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

Hess et al. BMC Microbiology 2011, 11:45 http://www.biomedcentral.com/1471-2180/11/45 RESEARCH ARTICLE Open Access Small RNA profiling of Dengue virus-mosquito interactions implicates the PIWI RNA pathway in anti-viral defense Ann M Hess1, Abhishek N Prasad2, Andrey Ptitsyn3, Gregory D Ebel2, Ken E Olson3,4, Catalin Barbacioru5, Cinna Monighetti5, Corey L Campbell6* Abstract Background: Small RNA (sRNA) regulatory pathways (SRRPs) are important to anti-viral defence in mosquitoes. To identify critical features of the virus infection process in Dengue serotype 2 (DENV2)-infected Ae. aegypti, we deepsequenced small non-coding RNAs. Triplicate biological replicates were used so that rigorous statistical metrics could be applied. Results: 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 noncoding RNAs (ncRNAs), such as tRNAs, spliceosomal U RNAs, and snoRNAs are highly enriched in DENV-infected samples at 2 and 4 dpi. Conclusions: 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. Background 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 * Correspondence: 6 Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, 80523, USA Full list of author information is available at the end of the article 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 (Ago2) dependent cleavage mechanism, whereas microRNAs (ca. 21-22 nts) are produced by a Dicer-1/ Loquacious/Ago1 dependent mechanism [8,9]. Intriguingly, components from these two pathways do not function exclusively from one another. Dicer-2 and an alternate spliceform of Loquacious interact to produce endogenous siRNAs (endo-siRNAs) [10,11]. This alternate pathway is also an important regulator of host gene expression and selfish genetic elements [12]. PIWI pathway products, piRNAs, 24-30 nts in length, are produced in a Dicer-independent manner [13]. Moreover, an additional sRNA size class has been described in the © 2011 Hess et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hess et al. BMC Microbiology 2011, 11:45 http://www.biomedcentral.com/1471-2180/11/45 anti-Ago2 antibody immunoprecipitation of unusually small RNAs (usRNAs) (ca. 13-19 nts) [14]. Triggers for SRRPs are only partially understood. The anti-viral and endo-siRNA pathways have a doublestranded RNA trigger which activates processing and loading of an 20-23 nt siRNA guide strand [15]. Once loaded, the RISC may be recycled. The miRNA pathway relies on microRNA-encoding genes that are processed in a DGCR8/Drosha-dependent manner [16]. In contrast to siRNAs, miRNAs, also 20-23 nts, bind to target transcripts with imperfect complementarity. PIWI pathway sRNA biogenesis is less understood but likely involves a single-stranded RNA trigger (reviewed in [7]). Mosquito-borne dengue virus is a human health threat in tropical urban areas and causes sporadic outbreaks in the southern US [17,18]. It is transmitted to humans by aedine mosquitoes and has bypassed the requirement for an enzootic amplification cycle, thus increasing the threat to public health. Arboviruses must successfully replicate in mosquitoes, escape anti-viral defense, and then invade salivary glands in order to be transmitted during blood feeding to subsequent hosts. Using radioisotopic detection, newly replicated Dengue virus serotype 2 (DENV2) genomes can be detected in Ae. aegypti Higg’s White Eye (HWE) midguts, the initial site of infection, as early as 4 days post infection (dpi), and viral interfering sRNAs (viRNAs) at 8 dpi [6,19]. The best described anti-viral RNAi pathway relies on a Dicer-2 dependent mechanism whereby the Ago2 endonuclease cleaves target RNAs [20]. Silencing of RNAi component transcripts Ago2, R2D2 and Dicer-2 in Ae. aegypti increases DENV2 titers; therefore these components play an important role in controlling arbovirus replication [3,6,21]. Another component of the RNAinduced Silencing Complex (RISC) is Tudor-SN (TSN), a transcriptional co-factor [3,22]. Given the presence of a functional RNAi pathway, it remains a mystery as to how arboviruses overcome anti-viral defense to establish persistent infections and perpetuate the arbovirus disease cycle. sRNAs represent the product of host mRNA or viral RNA cleavage in an RNAi-specific manner. Detection and characterization of RNAi pathway degradation products in arbovirus-infected mosquitoes lends insight into the interplay between virus and vector at a level of sensitivity not formerly possible. The goals of this study were to a) characterize changes in viRNA production and b) to identify host processes that are differentially regulated by RNAi over the course of infection. DENV2 Jamaica 1409 (JAM1409) was used to infect its natural mosquito vector, Aedes aegypti. Most current RNA deep sequencing studies use duplicate technical replicates. By using triplicate biological replicates, deep sequencing and rigorous statistical metrics similar to those used for Page 2 of 12 microarrays, we identify products of RNAi pathway activity that are altered in DENV2-infected mosquitoes. The resulting (...truncated)