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

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 deep-sequenced 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 non-coding 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.

Figure 1

Antiviral RNAi components are expressed and active in Ae. aegypti. A) Ago2 associates with a high MW complex in hemolymph and fat body prior to a blood-feeding. HWE strain hemolymph (collected through proboscis) or hemolymph collected with fat body before and 1 day following a blood meal. About 30 μg protein was separated on a 3-10% Blue Native gel and subjected to immunoblot analysis using anti-Ago2 antibody. 'H', hemolymph, 'H/F', hemolymph with fat body. Size markers show position of proteins of known molecular weight (not shown). B) Silver stained gel shows loading control. C) RNAi component transcripts are modulated during DENV2 infection. Relative changes in DENV2-infected HWE midgut transcript levels detected by qRT-PCR. Significant changes over controls are marked with asterisks (p ≤ 0.05, Mann-Whitney U test); error bars depict standard error of three biological replicates. Pools of 5 midguts were used in each replicate. Relative transcript levels were calculated using the delta-delta Ct method, using ribosomal protein S7 as a reference standard. Enrichment is relative to that of un-infected blood-fed control mosquitoes. D) Western blot of immunoprecipitated products (IP) from pools of 20 DENV2-infected RexD mosquitoes. 'UN', Un-infected blood-fed control mosquitoes collected at 2 dpf (days post-feeding), probed with non-immune serum; 'U', un-infected blood-fed mosquito Ago2 antibody IP; 'DN', Dengue/blood-fed mosquitoes collected at 2 dpi, probed with non-immune serum; 'D', Dengue/blood-fed mosquito Ago2 antibody IP. Size markers show approximate molecular weight of bands shown.

Figure 2

viRNA profiles change over the course of DENV2 infection. A) 2 days post-infection (dpi). B) 4 dpi. C) 9 dpi. Top panel shows mean mapped reads and count distribution along DENV2 genome for a representative library at each time point. Bottom panel shows mean viRNA distribution by sRNA size group. Blue and red bars indicate sense and anti-sense viRNAs, respectively.

Figure 3

sRNAs mapped to host target RNAs are enriched or depleted during DENV2 infection and show an inverse relationship to transcript levels. A) Enriched sRNAs categorized by target functional group in DENV2-infected samples over un-infected blood-fed controls. B) Depleted sRNAs categorized by target functional group in DENV2-infected samples over controls. C) Enriched sRNAs at 2 dpi categorized by sRNA size group. Targets of unknown function are not shown. D) Depleted sRNAs at 2 dpi categorized by sRNA size group. Targets of unknown function are not shown. 'ncRNA', non-coding RNAs, 'CSR', chemo-sensory receptor, 'TRP', transport (signal transduction, ion transport, transmembrane transport), 'PRO', protease, 'ReDox', oxidative reductive components not associated with the mitochondria, 'TT', Transcription/Translation mRNAs, 'MIT' mitochondrial function, 'LIPID_MET' Lipid_Metabolism, 'MET', general metabolism, 'IMM', immunity, 'DIV', diverse function, 'CYT/STR', cytoskeletal/structural. E) Selected target mRNAs were subjected to qRT-PCR analysis in pooled midguts. Bars represent percent change in 2 dpi DENV-2 infected RexD Ae. aegypti midguts versus un-infected control midguts from the same time-point. The Delta-delta Ct analytical method was applied and ribosomal protein S7 was used as reference standard. Target transcripts not maintaining the expected inverse relationship with sRNA profiles are marked with an asterisk.

Figure 4

Predicted alterations in oxidative phosphorylation pathway components in DENV2-infected mosquitoes at 2 dpi. Differences in sRNA profiles were compared for un-infected controls and DENV2-infected mosquitoes at 2 dpi. Over-represented sRNA targets are revealed in the oxidative phosphorylation pathway, particularly among components of respiratory complex III. Direction and relative scale of sRNA counts for a given target are marked by red bar indicators near the corresponding target genes. Bar 1 indicates un-infected controls; Bar 2 indicates DENV2-infected pools. The legend to GeneGo Metacore pathway maps is given in Additional File 4.