No abstract

The siRNA pathway is an essential antiviral mechanism in insects. Whether other RNA interference pathways are involved in antiviral defense remains unclear. Here, we report in cells derived from the two main vectors for arboviruses, Aedes albopictus and Aedes aegypti, the production of viral small RNAs that exhibit the hallmarks of ping-pong derived piwi-associated RNAs (piRNAs) after infection with positive or negative sense RNA viruses. Furthermore, these cells produce endogenous piRNAs that mapped to transposable elements. Our results show that these mosquito cells can initiate de novo piRNA production and recapitulate the ping-pong dependent piRNA pathway upon viral infection. The mechanism of viral-piRNA production is discussed.

RNA viruses in insects are targets of an RNA interference (RNAi)-based antiviral immune response, in which viral replication intermediates or viral dsRNA genomes are processed by Dicer-2 (Dcr-2) into viral small interfering RNAs (vsiRNAs). Whether dsDNA virus infections are controlled by the RNAi pathway remains to be determined. Here, we analyzed the role of RNAi in DNA virus infection using Drosophila melanogaster infected with Invertebrate iridescent virus 6 (IIV-6) as a model. We show that Dcr-2 and Argonaute-2 mutant flies are more sensitive to virus infection, suggesting that vsiRNAs contribute to the control of DNA virus infection. Indeed, small RNA sequencing of IIV-6-infected WT and RNAi mutant flies identified abundant vsiRNAs that were produced in a Dcr-2-dependent manner. We observed a highly uneven distribution with strong clustering of vsiRNAs to small defined regions (hotspots) and modest coverage at other regions (coldspots). vsiRNAs mapped in similar proportions to both strands of the viral genome, suggesting that long dsRNA derived from convergent overlapping transcripts serves as a substrate for Dcr-2. In agreement, strand-specific RT-PCR and Northern blot analyses indicated that antisense transcripts are produced during infection. Moreover, we show that vsiRNAs are functional in silencing reporter constructs carrying fragments of the IIV-6 genome. Together, our data indicate that RNAi provides antiviral defense against dsDNA viruses in animals. Thus, RNAi is the predominant antiviral defense mechanism in insects that provides protection against all major classes of viruses.insect immunity | Iridoviridae | siRNA | antiviral immunity | small RNA profiling | next-generation sequencing

The exosome is an exoribonuclease complex involved in the degradation and maturation of a wide variety of RNAs. The nine‐subunit core of the eukaryotic exosome is catalytically inactive and may have an architectural function and mediate substrate binding. In Saccharomyces cerevisiae, the associated Dis3 and Rrp6 provide the exoribonucleolytic activity. The human exosome‐associated Rrp6 counterpart contributes to its activity, whereas the human Dis3 protein is not detectably associated with the exosome. Here, a proteomic analysis of immunoaffinity‐purified human exosome complexes identified a novel exosome‐associated exoribonuclease, human Dis3‐like exonuclease 1 (hDis3L1), which was confirmed to associate with the exosome core by co‐immunoprecipitation. In contrast to the nuclear localization of Dis3, hDis3L1 exclusively localized to the cytoplasm. The hDis3L1 isolated from transfected cells degraded RNA in an exoribonucleolytic manner, and its RNB domain seemed to mediate this activity. The siRNA‐mediated knockdown of hDis3L1 in HeLa cells resulted in elevated levels of poly(A)‐tailed 28S rRNA degradation intermediates, indicating the involvement of hDis3L1 in cytoplasmic RNA decay. Taken together, these data indicate that hDis3L1 is a novel exosome‐associated exoribonuclease in the cytoplasm of human cells.