Visualisation of direct interaction of MDA5 and the dsRNA replicative intermediate form of positive strand RNA viruses

Triantafilou, Kathy; Vakakis, Emmanouil; Kar, Satwik; Richer, Edward; Evans, Gareth Lyndon; Triantafilou, Martha

doi:10.1242/jcs.103887

Cited by 76 publications

(82 citation statements)

References 26 publications

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“…The monoclonal anti-dsRNA J2 antibody binds dsRNAs at least 40 bp long in a sequence-independent fashion (12)(13)(14). The J2 dsRNA monoclonal antibody was previously used to detect viral dsRNA by dsRNA immunoblotting, immunohistochemistry, ELISA, and immunoprecipitation in eukaryotic cells (13)(14)(15)(16)(17)(18)(19)(20). In this study, we used the J2 dsRNA antibody in an immunoprecipitation assay to enrich dsRNA from total RNA extracted from E. coli.…”

Section: Resultsmentioning

confidence: 99%

The double-stranded transcriptome of Escherichia coli

Lybecker

Zimmermann

Bilusic

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

103

146

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Advances in high-throughput transcriptome analyses have revealed hundreds of antisense RNAs (asRNAs) for many bacteria, although few have been characterized, and the number of functional asRNAs remains unknown. We have developed a genomewide high-throughput method to identify functional asRNAs in vivo. Most mechanisms of gene regulation via asRNAs require an RNA-RNA interaction with its target RNA, and we hypothesized that a functional asRNA would be found in a double strand (dsRNA), duplexed with its cognate RNA in a single cell. We developed a method of isolating dsRNAs from total RNA by immunoprecipitation with a ds-RNA specific antibody. Total RNA and immunoprecipitated dsRNA from Escherichia coli RNase III WT and mutant strains were deep-sequenced. A statistical model was applied to filter for biologically relevant dsRNA regions, which were subsequently categorized by location relative to annotated genes. A total of 316 potentially functional asRNAs were identified in the RNase III mutant strain and are encoded primarily opposite to the 5′ ends of transcripts, but are also found opposite ncRNAs, gene junctions, and the 3′ ends. A total of 21 sense/antisense RNA pairs identified in dsRNAs were confirmed by Northern blot analyses. Most of the RNA steady-state levels were higher or detectable only in the RNase III mutant strain. Taken together, our data indicate that a significant amount of dsRNA is formed in the cell, that RNase III degrades or processes these dsRNAs, and that dsRNA plays a major role in gene regulation in E. coli.T he advent and development of high-throughput sequencing technologies has uncovered the presence of widespread antisense transcription in many bacteria, with the number of annotated genes associated with antisense RNA (asRNA) differing greatly among bacterial species (1, 2). asRNAs are encoded on the DNA strand opposite an annotated gene and overlap a portion of a gene or the entire gene, or span multiple genes with perfect complementarity. asRNAs range in size from tens to thousands of nucleotides. Although numerous chromosomally encoded asRNAs have been identified, few have been confirmed by traditional methods or functionally characterized. Raghavan et al. (3) reported that few asRNAs are conserved between Escherichia coli and Salmonella enterica, and that the predicted promoter sequences of the asRNAs are not conserved between these species, suggesting that most asRNA transcripts are products of spurious transcription and are not biologically functional RNAs.The majority of functionally characterized asRNAs are found on plasmids, phages, and transposons (4, 5). The mode of regulation by asRNAs can be classified according to molecular mechanism as transcription interference, transcription attenuation, alteration of transcript stability, and translation inhibition (1, 2, 6). With the exception of transcription interference, a physical RNA-RNA interaction between the sense RNAs and asRNAs is necessary for all of these mechanisms, requiring that both RNAs be expressed in the same...

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Section: Resultsmentioning

confidence: 99%

The double-stranded transcriptome of Escherichia coli

Lybecker

Zimmermann

Bilusic

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

103

146

View full text Add to dashboard Cite

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“…46 The viral ligand for MDA5 is long higher order dsRNA or dsRNA replication intermediates. 21; 47; 48; 49; 50 Interestingly, during infection of West Nile virus, an RNA virus also in the Flaviviridae, both RIG-I and MDA5 contribute to the antiviral response. In this case, RIG-I is important early after infection, while MDA5 is important for signaling during later times of infection.…”

Section: How Is Hcv Sensed As Non-self?mentioning

confidence: 99%

Activation and Evasion of Antiviral Innate Immunity by Hepatitis C Virus

Horner

2014

Journal of Molecular Biology

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Hepatitis C virus (HCV) chronically infects 130-170 million people worldwide and is a major public health burden. HCV is an RNA virus that infects hepatocytes within liver, and this infection is sensed as non-self by the intracellular innate immune response to program antiviral immunity to HCV. HCV encodes several strategies to evade this antiviral response, and this evasion of innate immunity plays a key role in determining viral persistence. This review discusses the molecular mechanisms of how the intracellular innate immune system detects HCV infection, including how HCV pathogen-associated molecular patterns are generated during infection and where they are recognized as foreign by the innate immune system. Further, this review highlights the key innate immune evasion strategies used by HCV to establish persistent infection within the liver, as well as how host genotype influences the outcome of HCV infection. Understanding these HCV-host interactions is key to understanding how to target HCV during infection and for the design of more effective HCV therapies at the immunological level.

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“…MDA5 ligands are less well defined, but MDA5 responds more specifically to long dsRNA that contain branched or higher order structures [6] and senses the absence of 2'-O-methylation at the 5′ cap structure of viral RNA [7]. Direct binding of viral RNA to MDA5 was not yet demonstrated, but colocalization of MDA5 and the replicative dsRNA of enteroviruses was observed using fluorescent imaging [8]. …”

Section: Introductionmentioning

confidence: 99%

Sustained Activation of Interferon Regulatory Factor 3 during Infection by Paramyxoviruses Requires MDA5

et al. 2014

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Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are the main cytosolic sensors of single-stranded RNA viruses, including paramyxoviruses, and are required to initiate a quick and robust innate antiviral response. Despite different ligand-binding properties, the consensus view is that RIG-I and MDA5 trigger common signal(s) to activate interferon regulatory factor 3 (IRF-3) and NF-κB, and downstream antiviral and proinflammatory cytokine expression. Here, we performed a thorough analysis of the temporal involvement of RIG-I and MDA5 in the regulation of IRF-3 during respiratory syncytial virus (RSV) infection. Based on specific RNA interference-mediated knockdown of RIG-I and MDA5 in A549 cells, we confirmed that RIG-I is critical for the initiation of IRF-3 phosphorylation, dimerization and downstream gene expression. On the other hand, our experiments yielded the first evidence that knockdown of MDA5 leads to early ubiquitination and proteasomal degradation of active IRF-3. Conversely, ectopic expression of MDA5 prolonged RIG-I-induced IRF-3 activation. Altogether, we provide novel mechanistic insight into the temporal involvement of RIG-I and MDA5 in the innate antiviral response. While RIG-I is essential for initial IRF-3 activation, engagement of induced MDA5 is essential to prevent early degradation of IRF-3, thereby sustaining IRF-3-dependent antiviral gene expression. MDA5 plays a similar role during Sendai virus infection suggesting that this model is not restricted to RSV amongst paramyxoviruses.

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Visualisation of direct interaction of MDA5 and the dsRNA replicative intermediate form of positive strand RNA viruses

Cited by 76 publications

References 26 publications

The double-stranded transcriptome of Escherichia coli

The double-stranded transcriptome of Escherichia coli

Activation and Evasion of Antiviral Innate Immunity by Hepatitis C Virus

Sustained Activation of Interferon Regulatory Factor 3 during Infection by Paramyxoviruses Requires MDA5

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