barrier, it remains unclear how peripheral immune cells contribute to the fight against intracerebral viruses. Here, we report that peripheral injection of an antibody against human Tim-3, an immune checkpoint inhibitor widely expressed on immune cells, markedly attenuated vesicular stomatitis virus (VSV) encephalitis, marked by decreased mortality and improved neuroethology in mice. Peripheral injection of Tim-3 antibody enhanced the recruitment of immune cells to the brain, increased the expression of major histocompatibility complex-I (MHC-I) on macrophages, and as a result, promoted the activation of VSV-specific CD8+ T cells. Depletion of macrophages abolished the peripheral injection-mediated protection against VSV encephalitis. Notably, for the first time, we found a novel post-translational modification of MHC-I by Tim-3, wherein, by enhancing the expression of MARCH9, Tim-3 promoted the proteasome-dependent degradation of MHC-I via K48-linked ubiquitination in macrophages. These results provide insights into the immune response against intracranial infections; thus, manipulating the peripheral immune cells with Tim-3 antibody to fight viruses in the brain may have potential applications for combating viral encephalitis.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Neutralizantes/administración & dosificación , Células Presentadoras de Antígenos/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encefalitis Viral/prevención & control , Receptor 2 Celular del Virus de la Hepatitis A/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Infecciones por Rhabdoviridae/prevención & control , Vesiculovirus/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Células Presentadoras de Antígenos/virología , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/virología , Chlorocebus aethiops , Modelos Animales de Enfermedad , Encefalitis Viral/inmunología , Encefalitis Viral/metabolismo , Encefalitis Viral/virología , Células HEK293 , Receptor 2 Celular del Virus de la Hepatitis A/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Interacciones Huésped-Patógeno , Humanos , Inyecciones Intraperitoneales , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Células RAW 264.7 , Infecciones por Rhabdoviridae/inmunología , Infecciones por Rhabdoviridae/metabolismo , Infecciones por Rhabdoviridae/virología , Ubiquitinación , Células Vero , Vesiculovirus/patogenicidad , Carga ViralRESUMEN
BACKGROUND: Oncolytic viruses reduce tumor burden in animal models and have generated promising results in clinical trials. However, it is likely that oncolytic viruses will be more effective when used in combination with other therapies. Current therapeutic approaches, including chemotherapeutics, come with dose-limiting toxicities. Another option is to combine oncolytic viruses with immunotherapeutic approaches. METHODS: Using experimental models of metastatic 4T1 breast cancer and ID8 ovarian peritoneal carcinomatosis, we examined natural killer T (NKT) cell-based immunotherapy in combination with recombinant oncolytic vesicular stomatitis virus (VSV) or reovirus. 4T1 mammary carcinoma cells or ID8 ovarian cancer cells were injected into syngeneic mice. Tumor-bearing mice were treated with VSV or reovirus followed by activation of NKT cells via the intravenous administration of autologous dendritic cells loaded with the glycolipid antigen α-galactosylceramide. The effects of VSV and reovirus on immunogenic cell death (ICD), cell viability and immunogenicity were tested in vitro. RESULTS: VSV or reovirus treatments followed by NKT cell activation mediated greater survival in the ID8 model than individual therapies. The regimen was less effective when the treatment order was reversed, delivering virus treatments after NKT cell activation. In the 4T1 model, VSV combined with NKT cell activation increased overall survival and decreased metastatic burden better than individual treatments. In contrast, reovirus was not effective on its own or in combination with NKT cell activation. In vitro, VSV killed a panel of tumor lines better than reovirus. VSV infection also elicited greater increases in mRNA transcripts for proinflammatory cytokines, chemokines, and antigen presentation machinery compared with reovirus. Oncolytic VSV also induced the key hallmarks of ICD (calreticulin mobilization, plus release of ATP and HMGB1), while reovirus only mobilized calreticulin. CONCLUSION: Taken together, these results demonstrate that oncolytic VSV and NKT cell immunotherapy can be effectively combined to decrease tumor burden in models of metastatic breast and ovarian cancers. Oncolytic VSV and reovirus induced differential responses in our models which may relate to differences in virus activity or tumor susceptibility.
Asunto(s)
Neoplasias de la Mama/terapia , Inmunoterapia Adoptiva , Células T Asesinas Naturales/trasplante , Viroterapia Oncolítica , Virus Oncolíticos/inmunología , Neoplasias Ováricas/terapia , Neoplasias Peritoneales/terapia , Reoviridae/inmunología , Vesiculovirus/inmunología , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Neoplasias de la Mama/virología , Línea Celular Tumoral , Chlorocebus aethiops , Terapia Combinada , Citocinas/metabolismo , Citotoxicidad Inmunológica , Femenino , Interacciones Huésped-Patógeno , Activación de Linfocitos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células T Asesinas Naturales/inmunología , Virus Oncolíticos/patogenicidad , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Neoplasias Ováricas/virología , Neoplasias Peritoneales/inmunología , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/virología , Reoviridae/patogenicidad , Células Vero , Vesiculovirus/patogenicidadRESUMEN
Type I interferons (IFNs) induce hundreds of IFN-stimulated genes (ISGs) in response to viral infection. Induction of these ISGs must be regulated for an efficient and controlled antiviral response, but post-transcriptional controls of these genes have not been well defined. Here, we identify a role for the RNA base modification N6-methyladenosine (m6A) in the regulation of ISGs. Using ribosome profiling and quantitative mass spectrometry, coupled with m6A-immunoprecipitation and sequencing, we identify a subset of ISGs, including IFITM1, whose translation is enhanced by m6A and the m6A methyltransferase proteins METTL3 and METTL14. We further determine that the m6A reader YTHDF1 increases the expression of IFITM1 in an m6A-binding-dependent manner. Importantly, we find that the m6A methyltransferase complex promotes the antiviral activity of type I IFN. Thus, these studies identify m6A as having a role in post-transcriptional control of ISG translation during the type I IFN response for antiviral restriction.
Asunto(s)
Adenosina/análogos & derivados , Biosíntesis de Proteínas , Procesamiento Postranscripcional del ARN , Transcripción Genética , Estomatitis Vesicular/genética , Vesiculovirus/patogenicidad , Células A549 , Adenosina/metabolismo , Animales , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Antivirales/farmacología , Chlorocebus aethiops , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Interferón beta/farmacología , Metiltransferasas/biosíntesis , Metiltransferasas/genética , Biosíntesis de Proteínas/efectos de los fármacos , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transcripción Genética/efectos de los fármacos , Células Vero , Estomatitis Vesicular/metabolismo , Estomatitis Vesicular/virología , Vesiculovirus/crecimiento & desarrollo , Replicación ViralRESUMEN
The heartland virus (HRTV) is a novel phlebovirus that causes severe infections in the USA and closely related to the severe fever thrombocytopenia syndrome virus (SFTSV), a causative agent for SFTS in Asia. The entry mechanisms of HRTV remain unclear. Here, we developed the pseudotyped vesicular stomatitis virus bearing the HRTV glycoprotein (GP) (HRTVpv), and the antigenicity and the entry mechanisms of HRTV were analyzed. HRTVpv was neutralized by anti-SFTSV Gc antibody, but not the anti-SFTSV Gn antibodies. Entry of HRTVpv to cells was inhibited by bafilomycin A1 and dynasore, and but it was enhanced in cells overexpressed with C-type lectins. Production of infectious HRTVpv and SFTSVpv was reduced by Nn-DNJ, α-glucosidase inhibitor. The entry of HRTV occurs via pH- and dynamin-dependent endocytosis. Furthermore, Nn-DNJ may be a possible therapeutic agent against HRTV and SFTSV.
Asunto(s)
Infecciones por Bunyaviridae/virología , Phlebovirus/patogenicidad , Estomatitis Vesicular/virología , Vesiculovirus/patogenicidad , Proteínas del Envoltorio Viral/metabolismo , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacología , Animales , Línea Celular , Cricetinae , Haplorrinos , Humanos , Ratones , Internalización del VirusRESUMEN
Innate immunity is the first-line defense against antiviral or antimicrobial infection. RIG-I and MDA5, which mediate the recognition of pathogen-derived nucleic acids, are essential for production of type I interferons (IFN). Here, we identified mitochondrion depolarization inducer carbonyl cyanide 3-chlorophenylhydrazone (CCCP) inhibited the response and antiviral activity of type I IFN during viral infection. Furthermore, we found that the PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin-protein ligase Parkin mediated mitophagy, thus negatively regulating the activation of RIG-I and MDA5. Parkin directly interacted with and catalyzed the K48-linked polyubiquitination and subsequent degradation of RIG-I and MDA5. Thus, we demonstrate that Parkin limits RLR-triggered innate immunity activation, suggesting Parkin as a potential therapeutic target for the control of viral infection.
Asunto(s)
Proteína 58 DEAD Box/metabolismo , Inmunidad Innata , Helicasa Inducida por Interferón IFIH1/metabolismo , Mitocondrias/inmunología , Receptores Inmunológicos/metabolismo , Virus Sendai/inmunología , Ubiquitina-Proteína Ligasas/metabolismo , Vesiculovirus/inmunología , Células A549 , Animales , Chlorocebus aethiops , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Hidrazonas/farmacología , Inmunidad Innata/efectos de los fármacos , Interferón Tipo I/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/virología , Mitofagia , Proteínas Quinasas/metabolismo , Células RAW 264.7 , Virus Sendai/genética , Virus Sendai/patogenicidad , Células THP-1 , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Desacopladores/farmacología , Células Vero , Vesiculovirus/genética , Vesiculovirus/patogenicidadRESUMEN
Filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic fever in humans and nonhuman primates with high mortality rates. There is no approved therapy against these deadly viruses. Antiviral drug development has been hampered by the requirement of a biosafety level (BSL)-4 facility to handle infectious EBOV and MARV because of their high pathogenicity to humans. In this study, we aimed to establish a surrogate animal model that can be used for anti-EBOV and -MARV drug screening under BSL-2 conditions by focusing on the replication-competent recombinant vesicular stomatitis virus (rVSV) pseudotyped with the envelope glycoprotein (GP) of EBOV (rVSV/EBOV) and MARV (rVSV/MARV), which has been investigated as vaccine candidates and thus widely used in BSL-2 laboratories. We first inoculated mice, rats, and hamsters intraperitoneally with rVSV/EBOV and found that only hamsters showed disease signs and succumbed within 4 days post-infection. Infection with rVSV/MARV also caused lethal infection in hamsters. Both rVSV/EBOV and rVSV/MARV were detected at high titers in multiple organs including the liver, spleen, kidney, and lungs of infected hamsters, indicating acute and systemic infection resulting in fatal outcomes. Therapeutic effects of passive immunization with an anti-EBOV neutralizing antibody were specifically observed in rVSV/EBOV-infected hamsters. Thus, this animal model is expected to be a useful tool to facilitate in vivo screening of anti-filovirus drugs targeting the GP molecule.
Asunto(s)
Modelos Animales de Enfermedad , Ebolavirus/genética , Marburgvirus/genética , Estomatitis Vesicular/virología , Vesiculovirus/genética , Proteínas del Envoltorio Viral/genética , Animales , Anticuerpos Antivirales/administración & dosificación , Cricetinae , Susceptibilidad a Enfermedades , Evaluación Preclínica de Medicamentos , Ebolavirus/inmunología , Mesocricetus , Ratones , Ratas , Vacunas Sintéticas , Estomatitis Vesicular/patología , Estomatitis Vesicular/prevención & control , Estomatitis Vesicular/terapia , Vesiculovirus/patogenicidad , Proteínas del Envoltorio Viral/inmunología , Carga ViralRESUMEN
Macrophages have been identified as key players within the tumor microenvironment, with classically (M1) and alternatively (M2) activated macrophages exhibiting anti-tumoral and pro-tumoral functions, respectively. The goal of this study was to determine the response of macrophage populations to infection with oncolytic vesicular stomatitis virus (VSV). THP-1 monocytes were differentiated into various macrophage subsets and infected with wild-type (rwt virus) or matrix (M) protein mutant (rM51R-M virus) strains of VSV. Monocytes and M2 macrophages were susceptible to infection and killing by both rwt and rM51R-M viruses. rM51R-M virus also increased expression of the M1 markers p-STAT1, CD80, and TNF-α in pro-tumoral M2 macrophages, suggesting reprogramming towards an M1-like pro-inflammatory state. Meanwhile, rwt virus was more effective than rM51R-M virus at replicating in M2 macrophages and at inhibiting the development of invasive podosome structures. This was all in contrast to anti-tumoral M1 macrophages, which remained resistant to VSV-induced cytopathic effects. These results indicate that macrophage populations are differentially susceptible to VSV and that rwt and rM51R-M viruses may modulate the tumor-promoting phenotype of M2 macrophages by distinct mechanisms.
Asunto(s)
Diferenciación Celular/inmunología , Macrófagos/clasificación , Macrófagos/virología , Virus Oncolíticos/inmunología , Vesiculovirus/inmunología , Humanos , Macrófagos/inmunología , Macrófagos/patología , Virus Oncolíticos/patogenicidad , Podosomas/virología , Células THP-1 , Vesiculovirus/patogenicidadRESUMEN
Lipid mediators (LMs) play a pivotal role in the induction and resolution of inflammation. To identify and elucidate their involvement during virus infection, multiple reaction monitoring (MRM) based liquid chromatography-tandem mass spectrometry lipidomic profiling of 62 lipid species was performed in this study. Results show that RAW264.7 macrophages differentially produce specific LMs signals depending on difference in virus pathogenicity. Integration of large-scale lipidomics with targeted gene expression data revealed mediators, such as RVD3, 18-HEPE, 11(12)-EET etc. correlated with the pathogenic phase of the infection. The herpes simplex virus (HSV)-induced keratitis model demonstrates that 11(12)-EET treatment represents a novel alternative for treating viral infection.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Antivirales/uso terapéutico , Córnea/virología , Inflamación/prevención & control , Queratitis Herpética/prevención & control , Ácido 8,11,14-Eicosatrienoico/uso terapéutico , Animales , Chlorocebus aethiops , Cromatografía Liquida , Inflamación/virología , Queratitis Herpética/virología , Lipidómica/métodos , Masculino , Ratones , Ratones Endogámicos BALB C , Células RAW 264.7 , Simplexvirus/patogenicidad , Espectrometría de Masas en Tándem , Células Vero , Vesiculovirus/patogenicidad , Replicación Viral/efectos de los fármacosRESUMEN
Type-I interferons (IFN-I) are used as common antiviral drugs for a range of viral diseases in clinic. However, the antiviral efficacy of IFN-I is largely restricted by negative regulators of IFN-I signaling in cells. Therefore, identification of intracellular inhibitors of IFN-I signaling is important for developing novel targets to improve IFN-I antiviral therapy. In this study, we report that the deubiquitinase ubiquitin-specific protease 7 (USP7) negatively regulates IFN-I-mediated antiviral activity. USP7 physically interacts with suppressor of cytokine signaling 1 (SOCS1) and enhances SOCS1 protein stability by deubiquitination effects, which in turn restricts IFN-I-induced activation of Janus kinase-signal transducer and activator of transcription 1 signaling. Interestingly, viral infection up-regulates USP7 and therefore facilitates viral immune evasion. Importantly, the USP7 small-molecule inhibitors P5091 and P22077 inhibit SOCS1 expression and enhance IFN-I antiviral efficacy. Our findings identify a novel regulator of IFN-I antiviral activity and reveal that USP7 inhibitors could be potential enhancement agents for improving IFN-I antiviral therapy.
Asunto(s)
Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Interferón-alfa/farmacología , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Tiofenos/farmacología , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Vesiculovirus/efectos de los fármacos , Células A549 , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Quinasas Janus/metabolismo , Unión Proteica , Estabilidad Proteica , Proteolisis , Transducción de Señal , Proteína 1 Supresora de la Señalización de Citocinas/genética , Factores de Tiempo , Peptidasa Específica de Ubiquitina 7/metabolismo , Ubiquitinación , Vesiculovirus/inmunología , Vesiculovirus/patogenicidadRESUMEN
Early and strong production of IFN-I by dendritic cells is important to control vesicular stomatitis virus (VSV), however mechanisms which explain this cell-type specific innate immune activation remain to be defined. Here, using a genome wide association study (GWAS), we identified Integrin alpha-E (Itgae, CD103) as a new regulator of antiviral IFN-I production in a mouse model of vesicular stomatitis virus (VSV) infection. CD103 was specifically expressed by splenic conventional dendritic cells (cDCs) and limited IFN-I production in these cells during VSV infection. Mechanistically, CD103 suppressed AKT phosphorylation and mTOR activation in DCs. Deficiency in CD103 accelerated early IFN-I in cDCs and prevented death in VSV infected animals. In conclusion, CD103 participates in regulation of cDC specific IFN-I induction and thereby influences immune activation after VSV infection.
Asunto(s)
Antígenos CD/metabolismo , Células Dendríticas/virología , Inmunidad Innata , Cadenas alfa de Integrinas/metabolismo , Interferón Tipo I/metabolismo , Estomatitis Vesicular/virología , Vesiculovirus/patogenicidad , Animales , Antígenos CD/genética , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Estudio de Asociación del Genoma Completo , Interacciones Huésped-Patógeno , Cadenas alfa de Integrinas/genética , Ratones de la Cepa 129 , Ratones Endogámicos AKR , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos NOD , Ratones Noqueados , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Estomatitis Vesicular/genética , Estomatitis Vesicular/inmunología , Estomatitis Vesicular/metabolismo , Vesiculovirus/crecimiento & desarrollo , Replicación ViralRESUMEN
Transcriptional responses to external stimuli remain poorly understood. Using global nuclear run-on followed by sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq), we show that CDK8 kinase activity promotes RNA polymerase II pause release in response to interferon-γ (IFN-γ), a universal cytokine involved in immunity and tumor surveillance. The Mediator kinase module contains CDK8 or CDK19, which are presumed to be functionally redundant. We implemented cortistatin A, chemical genetics, transcriptomics, and other methods to decouple their function while assessing enzymatic versus structural roles. Unexpectedly, CDK8 and CDK19 regulated different gene sets via distinct mechanisms. CDK8-dependent regulation required its kinase activity, whereas CDK19 governed IFN-γ responses through its scaffolding function (i.e., it was kinase independent). Accordingly, CDK8, not CDK19, phosphorylates the STAT1 transcription factor (TF) during IFN-γ stimulation, and CDK8 kinase inhibition blocked activation of JAK-STAT pathway TFs. Cytokines such as IFN-γ rapidly mobilize TFs to "reprogram" cellular transcription; our results implicate CDK8 and CDK19 as essential for this transcriptional reprogramming.
Asunto(s)
Quinasa 8 Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Fibroblastos/efectos de los fármacos , Interferón gamma/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Quinasa 8 Dependiente de Ciclina/genética , Quinasas Ciclina-Dependientes/genética , Fibroblastos/enzimología , Fibroblastos/virología , Células HCT116 , Interacciones Huésped-Patógeno , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , ARN Polimerasa II/metabolismo , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , Vesiculovirus/patogenicidadRESUMEN
Vesicular stomatitis (VS) is a notifiable disease of livestock affecting cattle, horses, pigs and humans. Vesicular stomatitis virus (VSV) serotypes Indiana and New Jersey are endemic to Central America; however, they also cause sporadic and scattered outbreaks in various countries in South and North America, including the USA. In order to develop an effective experimental challenge model for VSV, we compared the pathogenicity of three VSV serotype Indiana isolates in 36 4-5 week-old pigs. Two bovine isolates of Central American origin and one equine isolate from the USA were used for the experimental infections. Each pig was inoculated with a single isolate by both the intradermal and intranasal routes. Clinical signs of VSV infection were recorded daily for 10 days post-inoculation (days p.i.). Nasal and tonsillar swab samples and blood were collected to monitor immune responses, virus replication and shedding. Post-challenge, characteristic signs of VS were observed, including vesicles on the nasal planum and coronary bands, lameness, loss of hoof walls and pyrexia. Pigs inoculated with the Central American isolates showed consistently more severe clinical signs in comparison to the pigs infected with the USA isolate. Genomic RNA was isolated from the original challenge virus stocks, sequenced and compared to VSV genomes available in GenBank. Comparative genome analysis demonstrated significant differences between the VSV isolate from the USA and the two Central American isolates. Our results indicate that the Central American isolates of VSV serotype Indiana used in this study are more virulent in swine than the USA VSV serotype Indiana isolate and represent good candidate challenge strains for future VSV studies.
Asunto(s)
Modelos Animales de Enfermedad , Estomatitis Vesicular/patología , Estomatitis Vesicular/virología , Vesiculovirus/crecimiento & desarrollo , Vesiculovirus/patogenicidad , Estructuras Animales/patología , Estructuras Animales/virología , Animales , Sangre/virología , Serogrupo , Porcinos , Vesiculovirus/clasificación , Virulencia , Replicación Viral , Esparcimiento de VirusRESUMEN
Host cell metabolism can be modulated by viral infection, affecting viral survival or clearance. Yet the cellular metabolism rewiring mediated by the N 6-methyladenosine (m6A) modification in interactions between virus and host remains largely unknown. Here we report that in response to viral infection, host cells impair the enzymatic activity of the RNA m6A demethylase ALKBH5. This behavior increases the m6A methylation on α-ketoglutarate dehydrogenase (OGDH) messenger RNA (mRNA) to reduce its mRNA stability and protein expression. Reduced OGDH decreases the production of the metabolite itaconate that is required for viral replication. With reduced OGDH and itaconate production in vivo, Alkbh5-deficient mice display innate immune response-independent resistance to viral exposure. Our findings reveal that m6A RNA modification-mediated down-regulation of the OGDH-itaconate pathway reprograms cellular metabolism to inhibit viral replication, proposing potential targets for controlling viral infection.
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Adenosina/análogos & derivados , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Inmunidad Innata , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Vesiculovirus/patogenicidad , Replicación Viral , Adenosina/química , Animales , Células Cultivadas , Reprogramación Celular , Humanos , Macrófagos Peritoneales/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Procesamiento Proteico-Postraduccional , Células RAW 264.7 , Interferencia de ARN , Estabilidad del ARN , ARN Mensajero/química , Succinatos , Células THP-1 , Vesiculovirus/fisiologíaRESUMEN
Colorectal cancer (CRC) is the third most common cancer in both men and women. Oncolytic viral-based therapy methods seem to be promising for CRC treatment. Vesicular stomatitis virus (VSV) is considered as a potent candidate in viral therapy for several tumors. VSV particles with mutated matrix (M) protein are capable of initiating cell death cascades while not being harmful to the immune system. In the current study, the effects of the VSV M-protein was investigated on the apoptosis of the colorectal cancer SW480 cell. Wild-type, M51R, and ΔM51 mutants VSV M-protein genes were cloned into the PCDNA3.1 vector and transfected into the SW480 cells. The results of the MTT assay, Western blotting, and Caspase 3, 8, and 9 measurement, illustrated that both wild and M51R mutant M-proteins can destroy the SW480 colorectal cancer cells. DAPI/TUNEL double-staining reconfirmed the apoptotic effects of the M-protein expression. The ΔM51 mutant M-protein is effective likewise M51R, somehow it can be considered as a safer substitution.
Asunto(s)
Apoptosis/fisiología , Vesiculovirus/metabolismo , Proteínas de la Matriz Viral/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Humanos , Proteínas Mutantes/genética , Mutación , Viroterapia Oncolítica/métodos , Estomatitis Vesicular/metabolismo , Vesiculovirus/patogenicidadRESUMEN
Tryptophanyl-tRNA synthetase (WRS) is one of the aminoacyl-tRNA synthetases (ARSs) that possesses noncanonical functions. Full-length WRS is released during bacterial infection and primes the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex to elicit innate immune responses. However, the role of WRS in viral infection remains unknown. Here, we show that full-length WRS is secreted by immune cells in the early phase of viral infection and functions as an antiviral cytokine. Treatment of cells with recombinant WRS protein promotes the production of inflammatory cytokines and type I interferons (IFNs) and curtails virus replication in THP-1 and Raw264.7 cells but not in TLR4-/- or MD2-/- bone marrow-derived macrophages (BMDMs). Intravenous and intranasal administration of recombinant WRS protein induces an innate immune response and blocks viral replication in vivo These findings suggest that secreted full-length WRS has a noncanonical role in inducing innate immune responses to viral infection as well as to bacterial infection.IMPORTANCE ARSs are essential enzymes in translation that link specific amino acids to their cognate tRNAs. In higher eukaryotes, some ARSs possess additional, noncanonical functions in the regulation of cell metabolism. Here, we report a novel noncanonical function of WRS in antiviral defense. WRS is rapidly secreted in response to viral infection and primes the innate immune response by inducing the secretion of proinflammatory cytokines and type I IFNs, resulting in the inhibition of virus replication both in vitro and in vivo Thus, we consider WRS to be a member of the antiviral innate immune response. The results of this study enhance our understanding of host defense systems and provide additional information on the noncanonical functions of ARSs.
Asunto(s)
Infecciones por Rhabdoviridae/inmunología , Triptófano-ARNt Ligasa/genética , Triptófano-ARNt Ligasa/metabolismo , Vesiculovirus/patogenicidad , Administración Intranasal , Administración Intravenosa , Animales , Línea Celular , Citocinas/metabolismo , Células HEK293 , Células HeLa , Humanos , Inmunidad Innata , Interferón Tipo I/metabolismo , Ratones , Células RAW 264.7 , Infecciones por Rhabdoviridae/genética , Células THP-1 , Triptófano-ARNt Ligasa/administración & dosificación , Vesiculovirus/inmunologíaRESUMEN
Among the most important sensors of extracellular danger signals, purinergic receptors have been demonstrated to play crucial roles in host defense against infection. However, the function of P2 receptors in viral infection has been little explored. Here we demonstrated that P2Y13 and its ligand ADP play an important role in protecting hosts from viral infections. First, we demonstrate that P2Y13, as a typical interferon-stimulated gene, is induced together with extracellular ADP during viral infection. Most importantly, extracellular ADP restricts the replication of different kinds of viruses, including vesicular stomatitis virus, Newcastle disease virus, herpes simplex virus 1, and murine leukemia virus. This kind of protection is dependent on P2Y13 but not P2Y1 or P2Y12, which are also considered as receptors for ADP. Furthermore, cyclic adenosine monophosphate and EPAC1 are downregulated by extracellular ADP through the P2Y13-coupled Gi alpha subunit. Accordingly, inhibition or deletion of EPAC1 significantly eliminates ADP/P2Y13-mediated antiviral activities. Taken together, our results show that P2Y13 and ADP play pivotal roles in the clearance of invaded virus and have the potential as antiviral targets.