Abstract
In the germline of animals, PIWI interacting (pi)RNAs protect the genome against the detrimental effects of transposon mobilization. In Drosophila, piRNA-mediated cleavage of transposon RNA triggers the production of responder piRNAs via ping-pong amplification. Responder piRNA 3′ end formation by the nuclease Zucchini is coupled to the production of downstream trailer piRNAs, expanding the repertoire of transposon piRNA sequences. In Aedes aegypti mosquitoes, piRNAs are generated from viral RNA, yet, it is unknown how viral piRNA 3′ ends are formed and whether viral RNA cleavage gives rise to trailer piRNA production. Here we report that in Ae. aegypti, virus- and transposon-derived piRNAs have sharp 3′ ends, and are biased for downstream uridine residues, features reminiscent of Zucchini cleavage of precursor piRNAs in Drosophila. We designed a reporter system to study viral piRNA 3′ end formation and found that targeting viral RNA by abundant endogenous piRNAs triggers the production of responder and trailer piRNAs. Using this reporter, we identified the Ae. aegypti orthologs of Zucchini and Nibbler, two nucleases involved in piRNA 3′ end formation. Our results furthermore suggest that autonomous piRNA production from viral RNA can be triggered and expanded by an initial cleavage event guided by genome-encoded piRNAs.
Highlights
Blood-feeding mosquitoes of the Aedes genus are responsible for the transmission of arthropod-borneviruses that cause severe diseases, such as dengue, Zika, chikungunya and yellow fever
We propose that, through trailer piRNA production, a single endogenous initiator piRNA can induce the production of an expanded pool of viral piRNAs, thereby enforcing autonomous piRNA production from viral RNA
Zuc uses a sequence motif to preferentially cleave upstream of uridine residues in vivo [39,40,41], piRNAs generated by Zuc generally have sharp 3 ends and the nucleotide directly downstream of the 3 end is biased towards uridine (+1U bias) [7,8]. We examined whether these characteristics were present in our previously generated small RNA deep sequencing libraries from Ae. aegypti Aag2 cells infected with Sindbis virus (SINV) [24]
Summary
Blood-feeding mosquitoes of the Aedes genus are responsible for the transmission of arthropod-borne (arbo)viruses that cause severe diseases, such as dengue, Zika, chikungunya and yellow fever. For efficient transmission to occur, arboviruses have to actively replicate in several mosquito tissues to eventually infect the salivary gland [1]. The cornerstone of antiviral immunity in insects is the small interfering (si)RNA pathway, in which viral double stranded (ds)RNA is cleaved by Dicer-2 into siRNAs [2]. These siRNAs provide sequence specificity to the endonuclease Argonaute 2 to direct the cleavage of single stranded viral transcripts. In Aedes mosquitoes, viral RNA is processed by a somatically active PIWI interacting (pi)RNA pathway, suggesting that two independent small RNA pathways act in parallel to combat viral infections [3]
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