Dataset Information

A Transgenic Flock House Virus Replicon Reveals an RNAi Independent Antiviral Mechanism Acting in Drosophila Follicular Somatic Cells.

ABSTRACT: The small interfering RNA (siRNA) pathway is the main and best studied invertebrate antiviral response. Other poorly characterized protein based antiviral mechanisms also contribute to the control of viral replication in insects. In addition, it remains unclear whether tissue specific factors contribute to RNA and protein-based antiviral immunity mechanisms. In vivo screens to identify such factors are challenging and time consuming. In addition, the scored phenotype is usually limited to survival and/or viral load. Transgenic viral replicons are valuable tools to overcome these limitations and screen for novel antiviral factors. Here we describe transgenic Drosophila melanogaster lines encoding a Flock House Virus-derived replicon (FHV?B2eGFP), expressing GFP as a reporter of viral replication. This replicon is efficiently controlled by the siRNA pathway in most somatic tissues, with GFP fluorescence providing a reliable marker for the activity of antiviral RNAi. Interestingly, in follicular somatic cells (FSC) of ovaries, this replicon is still partially repressed in an siRNA independent manner. We did not detect replicon derived Piwi-interacting RNAs in FSCs and identified 31 differentially expressed genes between restrictive and permissive FSCs. Altogether, our results uncovered a yet unidentified RNAi-independent mechanism controlling FHV replication in FSCs of ovaries and validate the FHV?B2eGFP replicon as a tool to screen for novel tissue specific antiviral mechanisms.

SUBMITTER: Martins N

PROVIDER: S-EPMC6385967 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Publications

A Transgenic Flock House Virus Replicon Reveals an RNAi Independent Antiviral Mechanism Acting in <i>Drosophila</i> Follicular Somatic Cells.

Martins Nelson N Lemoine Aurélie A Santiago Estelle E Paro Simona S Imler Jean-Luc JL Meignin Carine C

G3 (Bethesda, Md.) 20190207 2

The small interfering RNA (siRNA) pathway is the main and best studied invertebrate antiviral response. Other poorly characterized protein based antiviral mechanisms also contribute to the control of viral replication in insects. In addition, it remains unclear whether tissue specific factors contribute to RNA and protein-based antiviral immunity mechanisms. <i>In vivo</i> screens to identify such factors are challenging and time consuming. In addition, the scored phenotype is usually limited to ...[more]

PMID: 30530643

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Project description:The larger segment (RNA 1) of the bipartite, positive-sense RNA genome of the nodavirus flock house virus encodes the viral RNA-dependent RNA polymerase. Two nonstructural viral proteins are made during the self-directed replication of this RNA: protein A (110 kDa), the translation product of RNA 1 itself, and protein B (11 kDa), the translation product of a subgenomic RNA (RNA 3) that is produced from RNA 1 during replication. To examine the roles of these proteins in RNA replication, specialized T7 transcription plasmids that contained wild-type or mutant copies of flock house virus RNA 1 cDNA were constructed and used in cells infected with the vaccinia virus-T7 RNA polymerase recombinant to make full-length transcripts that directed their own replication. Sequences in the primary transcripts that extended beyond the ends of the authentic RNA 1 sequence inhibited self-directed RNA replication, but plasmids that were constructed to minimize these terminal extensions produced primary transcripts that replicated as abundantly as authentic RNA 1. Truncation or mutation of the open reading frame for protein A eliminated self-directed replication, although the mutant RNA 1 remained a competent template for replication by wild-type protein A supplied in trans. These results showed that protein A was essential for RNA replication and that the process was not inseparably coupled to complete translation of the template. In contrast, protein B could be eliminated without inhibiting replication by mutations that disrupted the second of the two overlapping open reading frames on RNA 3. Furthermore, a mutant of RNA 1 in which the first nucleotide of the RNA 3 region was changed from G to U replicated at levels as high as those of the wild type without making either RNA 3 or protein B. However, diminishing replication levels were observed during subsequent replicative passages of RNA from both the mutants that could not make protein B. Roles for this protein that could account for the subtle phenotype of these mutants are discussed.

Dataset Information

A Transgenic Flock House Virus Replicon Reveals an RNAi Independent Antiviral Mechanism Acting in Drosophila Follicular Somatic Cells.

Publications

A Transgenic Flock House Virus Replicon Reveals an RNAi Independent Antiviral Mechanism Acting in <i>Drosophila</i> Follicular Somatic Cells.

Similar Datasets

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