The project initially focused on chikungunya virus, but Zika virus was included at a later stage for comparative purposes and due to the occurrence of the Zika virus epidemic.
(i) Our findings showed that the infection of Aedes aegypti mosquito derived Aag2 cells with either virus, CHIKV or ZIKV, resulted in the production of virus-specific siRNAs and piRNAs, as characterized by small RNA sequencing and bioinformatic analysis. In case of both viruses, the siRNAs with size of 21 nt mapped along the genomic and antigenomic strands. However, differences were observed between the produced virus specific piRNAs. Chikungunya virus specific piRNAs mapped to the subgenomic region of virus genome and these piRNAs carry characteristic signatures similar to other cellular piRNAs: sense piRNAs having nucleotide A at position 10 and antisense having U at position 1. Zika virus specific piRNAs were unique: piRNAs mapped to only one location on the genomic strand and these piRNAs lacked the characteristic signature, hence these are rather piRNA-like. By knocking down the primary proteins of the piRNA pathway individually, we found that the key proteins involved in the production of piRNAs did not affect chikungunya or Zika virus replication, unlike the case with siRNA producing proteins. Interestingly, one PIWI protein was found to be antiviral against both viruses: Piwi4.
(ii) It was further established that the key proteins for alphavirus-specific piRNA production are Ago3, Piwi5, and Piwi6. In case of ZIKV, Ago3 binds the ZIKV specific piRNA-like molecules, expanding the knowledge on flavivirus infection in mosquito cells in general. Additional studies on antiviral Piwi4 indicated that it interacts with key proteins in siRNA pathway (Ago2, Dcr2) and key proteins in piRNA pathway (Ago3, Piwi5, Piwi6), but its antiviral function is not directly linked to either classes of the virus specific small RNAs (siRNAs or piRNAs), indicating that mosquitoes have evolved additional layer of defence against viruses, which is different from the defence mechanisms of fruitfly, the model insect for molecular biology studies.
(iii) Moreover, quantitative proteomic study performed to find interaction partners of Piwi4 showed that it is complexed with multiple of proteins, including ribosomal proteins. Some of the Piwi4 partner do not have orthologues in other species. Further research indicated that some of the identified Piwi4 interactors are also antiviral and could be good targets to use for the control of vector-based control strategies.
