Pan-flavivirus analysis reveals that the insect-specific Kamiti River virus produces a new subgenomic RNA and high amounts of 3’ UTR-derived siRNAs

Abstract

ABSTRACTFlaviviruses subvert the host RNA decay machinery to produce subgenomic flavivirus RNA (sfRNA), products of the 5’-3’ exoribonuclease XRN1/Pacman stalling on secondary RNA structures in the 3’ untranslated region (UTR) of the viral genome. The classical insect-specific flavivirus (cISF) Kamiti River virus (KRV) has a unique 1.2 kb long 3’ UTR, of which only 40% is homologous to its closest family member, cell-fusing agent virus (CFAV). We mapped the 5’ end of KRV sfRNAs and found that KRV produces high copy numbers of a long, 1017 nt sfRNA1 and a short, 421 nt sfRNA2, corresponding to two predicted XRN1-resistant elements. Furthermore, we identified a new positive and negative sense 1.5 kb subgenomic RNA species that is colinear with the 3’ region of the NS5 gene and the viral 3’ UTR, which we tentatively named subgenomic cISF RNA (cifRNA). Expression of both sfRNA1 and sfRNA2 was reduced in Pacman deficient Aedes albopictus mosquito cells, while expression of the longer cifRNA was Pacman-independent. Interestingly, a pan-Flavivirus small RNA analysis in Aedes albopictus cells revealed that nearly all KRV-derived siRNAs mapped to the 3’ UTR region and that these siRNAs are produced in high quantity. 3’ UTR-biased siRNA production appeared to be conserved in other cISFs, albeit to a lesser extent, whereas siRNAs were evenly distributed across the viral genome of other representatives of the Flavivirus genus. We suggest that cISFs and particularly KRV developed a unique mechanism to produce high amounts of siRNA as a decoy of the antiviral RNAi response.IMPORTANCEThe Flavivirus genus contains diverse mosquito viruses ranging from insect-specific viruses circulating exclusively in mosquito populations to mosquito-borne viruses that cause disease in humans and animals. Studying the mechanisms of virus replication and antiviral immunity in mosquitoes is important to understand arbovirus transmission and may inform the development of disease control strategies. In insects, RNA interference (RNAi) provides broad antiviral activity, constituting the main immune response against viruses. Comparing the RNAi response across members of the Flavivirus genus, we found that all flaviviruses are targeted by RNAi. However, the insect-specific Kamiti River virus was unique in that small interfering RNAs are highly skewed towards its uniquely long 3’ untranslated region. Moreover, we found that Kamiti River virus produces a new subgenomic RNA species in addition to subgenomic flavivirus RNAs previously observed for other flaviviruses. These results suggest that mosquito-specific viruses have evolved unique mechanisms for genome replication and immune evasion.