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ISG15 facilitates cellular antiviral response to dengue and west nile virus infection in vitro.

ABSTRACT: Dengue virus (DENV) and West Nile virus (WNV), close siblings of the Flaviviridae family, are the causative agents of Dengue hemorraghic shock or West Nile meningoencephalitis respectively. Vaccines against these two flaviviruses are currently unavailable. Interferon- Stimulated Gene 15 (ISG15), encoding an ubiquitin-like protein, is significantly induced by type I interferons or viral infections. Its roles in viral infections, however, vary with viruses, being either anti- or pro-viral. The exact roles of ISG15 in DENV and WNV infections remain unknown. In the current study, we evaluated the relevancies of ISG15 to DENV and WNV infection of a mouse macrophage cell line RAW264.7.Quantitative PCR showed that mouse Isg15 was dramatically induced in DENV or WNV- infected RAW264.7 cells compared with non-infected cells. Isg15 and two other Jak-Stat related genes, Socs1 and Socs3, were silenced using siRNA mediated RNA interference. The intracellular DENV and WNV loads, as determined by quantitative PCR, were significantly higher in Isg15 silenced cells than control cells. The expression levels of interferon beta 1 (Ifnb1) were increased significantly in Isg15, Socs1 or Socs3 siRNA treated cells. Further investigation indicated that protein modification by ISG15, so called ISGylation, was significantly enhanced in DENV-infected cells compared to that in non-infected cells.These findings suggest that ISG15 plays an anti-DENV/WNV function via protein ISGylation.

SUBMITTER: Dai J

PROVIDER: S-EPMC3215395 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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ISG15 facilitates cellular antiviral response to dengue and west nile virus infection in vitro.

Dai Jianfeng J Pan Wen W Wang Penghua P

Virology journal 20111013

<h4>Background</h4>Dengue virus (DENV) and West Nile virus (WNV), close siblings of the Flaviviridae family, are the causative agents of Dengue hemorraghic shock or West Nile meningoencephalitis respectively. Vaccines against these two flaviviruses are currently unavailable. Interferon- Stimulated Gene 15 (ISG15), encoding an ubiquitin-like protein, is significantly induced by type I interferons or viral infections. Its roles in viral infections, however, vary with viruses, being either anti- or ...[more]

PMID: 21992229

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Project description:West Nile virus (WNV) is the most prevalent mosquito-borne virus in the United States with approximately 2,000 cases each year. There are currently no approved human vaccines and a lack of prophylactic and therapeutic treatments. Understanding host responses to infection may reveal potential intervention targets to reduce virus replication and disease progression. The use of Drosophila melanogaster as a model organism to understand innate immunity and host antiviral responses is well-established. Previous studies revealed that insulin-mediated signaling regulates WNV infection in invertebrates by regulating canonical antiviral pathways. Because insulin signaling is well-conserved across insect and mammalian species, we sought to determine if results using D. melanogaster can be extrapolated for the analysis of orthologous pathways in humans. Here, we identify insulin-mediated endothelin signaling using the D. melanogaster model and evaluate an orthologous pathway in human cells during WNV infection. We demonstrate that endothelin signaling reduces WNV replication through the activation of canonical antiviral signaling. Taken together, our findings show that endothelin-mediated antiviral immunity is broadly conserved across species and reduces replication of viruses that can cause severe human disease. IMPORTANCE Arboviruses, particularly those transmitted by mosquitoes, pose a significant threat to humans and are an increasing concern because of climate change, human activity, and expanding vector-competent populations. West Nile virus is of significant concern as the most frequent mosquito-borne disease transmitted annually within the continental United States. Here, we identify a previously uncharacterized signaling pathway that impacts West Nile virus infection, namely endothelin signaling. Additionally, we demonstrate that we can successfully translate results obtained from D. melanogaster into the more relevant human system. Our results add to the growing field of insulin-mediated antiviral immunity and identify potential biomarkers or intervention targets to better address West Nile virus infection and severe disease.

Project description:West Nile virus (WNV) is the most prevalent mosquito-borne virus in the United States with approximately 2,000 cases each year. There are currently no approved human vaccines and a lack of prophylactic and therapeutic treatments. Understanding host responses to infection may reveal potential intervention targets to reduce virus replication and disease progression. The use of Drosophila melanogaster as a model organism to understand innate immunity and host antiviral responses is well established. Previous studies revealed that insulin-mediated signaling regulates WNV infection in invertebrates by regulating canonical antiviral pathways. Because insulin signaling is well-conserved across insect and mammalian species, we sought to determine if results using D. melanogaster can be extrapolated for the analysis of orthologous pathways in humans. Here, we identify insulin-mediated endothelin signaling using the D. melanogaster model and evaluate an orthologous pathway in human cells during WNV infection. We demonstrate that endothelin signaling reduces WNV replication through the activation of canonical antiviral signaling. Taken together, our findings show that endothelin-mediated antiviral immunity is broadly conserved across species and reduces replication of viruses that can cause severe human disease.ImportanceArboviruses, particularly those transmitted by mosquitoes, pose a significant threat to humans and are an increasing concern because of climate change, human activity, and expanding vector-competent populations. West Nile virus is of significant concern as the most frequent mosquito-borne disease transmitted annually within the continental United States. Here, we identify a previously uncharacterized signaling pathway that impacts West Nile virus infection, namely endothelin signaling. Additionally, we demonstrate that we can successfully translate results obtained from D. melanogaster into the more relevant human system. Our results add to the growing field of insulin-mediated antiviral immunity and identifies potential biomarkers or intervention targets to better address West Nile virus infection and severe disease.

Project description:Purpose: Several members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways including the antiviral type I interferon (IFN-I) system. Therefore, we aimed to investigate the mechanism of IFN-I regulation upon West Nile virus infection using TRIM6 knockout (TRIM6-KO) human airway epithelial cells (A549). Methods: RNAs were extracted from wild-type (WT) and TRIM6KO A549 cells infected with WNV or mock-treated. RNA quality was assessed by visualization of 18S and 28S RNA bands using an Agilent BioAnalyzer 2100 (Agilent Technologies, CA); the electropherograms were used to calculate the 28S/18S ratio and the RNA Integrity Number. Poly-A+ RNA was enriched from total RNA (1 μg) using oligo dT-attached magnetic beads. First and second strand synthesis, adapter ligation, and amplification of the library were performed using the Illumina TruSeq RNA Sample Preparation kit as recommended by the manufacturer (Illumina, Inc). Library quality was evaluated using an Agilent DNA-1000 chip on an Agilent 2100 Bioanalyzer. Quantification of library DNA templates was performed using qPCR and a known-size reference standard. Cluster formation of the library DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation using conditions recommended by the manufacturer. Paired end 50 base sequencing by synthesis was performed using TruSeq SBS kit v3 (Illumina) on an Illumina HiSeq 1500 using protocols defined by the manufacturer. The alignment of NGS sequence reads was performed using the Spliced Transcript Alignment to a Reference (STAR) program, version 2.5.1b, using default parameters. We used the human hg38 assembly as a reference with the UCSC gene annotation file. The –quantMode GeneCounts option of STAR provided read counts per gene, which were input into the DESeq2 (version 1.12.1) differential expression analysis program to determine expression levels and differentially expressed genes. Processed data are presented as reads per kilobase transcript per million (RPKM). Results: Using next generation sequencing, we identified VAMP8 as a factor involved in this TRIM6-mediated antiviral response. In support, VAMP8 knockdown resulted in reduced Jak1 and STAT1 phosphorylation and impaired induction of several ISGs following WNV infection or IFNβ treatment. Conclusions: Our results provide evidence that TRIM6 contributes to the antiviral response against WNV and identified VAMP8 as a novel regulator of the IFN-I system

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ISG15 facilitates cellular antiviral response to dengue and west nile virus infection in vitro.

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ISG15 facilitates cellular antiviral response to dengue and west nile virus infection in vitro.

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