Antiviral RNA Interference Responses Induced by Semliki Forest Virus Infection of Mosquito Cells: Characterization, Origin, and Frequency-Dependent Functions of Virus-Derived Small Interfering RNAs

Abstract

RNA interference (RNAi) is an important mosquito defense mechanism against arbovirus infection. In this paper we study the processes underlying antiviral RNAi in Aedes albopictus-derived U4.4 mosquito cells infected with Semliki Forest virus (SFV) (Togaviridae; Alphavirus). The production of virus-derived small interfering RNAs (viRNAs) from viral double-stranded RNA (dsRNA) is a key event in this host response. dsRNA could be formed by RNA replication intermediates, by secondary structures in RNA genomes or antigenomes, or by both. Which of these dsRNAs is the substrate for the generation of viRNAs is a fundamental question. Here we used deep sequencing of viRNAs and bioinformatic analysis of RNA secondary structures to gain insights into the characteristics and origins of viRNAs. An asymmetric distribution of SFV-derived viRNAs with notable areas of high-level viRNA production (hot spots) and no or a low frequency of viRNA production (cold spots) along the length of the viral genome with a slight bias toward the production of genome-derived viRNAs over antigenome-derived viRNAs was observed. Bioinformatic analysis suggests that hot spots of viRNA production are rarely but not generally associated with putative secondary structures in the SFV genome, suggesting that most viRNAs are derived from replicative dsRNA. A pattern of viRNAs almost identical to those of A. albopictus cells was observed for Aedes aegypti-derived Aag2 cells, suggesting common mechanisms that lead to viRNA production. Hot-spot viRNAs were found to be significantly less efficient at mediating antiviral RNAi than cold-spot viRNAs, pointing toward a nucleic acid-based viral decoy mechanism to evade the RNAi response.

The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery

“…In several studies in insects, the polarity of the vsiRNA population deviates strongly from the highly skewed distribution of positive strand (+) over negative (−) viral RNAs that is generally observed in (+) RNA virus infection. Indeed, vsiRNAs mapped in similar proportions to (+) and (−) viral RNA strands in Aedes aegypti, Aedes albopictus, and Culex pipiens mosquitoes infected with a number of arthropodborne viruses, including Sindbis virus, Semliki Forest virus, West Nile virus, Dengue virus, and Chikungunya virus, as well as in Drosophila infected with (+) RNA viruses from different families (5,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). In addition, in infections of Drosophila with the negative-strand RNA virus vesicular stomatitis virus, similar numbers of (+) over (−) vsiRNAs were recovered (14).…”

Antiviral responses of arthropod vectors: an update on recent advances

“…Some (or possibly all) flaviviruses have the ability to suppress RNAi [59,101]; thus it is not surprising that other innate immunity pathways such as JAK/STAT and Toll are involved in controlling flavivirus infection in mosquitoes [92,115,130]. In contrast, alphaviruses are not known to actively suppress RNAi and use only decoy mechanisms to evade the RNAi response [111]. It is possible that antiviral RNAi is thus more efficient in controlling alphavirus replication and innate immunity signalling pathways may play only a small role in controlling alphavirus replication in mosquitoes.…”

“…Another observation indicating that the origin of virus-specific piRNAs is likely to be viral ssRNA of genomic or subgenomic origin is that the majority of piRNAs derived from SFV, SINV and CHIKV map to the coding strand in the 5 0 end of the subgenomic mRNA or to a lesser extent to the 5 0 end of the genomic mRNA [82,102,123]. The mapping of virus-derived piRNAs to specific regions in all three alphaviruses, as opposed to a more general distribution of 21 nt viRNAs across genome and antigenome [111], suggests that specific viral sequences or secondary structures are the source of virus-derived piRNAs. Future studies should elucidate how dsRNA and viral ssRNA contribute to the induction and generation of piRNAs.…”

“…For a summary of known suppressors of RNAi expressed by insect and plant viruses, see http://viralzone.expasy.org/all_by_protein/891.html [77]. One interesting observation is that while some arboviruses do have RNAi evasion strategies [111], they generally do not encode efficient suppressors of RNAi. Strong suppression of the antiviral RNAi response could be detrimental to vector survival [24,84] and thus to the transmission of the arbovirus.…”

“…While the generation of exogenous siRNAs has been implicated in antiviral defences in arthropods, miRNAs and piRNAs have mainly been associated with regulation of gene expression and control of transposons in germlines (reviewed by [5,55]); however, recently their role in virus infection has become more apparent. Numerous small RNA profiles of arbovirus-infected mosquitoes or mosquito cells have been published [45,68,84,111,123] and have provided insights into the importance of the different small RNA pathways during arbovirus infection. Knowledge gained about antiviral RNAi over the last decade has also led to new techniques for virus discovery.…”