ABSTRACT
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.
Subject(s)
Mice, Knockout , Neurons , RNA-Binding Proteins , Vesicular Stomatitis , Animals , Mice , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Neurons/virology , Neurons/metabolism , Vesicular Stomatitis/virology , Vesicular Stomatitis/immunology , Vesicular Stomatitis/genetics , Virus Replication , Vesiculovirus/immunology , Vesiculovirus/genetics , Mice, Inbred C57BL , Vesicular stomatitis Indiana virus/immunology , Vesicular stomatitis Indiana virus/genetics , Apoptosis Regulatory ProteinsABSTRACT
We present the first complete stochastic model of vesicular stomatitis virus (VSV) intracellular replication. Previous models developed to capture VSV's intracellular replication have either been ODE-based or have not represented the complete replicative cycle, limiting our ability to understand the impact of the stochastic nature of early cellular infections on virion production between cells and how these dynamics change in response to mutations. Our model accurately predicts changes in mean virion production in gene-shuffled VSV variants and can capture the distribution of the number of viruses produced. This model has allowed us to enhance our understanding of intercellular variability in virion production, which appears to be influenced by the duration of the early phase of infection, and variation between variants, arising from balancing the time the genome spends in the active state, the speed of incorporating new genomes into virions, and the production of viral components. Being a stochastic model, we can also assess other effects of mutations beyond just the mean number of virions produced, including the probability of aborted infections and the standard deviation of the number of virions produced. Our model provides a biologically interpretable framework for studying the stochastic nature of VSV replication, shedding light on the mechanisms underlying variation in virion production. In the future, this model could enable the design of more complex viral phenotypes when attenuating VSV, moving beyond solely considering the mean number of virions produced.
Subject(s)
Vesicular Stomatitis , Animals , Vesicular Stomatitis/genetics , Vesicular stomatitis Indiana virus/genetics , Virion/genetics , Virus Replication/genetics , MutationABSTRACT
Viruses that are transmitted by arthropods, or arboviruses, have evolved to successfully navigate both the invertebrate and vertebrate hosts, including their immune systems. Biting midges transmit several arboviruses including vesicular stomatitis virus (VSV). To study the interaction between VSV and midges, we characterized the transcriptomic responses of VSV-infected and mock-infected Culicoides sonorensis cells at 1, 8, 24, and 96 h post inoculation (HPI). The transcriptomic response of VSV-infected cells at 1 HPI was significant, but by 8 HPI there were no detectable differences between the transcriptome profiles of VSV-infected and mock-infected cells. Several genes involved in immunity were upregulated (ATG2B and TRAF4) or downregulated (SMAD6 and TOLL7) in VSV-treated cells at 1 HPI. These results indicate that VSV infection in midge cells produces an early immune response that quickly wanes, giving insight into in vivo C. sonorensis VSV tolerance that may underlie their permissiveness as vectors for this virus.
Subject(s)
Arboviruses , Ceratopogonidae , Vesicular Stomatitis , Animals , Transcriptome , Ceratopogonidae/genetics , Vesicular Stomatitis/genetics , Insect Vectors , Vesiculovirus/genetics , Arboviruses/genetics , Vesicular stomatitis Indiana virus/geneticsABSTRACT
The phospho- (P) protein, the co-factor of the RNA polymerase large (L) protein, of vesicular stomatitis virus (VSV, a prototype of nonsegmented negative-strand RNA viruses) plays pivotal roles in transcription and replication. However, the precise mechanism underlying the transcriptional transactivation by the P protein has remained elusive. Here, using an in vitro transcription system and a series of deletion mutants of the P protein, we mapped a region encompassing residues 51-104 as a transactivation domain (TAD) that is critical for terminal de novo initiation, the initial step of synthesis of the leader RNA and anti-genome/genome, with the L protein. Site-directed mutagenesis revealed that conserved amino acid residues in three discontinuous L-binding sites within the TAD are essential for the transactivation activity of the P protein or important for maintaining its full activity. Importantly, relative inhibitory effects of TAD point mutations on synthesis of the full-length leader RNA and mRNAs from the 3'-terminal leader region and internal genes, respectively, of the genome were similar to those on terminal de novo initiation. Furthermore, any of the examined TAD mutations did not alter the gradient pattern of mRNAs synthesized from internal genes, nor did they induce the production of readthrough transcripts. These results suggest that these TAD mutations impact mainly terminal de novo initiation but rarely other steps (e.g., elongation, termination, internal initiation) of single-entry stop-start transcription. Consistently, the mutations of the essential or important amino acid residues within the P TAD were lethal or deleterious to VSV replication in host cells. IMPORTANCE RNA-dependent RNA polymerase L proteins of nonsegmented negative-strand RNA viruses belonging to the Mononegavirales order require their cognate co-factor P proteins or their counterparts for genome transcription and replication. However, exact roles of these co-factor proteins in modulating functions of L proteins during transcription and replication remain unknown. In this study, we revealed that three discrete L-binding motifs within a transactivation domain of the P protein of vesicular stomatitis virus, a prototypic nonsegmented negative-strand RNA virus, are required for terminal de novo initiation mediated by the L protein, which is the first step of synthesis of the leader RNA as well as genome/anti-genome.
Subject(s)
Vesicular Stomatitis , Animals , Vesicular Stomatitis/genetics , Transcriptional Activation , RNA, Viral/genetics , RNA, Viral/metabolism , Vesiculovirus/metabolism , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/metabolism , RNA, Messenger/genetics , Amino Acids/genetics , Transcription, Genetic , Virus Replication/geneticsABSTRACT
As a single-stranded RNA virus, vesicular stomatitis virus (VSV) causes influenza-like clinical symptoms in infected individuals. Type-I interferon signaling pathway plays a vital role in inhibiting VSV replication. It has been shown that RNF114 (RING finger protein 114) acts as an E3 ubiquitin ligase to regulate the type-I interferon signaling pathway. In contrast, the effects of RNF114 from Chinese sturgeon or sea perch remain controversial. In the present study, we reported the effect of human RNF114 on VSV infection. Overexpression of RNF114 promoted VSV replication, while depletion of RNF114 reduced viral replication. We further found that RNF114 inhibited type-I interferon production via interacting with mitochondrial antiviral signaling protein (MAVS). Moreover, in vivo experiments demonstrated that RNF114 could also accelerate VSV replication and virus-induced inflammation in lung tissues. Collectively, our findings supported that RNF114 negatively regulated the type-I interferon signaling pathway during VSV replication, providing novel and favorable insights into clinical treatment of VSV infection.
Subject(s)
Interferon Type I , Intracellular Signaling Peptides and Proteins/metabolism , Vesicular Stomatitis , Animals , Humans , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Vesicular Stomatitis/genetics , Vesicular Stomatitis/metabolism , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/metabolism , Vesiculovirus , Virus ReplicationABSTRACT
The matrix (M) protein of vesicular stomatitis virus (VSV) has a complex role in infection and immune evasion, particularly with respect to suppression of Type I interferon (IFN). Viral strains bearing the wild-type (wt) M protein are able to suppress Type I IFN responses. We recently reported that the 22-25 strain of VSV encodes a wt M protein, however its sister plaque isolate, strain 22-20, carries a M[MD52G] mutation that perturbs the ability of the M protein to block NFκB, but not M-mediated inhibition of host transcription. Therefore, although NFκB is activated in 22-20 infected murine L929 cells infected, no IFN mRNA or protein is produced. To investigate the impact of the M[D52G] mutation on immune evasion by VSV, we used transcriptomic data from L929 cells infected with wt, 22-25, or 22-20 to define parameters in a family of executable logical models with the aim of discovering direct targets of viruses encoding a wt or mutant M protein. After several generations of pruning or fixing hypothetical regulatory interactions, we identified specific predicted targets of each strain. We predict that wt and 22-25 VSV both have direct inhibitory actions on key elements of the NFκB signaling pathway, while 22-20 fails to inhibit this pathway.
Subject(s)
Computational Biology/methods , Fibroblasts/metabolism , Mutant Proteins/metabolism , NF-kappa B/metabolism , Transcriptome , Vesicular Stomatitis/metabolism , Viral Matrix Proteins/metabolism , Animals , Fibroblasts/virology , Interferon Type I/metabolism , Mice , Mutant Proteins/genetics , NF-kappa B/genetics , Vesicular Stomatitis/genetics , Vesicular Stomatitis/virology , Vesicular stomatitis Indiana virus/physiology , Viral Matrix Proteins/geneticsABSTRACT
The ongoing pandemic of coronavirus (CoV) disease 2019 (COVID-19) continues to exert a significant burden on health care systems worldwide. With limited treatments available, vaccination remains an effective strategy to counter transmission of severe acute respiratory syndrome CoV 2 (SARS-CoV-2). Recent discussions concerning vaccination strategies have focused on identifying vaccine platforms, number of doses, route of administration, and time to reach peak immunity against SARS-CoV-2. Here, we generated a single-dose, fast-acting vesicular stomatitis virus (VSV)-based vaccine derived from the licensed Ebola virus (EBOV) vaccine rVSV-ZEBOV, expressing the SARS-CoV-2 spike protein and the EBOV glycoprotein (VSV-SARS2-EBOV). Rhesus macaques vaccinated intramuscularly (i.m.) with a single dose of VSV-SARS2-EBOV were protected within 10 days and did not show signs of COVID-19 pneumonia. In contrast, intranasal (i.n.) vaccination resulted in limited immunogenicity and enhanced COVID-19 pneumonia compared to results for control animals. While both i.m. and i.n. vaccination induced neutralizing antibody titers, only i.m. vaccination resulted in a significant cellular immune response. RNA sequencing data bolstered these results by revealing robust activation of the innate and adaptive immune transcriptional signatures in the lungs of i.m. vaccinated animals only. Overall, the data demonstrate that VSV-SARS2-EBOV is a potent single-dose COVID-19 vaccine candidate that offers rapid protection based on the protective efficacy observed in our study. IMPORTANCE The vesicular stomatitis virus (VSV) vaccine platform rose to fame in 2019, when a VSV-based Ebola virus (EBOV) vaccine was approved by the European Medicines Agency and the U.S. Food and Drug Administration for human use against the deadly disease. Here, we demonstrate the protective efficacy of a VSV-EBOV-based COVID-19 vaccine against challenge in nonhuman primates (NHPs). When a single dose of the VSV-SARS2-EBOV vaccine was administered intramuscularly (i.m.), the NHPs were protected from COVID-19 within 10 days. In contrast, if the vaccine was administered intranasally, there was no benefit from the vaccine and the NHPs developed pneumonia. The i.m. vaccinated NHPs quickly developed antigen-specific IgG, including neutralizing antibodies. Transcriptional analysis highlighted the development of protective innate and adaptive immune responses in the i.m. vaccination group only.
Subject(s)
COVID-19 Vaccines , COVID-19 , Ebola Vaccines , Ebolavirus , Macaca mulatta , Vesicular Stomatitis , Animals , Antibodies, Viral/genetics , Antibodies, Viral/immunology , COVID-19/prevention & control , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/therapeutic use , Ebola Vaccines/genetics , Ebola Vaccines/immunology , Ebola Vaccines/therapeutic use , Ebolavirus/genetics , Ebolavirus/immunology , Hemorrhagic Fever, Ebola/genetics , Hemorrhagic Fever, Ebola/immunology , Hemorrhagic Fever, Ebola/prevention & control , Macaca mulatta/immunology , SARS-CoV-2 , Vaccination/methods , Vesicular Stomatitis/genetics , Vesicular Stomatitis/immunology , Vesicular Stomatitis/prevention & control , Vesiculovirus/geneticsABSTRACT
Interferon regulatory factor 3 (IRF3) is an essential transductor for initiation of many immune responses. Here, we show that lncRNA-ISIR directly binds IRF3 to promote its phosphorylation, dimerization, and nuclear translocation, along with enhanced target gene productions. In vivo lncRNA-ISIR deficiency results in reduced IFN production, uncontrolled viral replication, and increased mortality. The human homolog, AK131315, also binds IRF3 and promotes its activation. More important, AK131315 expression is positively correlated with type I interferon (IFN-I) level and severity in patients with lupus. Mechanistically, in resting cells, IRF3 is bound to suppressor protein Flightless-1 (Fli-1), which keeps its inactive state. Upon infection, IFN-I-induced lncRNA-ISIR binds IRF3 at DNA-binding domain in cytoplasm and removes Fli-1's association from IRF3, consequently facilitating IRF3 activation. Our results demonstrate that IFN-I-inducible lncRNA-ISIR feedback strengthens IRF3 activation by removing suppressive Fli-1 in immune responses, revealing a method of lncRNA-mediated modulation of transcription factor (TF) activation.
Subject(s)
Interferon Regulatory Factor-3/metabolism , Lupus Erythematosus, Systemic/metabolism , Macrophages, Peritoneal/metabolism , RNA, Long Noncoding/metabolism , Vesicular Stomatitis/metabolism , Animals , Case-Control Studies , Chlorocebus aethiops , Disease Models, Animal , Gene Silencing , HEK293 Cells , Humans , Interferon Regulatory Factor-3/genetics , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/virology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Proto-Oncogene Protein c-fli-1/genetics , Proto-Oncogene Protein c-fli-1/metabolism , RAW 264.7 Cells , RNA, Long Noncoding/genetics , Receptor, Interferon alpha-beta/genetics , Receptor, Interferon alpha-beta/metabolism , Vero Cells , Vesicular Stomatitis/genetics , Vesicular Stomatitis/immunology , Vesicular Stomatitis/virology , Vesicular stomatitis Indiana virus/immunology , Vesicular stomatitis Indiana virus/pathogenicityABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Immunity, Innate , Tripartite Motif Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Vesicular Stomatitis/metabolism , Vesiculovirus/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Animals , Chlorocebus aethiops , Cytokines/genetics , Cytokines/immunology , Cytokines/metabolism , HEK293 Cells , HaCaT Cells , Host-Pathogen Interactions , Humans , Mice, Knockout , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Signal Transduction , THP-1 Cells , Tripartite Motif Proteins/genetics , Tripartite Motif Proteins/immunology , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/immunology , Ubiquitination , Vero Cells , Vesicular Stomatitis/genetics , Vesicular Stomatitis/immunology , Vesicular Stomatitis/virology , Vesiculovirus/pathogenicityABSTRACT
Interferons induce cell-intrinsic responses associated with resistance to viral infection. To overcome the suppressive action of interferons and their effectors, viruses have evolved diverse mechanisms. Using vesicular stomatitis virus (VSV), we report that the host cell N6-adenosine messenger RNA (mRNA) cap methylase, phosphorylated C-terminal domain interacting factor 1 (PCIF1), attenuates the antiviral response. We employed cell-based and in vitro biochemical assays to demonstrate that PCIF1 efficiently modifies VSV mRNA cap structures to m7Gpppm6Am and define the substrate requirements for this modification. Functional assays revealed that the PCIF1-dependent modification of VSV mRNA cap structures is inert with regard to mRNA stability, translation, and viral infectivity but attenuates the antiviral effects of the treatment of cells with interferon-ß. Cells lacking PCIF1 or expressing a catalytically inactive PCIF1 exhibit an augmented inhibition of viral replication and gene expression following interferon-ß treatment. We further demonstrate that the mRNA cap structures of rabies and measles viruses are also modified by PCIF1 to m7Gpppm6Am This work identifies a function of PCIF1 and cap-proximal m6Am in attenuation of the host response to VSV infection that likely extends to other viruses.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Interferon-beta/immunology , Nuclear Proteins/metabolism , RNA Caps/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , Vesicular Stomatitis/immunology , Vesicular stomatitis Indiana virus/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Host-Pathogen Interactions , Humans , Interferon-beta/genetics , Methylation , Nuclear Proteins/genetics , Nuclear Proteins/immunology , RNA Caps/genetics , RNA Stability , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Viral/chemistry , RNA, Viral/genetics , Vesicular Stomatitis/genetics , Vesicular Stomatitis/metabolism , Vesicular Stomatitis/virology , Vesicular stomatitis Indiana virus/chemistry , Vesicular stomatitis Indiana virus/genetics , Virus ReplicationABSTRACT
Rationale: Hosts defend against viral infection by sensing viral pathogen-associated molecular patterns and activating antiviral innate immunity through TBK1-IRF3 signaling. However, the underlying molecular mechanism remains unclear. Methods: SiRNAs targeting Sirt1-7 were transfected into macrophages to screen the antiviral function. Sirt5 deficient mice or macrophages were subjected to viral infection to assess in vivo and in vitro function of Sirt5 by detecting cytokines, viral replicates and survival rate. Immunoprecipitation, WesternBlot and luciferase reporter assay were used to reveal molecular mechanism. Results: In this study, we functionally screened seven Sirtuin family members, and found that Sirtuin5 (Sirt5) promotes antiviral signaling and responses. Sirt5 deficiency leads to attenuated antiviral innate immunity in vivo and in vitro upon viral infection by decreasing TBK1-IRF3 activation and type I IFN production. Sirt5 overexpression increased antiviral innate immunity. Mechanism investigation revealed that Sirt5 interacts with DDX3 and demalonylates DDX3, which is critical for TBK1-IRF3 activation. Mutation of the demalonylation lysine sites (K66, K130, and K162) of DDX3 increased ifnß transcription. Furthermore, the acetylation on lysine 118 of DDX3 positively regulated ifnß transcription, whereas Sirt5 could not deacetylate this site. Conclusion: Sirt5 promotes anti- RNA and DNA virus innate immune responses by increasing TBK1 signaling through demalonylating DDX3, which identifies a novel regulatory pathway of antiviral innate immune response.
Subject(s)
DEAD-box RNA Helicases/immunology , Immunity, Innate , Macrophages/immunology , Sirtuins/immunology , Vesicular Stomatitis/immunology , Vesicular stomatitis Indiana virus/immunology , Animals , DEAD-box RNA Helicases/genetics , HEK293 Cells , Humans , Lipoylation/genetics , Lipoylation/immunology , Macrophages/virology , Mice , Mice, Knockout , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/immunology , RAW 264.7 Cells , Sirtuins/genetics , Vesicular Stomatitis/genetics , Vesicular stomatitis Indiana virus/geneticsABSTRACT
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.
Subject(s)
Adenosine/analogs & derivatives , Protein Biosynthesis , RNA Processing, Post-Transcriptional , Transcription, Genetic , Vesicular Stomatitis/genetics , Vesiculovirus/pathogenicity , A549 Cells , Adenosine/metabolism , Animals , Antigens, Differentiation/biosynthesis , Antigens, Differentiation/genetics , Antiviral Agents/pharmacology , Chlorocebus aethiops , HEK293 Cells , Host-Pathogen Interactions , Humans , Interferon-beta/pharmacology , Methyltransferases/biosynthesis , Methyltransferases/genetics , Protein Biosynthesis/drug effects , RNA Processing, Post-Transcriptional/drug effects , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Transcription, Genetic/drug effects , Vero Cells , Vesicular Stomatitis/metabolism , Vesicular Stomatitis/virology , Vesiculovirus/growth & development , Virus ReplicationABSTRACT
Vesicular stomatitis virus (VSV) is an archetypal member of Mononegavirales which causes important diseases in cattle, horses and pigs. The matrix protein (M) of VSV plays critical roles in the replication, assembly/budding and pathogenesis of VSV. To further investigate the role of M during viral growth, we used a two-hybrid system to screen for host factors that interact with the M protein. Here, NADH: ubiquinone oxidoreductase complex assembly factor 4 (Ndufaf4) was identified as an M-binding partner, and this interaction was confirmed by yeast cotransformation and GST pulldown assays. The globular domain of M was mapped and shown to be critical for the M-Ndufaf4 interaction. Two double mutations (E156A/H157A, D180A/E181A) in M impaired the M-Ndufaf4 interaction. Overexpression of Ndufaf4 inhibited VSV propagation, and knockdown of Ndufaf4 by short hairpin RNA (shRNA) markedly promoted VSV replication. Finally, we also demonstrate that the anti-VSV effect of Ndufaf4 is independent of activation of the type I IFN response. These results indicated that Ndufaf4 might exploit other mechanisms to affect VSV replication. In summary, we identify Ndufaf4 as a potential target for the inhibition of VSV propagation. These results provided further insight into the study of VSV pathogenesis.
Subject(s)
Calmodulin-Binding Proteins/genetics , Host-Pathogen Interactions/genetics , Vesicular Stomatitis/genetics , Viral Matrix Proteins/genetics , Animals , Cattle , Horses/virology , Humans , Mutation/genetics , Swine/virology , Vesicular Stomatitis/virology , Virus Replication/geneticsABSTRACT
We describe the production of single-cycle (sc) and replication-competent recombinant vesicular stomatitis viruses (rcVSVs) displaying heterologous envelope glycoproteins (Envs) on their surface. We prepare scVSVs by transiently expressing HIV-1 Envs or SARS-CoV-2 spike followed by infection of the cells with scVSV particles, which do not carry the vsv-g gene. To prepare rcVSVs, we replace the vsv-g with a specific env-encoding gene, transfect cells with multiple plasmids for production of the genomic RNA and viral proteins, and rescue replication-competent viruses.
Subject(s)
Recombinant Proteins , Spike Glycoprotein, Coronavirus , Vesicular Stomatitis/genetics , env Gene Products, Human Immunodeficiency Virus , Animals , COVID-19/virology , Cell Line , Cricetinae , HIV-1/genetics , Humans , Protein Engineering , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , env Gene Products, Human Immunodeficiency Virus/chemistry , env Gene Products, Human Immunodeficiency Virus/genetics , env Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
The matrix (M) protein of vesicular stomatitis virus (VSV) plays a key role in immune evasion. While VSV has been thought to suppress the interferon (IFN) response primarily by inhibiting host cell transcription and translation, our recent findings indicate that the M protein also targets NF-κB activation. Therefore, the M protein may utilize two distinct mechanisms to limit expression of antiviral genes, inhibiting both host gene expression and NF-κB activation. Here we characterize a recently reported mutation in the M protein [M(D52G)] of VSV isolate 22-20, which suppressed IFN mRNA and protein production despite activating NF-κB. 22-20 inhibited reporter gene expression from multiple promoters, suggesting that 22-20 suppressed the IFN response via M-mediated inhibition of host cell transcription. We propose that suppression of the IFN response and regulation of NF-κB are independent, genetically separable functions of the VSV M protein.
Subject(s)
Interferon-beta/immunology , NF-kappa B/immunology , Vesicular Stomatitis/immunology , Vesicular stomatitis Indiana virus/immunology , Viral Matrix Proteins/immunology , Animals , Cell Line , Gene Expression Regulation , Host-Pathogen Interactions , Humans , Interferon-beta/genetics , Mice , NF-kappa B/genetics , Vesicular Stomatitis/genetics , Vesicular Stomatitis/virology , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/physiology , Viral Matrix Proteins/geneticsABSTRACT
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.
Subject(s)
Antigens, CD/metabolism , Dendritic Cells/virology , Immunity, Innate , Integrin alpha Chains/metabolism , Interferon Type I/metabolism , Vesicular Stomatitis/virology , Vesiculovirus/pathogenicity , Animals , Antigens, CD/genetics , Cells, Cultured , Dendritic Cells/immunology , Dendritic Cells/metabolism , Disease Models, Animal , Genome-Wide Association Study , Host-Pathogen Interactions , Integrin alpha Chains/genetics , Mice, 129 Strain , Mice, Inbred AKR , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Inbred NOD , Mice, Knockout , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Receptor, Interferon alpha-beta/genetics , Receptor, Interferon alpha-beta/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Vesicular Stomatitis/genetics , Vesicular Stomatitis/immunology , Vesicular Stomatitis/metabolism , Vesiculovirus/growth & development , Virus ReplicationABSTRACT
Vesicular stomatitis Indiana virus (VSIV), formerly known as vesicular stomatitis virus (VSV) Indiana (VSVIND), is a model virus that is exceptionally sensitive to the inhibitory action of interferons (IFNs). Interferons induce an antiviral state by stimulating the expression of hundreds of interferon-stimulated genes (ISGs). These ISGs can constrain viral replication, limit tissue tropism, reduce pathogenicity, and inhibit viral transmission. Since VSIV is used as a backbone for multiple oncolytic and vaccine strategies, understanding how ISGs restrict VSIV not only helps in understanding VSIV-induced pathogenesis but also helps us evaluate and understand the safety and efficacy of VSIV-based therapies. Thus, there is a need to identify and characterize the ISGs that possess anti-VSIV activity. Using arrayed ISG expression screening, we identified TRIM69 as an ISG that potently inhibits VSIV. This inhibition was highly specific as multiple viruses, including influenza A virus, HIV-1, Rift Valley fever virus, and dengue virus, were unaffected by TRIM69. Indeed, just one amino acid substitution in VSIV can govern sensitivity/resistance to TRIM69. Furthermore, TRIM69 is highly divergent in human populations and exhibits signatures of positive selection that are consistent with this gene playing a key role in antiviral immunity. We propose that TRIM69 is an IFN-induced inhibitor of VSIV and speculate that TRIM69 could be important in limiting VSIV pathogenesis and might influence the specificity and/or efficacy of vesiculovirus-based therapies.IMPORTANCE Vesicular stomatitis Indiana virus (VSIV) is a veterinary pathogen that is also used as a backbone for many oncolytic and vaccine strategies. In natural and therapeutic settings, viral infections like VSIV are sensed by the host, and as a result the host cells make proteins that can protect them from viruses. In the case of VSIV, these antiviral proteins constrain viral replication and protect most healthy tissues from virus infection. In order to understand how VSIV causes disease and how healthy tissues are protected from VSIV-based therapies, it is crucial that we identify the proteins that inhibit VSIV. Here, we show that TRIM69 is an antiviral defense that can potently and specifically block VSIV infection.
Subject(s)
Host-Pathogen Interactions , Tripartite Motif Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Vesicular Stomatitis/metabolism , Vesicular Stomatitis/virology , Vesicular stomatitis Indiana virus/physiology , Virus Replication , Alleles , Amino Acid Sequence , Animals , Antiviral Agents/pharmacology , Dengue Virus/physiology , Disease Resistance , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Interferons/metabolism , Interferons/pharmacology , Multigene Family , Phosphorylation , Signal Transduction , Tripartite Motif Proteins/chemistry , Tripartite Motif Proteins/genetics , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Vesicular Stomatitis/genetics , Vesicular Stomatitis/immunologyABSTRACT
Precise control of interferons (IFNs) is crucial to maintain immune homeostasis. Here, we demonstrated that homeodomain-interacting protein kinase 2 (HIPK2) was required for the production of type I IFNs in response to RNA virus infection. HIPK2 deficiency markedly impaired IFN production in macrophages after vesicular stomatitis virus (VSV) infection, and HIPK2-deficient mice were more susceptible to lethal VSV disease than were wild-type mice. After VSV infection, HIPK2 was cleaved by active caspases, which released a hyperactive, N-terminal fragment that translocated to the nucleus and further augmented antiviral responses. In part, HIPK2 interacted with ELF4 and promoted its phosphorylation at Ser369, which enabled Ifn-b transcription. In addition, HIPK2 production was stimulated by type I IFNs to further enhance antiviral immunity. These data suggest that the kinase activity and nuclear localization of HIPK2 are essential for the production of type I IFNs.
Subject(s)
Cell Nucleus/immunology , Interferon Type I/immunology , Protein Serine-Threonine Kinases/immunology , Vesicular Stomatitis/immunology , Vesiculovirus/immunology , Animals , Cell Nucleus/genetics , Cell Nucleus/virology , Chlorocebus aethiops , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , HEK293 Cells , HeLa Cells , Humans , Interferon Type I/genetics , Mice , Mice, Knockout , Protein Serine-Threonine Kinases/genetics , Transcription Factors/genetics , Transcription Factors/immunology , Vero Cells , Vesicular Stomatitis/genetics , Vesicular Stomatitis/pathologyABSTRACT
Intron retention (IR) has emerged as an important mechanism of gene expression control, but the factors controlling IR events remain poorly understood. We observed consistent IR in one intron of the Irf7 gene and identified BUD13 as an RNA-binding protein that acts at this intron to increase the amount of successful splicing. Deficiency in BUD13 was associated with increased IR, decreased mature Irf7 transcript and protein levels, and consequently a dampened type I interferon response, which compromised the ability of BUD13-deficient macrophages to withstand vesicular stomatitis virus (VSV) infection. Global analysis of BUD13 knockdown and BUD13 cross-linking to RNA revealed a subset of introns that share many characteristics with the one found in Irf7 and are spliced in a BUD13-dependent manner. Deficiency of BUD13 led to decreased mature transcript from genes containing such introns. Thus, by acting as an antagonist to IR, BUD13 facilitates the expression of genes at which IR occurs.
Subject(s)
Interferon Regulatory Factor-7/metabolism , Interferon Type I/metabolism , Introns , Macrophages/metabolism , RNA-Binding Proteins/metabolism , Vesicular Stomatitis/metabolism , Vesicular stomatitis Indiana virus/pathogenicity , Animals , Binding Sites , Chlorocebus aethiops , GC Rich Sequence , HEK293 Cells , Host-Pathogen Interactions , Humans , Interferon Regulatory Factor-7/genetics , Interferon Type I/immunology , Macrophages/immunology , Macrophages/virology , Mice, Inbred C57BL , Protein Binding , RNA Splice Sites , RNA Splicing , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Vero Cells , Vesicular Stomatitis/genetics , Vesicular Stomatitis/immunology , Vesicular Stomatitis/virology , Vesicular stomatitis Indiana virus/immunologyABSTRACT
Vesicular stomatitis virus (VSV) is an oncolytic rhabdovirus and its glycoprotein G is widely used to pseudotype other viruses for gene therapy. Low-density lipoprotein receptor (LDL-R) serves as a major entry receptor for VSV. Here we report two crystal structures of VSV G in complex with two distinct cysteine-rich domains (CR2 and CR3) of LDL-R, showing that their binding sites on G are identical. We identify two basic residues on G, which are essential for its interaction with CR2 and CR3. Mutating these residues abolishes VSV infectivity even though VSV can use alternative receptors, indicating that all VSV receptors are members of the LDL-R family. Collectively, our data suggest that VSV G has specifically evolved to interact with receptor CR domains. These structural insights into the interaction between VSV G and host cell receptors provide a basis for the design of recombinant viruses with an altered tropism.