Small RNA profiling of Dengue virus-mosquito interactions implicates the PIWI RNA pathway in anti-viral defense

In addition to virus-derived siRNAs (20-23 nts) previously reported for other arbovirus-infected mosquitoes, we show that PIWI pathway sRNAs (piRNAs) (24-30 nts) and unusually small RNAs (usRNAs) (13-19 nts) are produced in DENV-infected mosquitoes. We demonstrate that a major catalytic enzyme of the siRNA pathway, Argonaute 2 (Ago2), co-migrates with a ~1 megadalton complex in adults prior to bloodfeeding. sRNAs were cloned and sequenced from Ago2 immunoprecipitations. Viral sRNA patterns change over the course of infection. Host sRNAs were mapped to the published aedine transcriptome and subjected to analysis using edgeR (Bioconductor). We found that sRNA profiles are altered early in DENV2 infection, and mRNA targets from mitochondrial, transcription/translation, and transport functional categories are affected. Moreover, small non-coding RNAs (ncRNAs), such as tRNAs, spliceosomal U RNAs, and snoRNAs are highly enriched in DENV-infected samples at 2 and 4 dpi.These data implicate the PIWI pathway in anti-viral defense. Changes to host sRNA profiles indicate that specific cellular processes are affected during DENV infection, such as mitochondrial function and ncRNA levels. Together, these data provide important progress in understanding the DENV2 infection process in Ae. aegypti.Small RNA (sRNA) regulatory pathways (SRRPs) control gene expression through a variety of mechanisms [1]. Components of the microRNA, small interfering (siRNA), and PIWI RNA pathways, three major SRRPs, are present in mosquitoes [2]. In each of these pathways, gene expression is regulated in the cleavage and degradation of mRNAs. Cellular processes as diverse as development, anti-viral defense and maintenance of the germline are controlled by these mechanisms [3-6]. In general, the size of the cleavage products reveals the pathway(s) by which degradation occurs [7]. In mosquitoes and other invertebrates, siRNAs of ca. 21-22 nts are expected to be produced by a Dicer-2/R2D2/Argonaute 2 (Ag