Defective Interfering Genomes and the Full-Length Viral Genome Trigger RIG-I After Infection With Vesicular Stomatitis Virus in a Replication Dependent Manner.
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ABSTRACT:
SUBMITTER: Linder A
PROVIDER: S-EPMC8119886 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We have generated replication-competent (VSV-C/E1/E2) and nonpropagating (VSVDeltaG-C/E1/E2) vesicular stomatitis virus (VSV) contiguously expressing the structural proteins of hepatitis C virus (HCV; core [C] and glycoproteins E1 and E2) and report on their immunogenicity in murine models. VSV-C/E1/E2 and VSVDeltaG-C/E1/E2 expressed high levels of HCV C, E1, and E2, which were authentically posttranslationally processed. Both VSV-expressed HCV E1-E2 glycoproteins were found to form noncovalently linked heterodimers and appeared to be correctly folded, as confirmed by coimmunoprecipitation analysis using conformationally sensitive anti-HCV-E2 monoclonal antibodies (MAbs). Intravenous or intraperitoneal immunization of BALB/c mice with VSV-C/E1/E2 or VSVDeltaG-C/E1/E2 resulted in significant and surprisingly comparable HCV core or E2 antibody responses compared to those of control mice. In addition, both virus types generated HCV C-, E1-, or E2-specific gamma interferon (IFN-gamma)-producing CD8(+) T cells, as determined by enzyme-linked immunospot (ELISPOT) analysis. Mice immunized with VSVDeltaG-C/E1/E2 were also protected against the formation of tumors expressing HCV E2 (CT26-hghE2t) and exhibited CT26-hghE2t-specific IFN-gamma-producing and E2-specific CD8(+) T-cell activity. Finally, recombinant vaccinia virus (vvHCV.S) expressing the HCV structural proteins replicated at significantly lower levels when inoculated into mice immunized with VSV-C/E1/E2 or VSVDeltaG-C/E1/E2, but not with control viruses. Our data therefore illustrate that potentially safer replication-defective VSV can be successfully engineered to express high levels of antigenically authentic HCV glycoproteins. In addition, this strategy may therefore serve in effective vaccine and immunotherapy-based approaches to the treatment of HCV-related disease.
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Project description:Vesicular stomatitis virus (VSV): Genome sequencing and assembly
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Project description:Using next-generation sequencing (NGS) combined with bioinformatics tools, we characterized two major 5’copy-back defective interfering (5’cb DI) genomes generated during SAD replication. Furthermore, we identified a specific interaction of 5’cb DI genomes and RIG-I that correlated with a high stimulation of the type I IFN signaling
2024-03-22 | GSE196051 | GEO
Project description:Attenuated measles virus (MV) is one of the most effective and safe vaccines available, making it an attractive candidate vector for preventing other infectious diseases. Yet the great capacity of this vaccine still needs to be understood at the molecular level. MV vaccine strains have different type I interferon (IFN)-inducing abilities that partially depend on the presence of 5' copy-back defective interfering genomes (DI-RNAs). DI-RNAs are pathogen-associated molecular patterns recognized by RIG-I-like receptors (RLRs) (RIG-I, MDA5, and LGP2) that activate innate immune signaling and shape the adaptive immune response. In this study, we characterized the DI-RNAs produced by various modified recombinant MVs (rMVs), including vaccine candidates, as well as wild-type MV. All tested rMVs produced 5' copy-back DI-RNAs that were different in length and nucleotide sequence but still respected the so-called "rule of six." We correlated the presence of DI-RNAs with a larger stimulation of the IFN-β pathway and compared their immunostimulatory potentials. Importantly, we revealed that encapsidation of DI-RNA molecules within the MV nucleocapsid abolished their immunoactive properties. Furthermore, we identified specific interactions of DI-RNAs with both RIG-I and LGP2 but not MDA5. Our results suggest that DI-RNAs produced by rMV vaccine candidates may indeed strengthen their efficiency by triggering RLR signaling.IMPORTANCE Having been administered to hundreds of millions of children, the live attenuated measles virus (MV) vaccine is the safest and most widely used human vaccine, providing high protection with long-term memory. Additionally, recombinant MVs carrying heterologous antigens are promising vectors for new vaccines. The great capacity of this vaccine still needs to be elucidated at the molecular level. Here we document that recombinant MVs produce defective interfering genomes that have high immunostimulatory properties via their binding to RIG-I and LGP2 proteins, both of which are cytosolic nonself RNA sensors of innate immunity. Defective interfering genome production during viral replication should be considered of great importance due to the immunostimulatory properties of these genomes as intrinsic adjuvants produced by the vector that increase recognition by the innate immune system.
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Project description:UnlabelledViruses are dependent on their host cells for replication and thus have evolved in intimate association with them. The identification of host factors required for viral infection has led to advances in both viral and cellular biology. Vesicular stomatitis virus (VSV), a negative-sense RNA virus, replicates in all eukaryotic cells in culture, suggesting that the host requirements for its replication are ubiquitous. In this study, we performed a genome-wide small interfering RNA screen of human cells in culture and identified multiple cellular genes that influence the entry and replication of VSV. From a list of >300 genes, we selected the most promising candidates to perform further analysis to assign their functions to either the entry or intracellular replication step of infection. We implicate 3 new factors in VSV entry and 20 new factors in viral gene expression. These proteins have diverse cellular roles, including S-adenosylmethionine synthesis, respiration, and host translation machinery, underscoring the intimate relationship between VSV and the host cell. Together, these results provide a curated list of genes required for VSV replication.ImportanceReplication of vesicular stomatitis virus (VSV) has long served as a model for understanding host-virus interactions and neuropathogenesis. We performed a genome-wide analysis of host factors and revealed genes critical for viral replication, including some involved in vesicular trafficking, cell cycling, and protein modification. Our results provide an enriched list of host factors that are required for specific stages of VSV entry and gene expression. This study may also potentially expand the repertoire of targets for antiviral therapy against negative-strand RNA viruses.
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Project description:Propagation-defective vesicular stomatitis virus (VSV) vectors that encode a truncated G protein (VSV-Gstem) or lack the G gene entirely (VSV-DeltaG) are attractive vaccine vectors because they are immunogenic, cannot replicate and spread after vaccination, and do not express many of the epitopes that elicit neutralizing anti-VSV immunity. To consider advancing non-propagating VSV vectors towards clinical assessment, scalable technology that is compliant with human vaccine manufacturing must be developed to produce clinical trial material. Accordingly, two propagation methods were developed for VSV-Gstem and VSV-DeltaG vectors encoding HIV gag that have the potential to support large-scale production. One method is based on transient expression of G protein after electroporating plasmid DNA into Vero cells and the second is based on a stable Vero cell line that contains a G gene controlled by a heat shock-inducible transcription unit. Both methods reproducibly supported production of 1 x 10(7) to 1 x 10(8) infectious units (I.U.s) of vaccine vector per milliliter. Results from these studies also showed that optimization of the G gene is necessary for abundant G protein expression from electroporated plasmid DNA or from DNA integrated in the genome of a stable cell line, and that the titers of VSV-Gstem vectors generally exceeded VSV-DeltaG.
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Project description:Infectious disease metagenomics is driven by the question: "what is causing the disease?" in contrast to classical metagenome studies which are guided by "what is out there?" In case of a novel virus, a first step to eventually establishing etiology can be to recover a full-length viral genome from a metagenomic sample. However, retrieval of a full-length genome of a divergent virus is technically challenging and can be time-consuming and costly. Here we discuss different assembly and fragment linkage strategies such as iterative assembly, motif searches, k-mer frequency profiling, coverage profile binning, and other strategies used to recover genomes of potential viral pathogens in a timely and cost-effective manner.
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Project description:Defective interfering (DI) genomes, or defective viral genomes (DVGs), are truncated viral genomes generated during replication of most viruses, including live viral vaccines. Among these, "panhandle" or copy-back (cb) and "hairpin" or snap-back (sb) DI genomes are generated during RNA virus replication. 5' cb/sb DI genomes are highly relevant for viral pathogenesis since they harbor immunostimulatory properties that increase virus recognition by the innate immune system of the host. We have developed DI-tector, a user-friendly and freely available program that identifies and characterizes cb/sb genomes from next-generation sequencing (NGS) data. DI-tector confirmed the presence of 5' cb genomes in cells infected with measles virus (MV). DI-tector also identified a novel 5' cb genome, as well as a variety of 3' cb/sb genomes whose existence had not previously been detected by conventional approaches in MV-infected cells. The presence of these novel cb/sb genomes was confirmed by RT-qPCR and RT-PCR, validating the ability of DI-tector to reveal the landscape of DI genome population in infected cell samples. Performance assessment using different experimental and simulated data sets revealed the robust specificity and sensitivity of DI-tector. We propose DI-tector as a universal tool for the unbiased detection of DI viral genomes, including 5' cb/sb DI genomes, in NGS data.
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Project description: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.
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