Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs

Abstract

In Aedes mosquitoes, infections with arthropod-borne viruses (arboviruses) trigger or modulate the expression of various classes of viral and host-derived small RNAs, including small interfering RNAs (siRNAs), PIWI interacting RNAs (piRNAs), and microRNAs (miRNAs). Viral siRNAs are at the core of the antiviral RNA interference machinery, one of the key pathways that limit virus replication in invertebrates. Besides siRNAs, Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs, the best studied examples being viruses from the Togaviridae or Bunyaviridae families. Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections. In addition, some viruses, mostly with a DNA genome, express their own miRNAs to regulate host and viral gene expression. Here, we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 (DENV), a member of the Flaviviridae family. Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways. Interestingly, besides the well-characterized DENV-derived siRNAs, a specific population of viral piRNAs was identified in infected Aag2 cells. Knockdown of Piwi5, Ago3 and, to a lesser extent, Piwi6 results in reduction of vpiRNA levels, providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs. In contrast, we do not find convincing evidence for the production of virus-derived miRNAs. Neither do we find that host miRNA expression is strongly changed upon DENV2 infection. Finally, our deep-sequencing analyses detect 30 novel Aedes miRNAs, complementing the repertoire of regulatory small RNAs in this important vector species.

PIWIs Go Viral: Arbovirus-Derived piRNAs in Vector Mosquitoes

“…Even infection with Sigma virus, which naturally infects Drosophila germ cells, does not give rise to vpiRNA production [70], despite ample expression of PIWI proteins in these cells. In sharp contrast, vpiRNAs are readily detected both in Aedes cell lines and in somatic tissues of adult Aedes mosquitoes upon infection with several arboviruses, including members of the Togaviridae [28–33], Flaviviridae [34–36], Bunyaviridae [28,29,37–39], and Reoviridae [37] (Table 1). Besides a typical size distribution of small RNAs around 24–30 nt, piRNAs from several viruses display the characteristic nucleotide bias indicative of ping-pong amplification (Box 1).…”

“…In addition, vpiRNAs from dengue virus ( Flavivirus genus, Flaviviridae family) and Sindbis virus ( Alphavirus genus, Togaviridae family) have been verified to be 2′- O methylated at the 3′ terminal nucleotide (Table 1), a modification that is present on all PIWI-loaded mature piRNAs (Box 1). PIWI-dependence of vpiRNAs has been established for dengue, Sindbis, and Semliki Forest virus ( Alphavirus genus, Togaviridae family) [31,32,36] and direct association with PIWI proteins has been demonstrated for Sindbis virus–derived piRNAs [32]. …”

“…The plots depict published genome profiles of Sindbis virus (SINV) [29], chikungunya virus (CHIKV) [30], Semliki Forest virus (SFV) [31], dengue virus serotype 2 (DENV2) [35,36], cell fusing agent virus (CFAV) [34], Rift Valley fever virus (RVFV) [38], and Schmallenberg virus (SBV) [37]. For alphaviruses, the position of the subgenomic promoter is depicted.…”

“…Knockdown of Piwi5 and Ago3 —and to a lesser extent, Piwi6 —results in reduced vpiRNA production from dengue virus serotype 2 in Aag2 cells [36]. The additional requirement of Piwi6 specifically for dengue virus piRNA biogenesis suggests that Aedes PIWI proteins have specialized in processing distinct RNA sources.…”

“…Therefore, the molecular mechanism by which Piwi4 exerts its antiviral activity remains to be investigated. Knockdown of Piwi5 and Ago3 in Aag2 cells results in profound decline in vpiRNA expression from Sindbis virus and dengue virus, but viral replication is not strongly affected [36]. Whether PIWI depletion in adult mosquitoes causes enhanced arbovirus replication remains to be shown.…”