Production of Virus-Derived Ping-Pong-Dependent piRNA-like Small RNAs in the Mosquito Soma

Abstract

The natural maintenance cycles of many mosquito-borne pathogens require establishment of persistent non-lethal infections in the invertebrate host. The mechanism by which this occurs is not well understood, but we have previously shown that an antiviral response directed by small interfering RNAs (siRNAs) is important in modulating the pathogenesis of alphavirus infections in the mosquito. However, we report here that infection of mosquitoes with an alphavirus also triggers the production of another class of virus-derived small RNAs that exhibit many similarities to ping-pong-dependent piwi-interacting RNAs (piRNAs). However, unlike ping-pong-dependent piRNAs that have been described previously from repetitive elements or piRNA clusters, our work suggests production in the soma. We also present evidence that suggests virus-derived piRNA-like small RNAs are capable of modulating the pathogenesis of alphavirus infections in dicer-2 null mutant mosquito cell lines defective in viral siRNA production. Overall, our results suggest that a non-canonical piRNA pathway is present in the soma of vector mosquitoes and may be acting redundantly to the siRNA pathway to target alphavirus replication.

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

“…The different replication strategies of these viruses may require different control mechanisms. The recent discovery of putative new antiviral responses such as melanisation and, particularly, the implication of piRNAs in mosquito antiviral immunity against a variety of arboviruses (CHIKV, SFV, SINV, DENV, LACV, RVFV, SBV and BTV) [45,68,82,102,103,123], show how complex the arthropod immune system is and how far there is still to go in understanding it. One of the questions that will surely be addressed in the future is how virus-derived piRNAs are generated and whether it is possible to use the knowledge gained to benefit vector control strategies.…”

“…Increased knowledge about the piRNA pathway in mosquitoes reveals many apparent differences from that in Drosophila (reviewed by [32]). Virus-derived piRNAs have been shown to be produced in whole Aedes mosquitoes upon infection with CHIKV [82] and DENV [45,104], in Aedes mosquito soma upon infection with CHIKV [82] and in the cell lines U4.4, C6/36 (both A. albopictus) and Aag2 (A. aegypti) upon infection with Sindbis virus (SINV) [123]. Virus-derived piRNAs were also observed in C6/36 cells infected with the bunyavirus La Crosse virus (LACV) [123].…”

“…The mechanism and origin of these virus-specific piRNAs remains unclear. Hoa et al [47] have shown that the PIWI protein Ago-3 plays a role in gene silencing mediated by dsRNA treatment in A. gambiae cells and Morazzani et al [82] suggested that dsRNA is responsible for the piRNA-like molecules induced by CHIKV in C6/36 cells. However, Schnettler et al [102] showed that transfection of dsRNA alone is not sufficient to induce production of piRNA-like molecules, thus providing evidence that ssRNA as a result of active virus replication is required, and Vodovar et al [123] also argue that piRNAs are derived from viral ssRNA generating primary piRNAs from the (-) strand and secondary piRNAs from the abundant (?)…”

“…sense mRNA. 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.…”