Ae. aegypti dR2D2 mutant, small RNA study following Mayaro virus challenge

An Aedes aegypti mutant strain with an R2D2 gene knockout was produced. R2D2 is a double-stranded RNA binding protein which heterodimerizes with Dicer-2 and facilitates siRNA loading to Argonaute 2. Dicer-2-R2D2 heterodimers cleave long dsRNA substrates, including those produced during alphavirus replication, into 21 bp siRNAs. R2D2 facilitates Argonaute 2 mediated RNA silencing through strand specific loading of siRNAs. Here, we produced an R2D2 mutant to explore the genes role in vector-pathogen interactions in a major arboviral vector species.An R2D2 mutant strain was produced by a transgene knockin into the native Ae. aegypti R2D2 locus with CRISPR/Cas9 mediated genome editing. Surprisingly, the R2D2 knockin was initially hereditary only in the male mosquitoes. Upon observation that the initial transgene insertion was in linkage to the dominant male determining factor Nix, a copycat strategy was utilized to produce a lab strain with R2D2 knockout transgene heredity through the female lineage. Subsequent reciprocal outcrossing experiments were performed to further explore the hereditary dynamics of R2D2 alleles.To study vector-arbovirus interactions, R2D2 mutant mosquitoes were challenged with a major tropical arbovirus, mayaro virus. R2D2 mutants developed higher viral titres as measured by qRT-PCR assays.Small RNA sequencing was conducted on whole body samples of mayaro virus challenged R2D2 mosquitoes and the control parental laboratory strain. Small RNA sequencing results showed no differences in the production of 21bp virus derived siRNAs in the heterozygous mutants. Interestingly, small RNA samples from infected R2D2 heterozygous mutants had increased virus-derived piRNAs.The study results show three major findings: 1) R2D2 is an essential gene, 2) R2D2 alleles are tightly sex-linked and 3) R2D2 is a rate limiting component in the processing of viral double stranded RNAs, with a doubling of the total viral RNA observed in mutants, and increased viral derived piRNAs, but not siRNA, observed in R2D2 knockout heterozygotes.