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Dicing of viral replication intermediates during silencing of latent Drosophila viruses.

ABSTRACT: Previous studies revealed roles for RNA interference (RNAi) in the immediate cellular response to viral infection in plants, nematodes and flies. However, little is known about how RNAi combats viruses during persistent or latent infections. Our analysis of small RNAs cloned from Drosophila cells latently infected with Flock House Virus (FHV) failed to reveal signatures of bulk degradation of the viral genome. Instead, this + strand virus specifically generated Dicer-2-dependent, 21-nucleotide siRNAs that derived in equal proportion from + and - strands. Curiously, luciferase reporters that are fully complementary to abundant viral siRNAs were poorly repressed. Moreover, although the viral siRNAs that were incorporated into an effector complex associated with Argonaute2, bulk FHV siRNAs in latently infected cells were not loaded into any Argonaute protein. Together, these data suggest that direct dicing of viral replication intermediates plays an important role in maintaining the latent viral state. In addition, the denial of bulk viral siRNAs from effector complexes suggests that criteria beyond the structural competency of RNA duplexes influence the assembly of functional silencing complexes.

SUBMITTER: Flynt A

PROVIDER: S-EPMC2663985 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Dicing of viral replication intermediates during silencing of latent Drosophila viruses.

Flynt Alex A Liu Na N Martin Raquel R Lai Eric C EC

Proceedings of the National Academy of Sciences of the United States of America 20090227 13

Previous studies revealed roles for RNA interference (RNAi) in the immediate cellular response to viral infection in plants, nematodes and flies. However, little is known about how RNAi combats viruses during persistent or latent infections. Our analysis of small RNAs cloned from Drosophila cells latently infected with Flock House Virus (FHV) failed to reveal signatures of bulk degradation of the viral genome. Instead, this + strand virus specifically generated Dicer-2-dependent, 21-nucleotide s ...[more]

PMID: 19251644

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Project description:Mosquito-borne viruses encompass a range of virus families, comprising a number of significant human pathogens (e.g., dengue viruses, West Nile virus, Chikungunya virus). Virulent strains of these viruses are continually evolving and expanding their geographic range, thus rapid and sensitive screening assays are required to detect emerging viruses and monitor their prevalence and spread in mosquito populations. Double-stranded RNA (dsRNA) is produced during the replication of many of these viruses as either an intermediate in RNA replication (e.g., flaviviruses, togaviruses) or the double-stranded RNA genome (e.g., reoviruses). Detection and discovery of novel viruses from field and clinical samples usually relies on recognition of antigens or nucleotide sequences conserved within a virus genus or family. However, due to the wide antigenic and genetic variation within and between viral families, many novel or divergent species can be overlooked by these approaches. We have developed two monoclonal antibodies (mAbs) which show co-localised staining with proteins involved in viral RNA replication in immunofluorescence assay (IFA), suggesting specific reactivity to viral dsRNA. By assessing binding against a panel of synthetic dsRNA molecules, we have shown that these mAbs recognise dsRNA greater than 30 base pairs in length in a sequence-independent manner. IFA and enzyme-linked immunosorbent assay (ELISA) were employed to demonstrate detection of a panel of RNA viruses from several families, in a range of cell types. These mAbs, termed monoclonal antibodies to viral RNA intermediates in cells (MAVRIC), have now been incorporated into a high-throughput, economical ELISA-based screening system for the detection and discovery of viruses from mosquito populations. Our results have demonstrated that this simple system enables the efficient detection and isolation of a range of known and novel viruses in cells inoculated with field-caught mosquito samples, and represents a rapid, sequence-independent, and cost-effective approach to virus discovery.

Dataset Information

Dicing of viral replication intermediates during silencing of latent Drosophila viruses.

Publications

Dicing of viral replication intermediates during silencing of latent Drosophila viruses.

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