UC Riverside

Infection of RNA viruses induces RNA interference (RNAi) - directed antiviral immunity in plants and invertebrates. However, it is not known if the RNAi machinery in the fission yeast Schizosaccharomyces pombe has an antiviral function. Replication of Flock House Virus (FHV) genomic RNA1 and its mutant deficient in the expression of the viral suppressor of RNAi (VSR) was examined in both wild type and RNA-defective mutant S. pombe strains. The results indicated that the primitive RNAi pathway in S. pombe was incompetent to inhibit viral RNA replication. Deep sequencing of small RNAs also did not detect production of virus-derived small interfering RNAs (siRNAs) following FHV replication in S. pombe.

Since most of the previous studies in Drosophila used cell culture and embryos, little is known about the population structure, biogenesis pathway and relative potency of virus-derived siRNAs in adult Drosophila melanogaster. Deep sequencing of small RNAs from wild type and RNAi-defective Drosophila mutants challenged by a VSR-deficient FHV mutant showed that virus-derived siRNAs were produced by the type III ribonuclease Dicer-2, were predominantly 21 nucleotides in length with an approximately equal ratio of positive and negative strands, and sufficiently potent to terminate virus infection in adult flies. Importantly, two dsRNA-binding proteins, Loquacious and R2D2, which act sequentially in the biogenesis and loading of siRNAs in the canonical RNAi pathway, appeared to function redundantly in antiviral RNAi, whereas loss of Argonaute-2 resulted in a strong bias for positive-strand viral siRNAs.

Further genetic studies revealed a prominent antiviral role for the PIWI gene, which encodes an Argonaute protein required for silencing transposons and repeat elements in the Drosophila germline. Notably, enhanced accumulation of FHV proteins in the epithelial sheath and follicle cells surrounding the developing egg chambers in the ovaries was illustrated in the Drosophila mutant heterozygous for PIWI. Consistently, presence of the piwi mutant allele resulted in significantly enhanced levels of FHV and Drosophila C virus in the progenies of virus-infected adult flies. Thus, Piwi may be essential for virus clearance in the developing oocytes, and hence prevent vertical transmission of certain RNA viruses in fruit flies.