Three lines of experimental evidence suggest that the B2 protein

Three lines of experimental evidence suggest that the B2 protein was functional in RNAi suppression when expressed during TE/3’2J/B2 virus infection. First, in vitro dicing experiments show inhibition siRNA accumulation in cell lysates derived from TE/3’2J/B2 virus-infected Aag2 cells. The presence of B2 protein inhibits the accumulation of biotinylated siRNAs, presumably by binding to the synthetic dsRNA and sequestering from Dicer-2. The presence of siRNAs in mock- and TE/3’2J/GFP-infected lysates provides evidence that Aag2 cells have a functional RNAi mechanism. Also, this shows that inhibition of siRNA accumulation is specific

to TE/3’2J/B2 virus infection. The second line of evidence comes from Northern blot analysis of small RNAs in mosquito cells. Considerably less SINV-specific siRNAs accumulated in cell CX-6258 in vivo culture 4SC-202 mw and P505-15 chemical structure mosquitoes infected with TE/3’2J/B2 virus compared to TE/3’2J and TE/3’2J/GFP virus infection. The dsRNA formed by viral replicative intermediates may be bound by B2 protein, protecting the dsRNA from detection by the RNAi

machinery. Finally, virus titers observed in Aag2 cells and adult Ae. aegypti mosquitoes were much higher when B2 protein was expressed during infection. This agrees with previous data showing that inhibition of the RNAi pathway allows for arboviruses to replicate more efficiently in mosquitoes [6, 7]. By injecting mosquitoes with dsRNA targeting Dicer-2 or Argonaute-2 after an infectious

bloodmeal, Campbell et al [6] were able to show that SINV titers in individual mosquitoes increased significantly by day four as compared to β-gal dsRNA injected controls. The same effect was not seen at day seven and the authors suggest this may be due to a stimulation of the antiviral response by this time point or degradation of the dsRNA triggers via decay [6]. A similar general phenomenon was seen with ONNV infection of An. gambiae mosquitoes, with a detectable increase in virus titer up to six days post infection [7]. This difference may be explained by the inoculation route as both dsRNA and ONNV were administered intrathoracically, bypassing any infection barriers 4-Aminobutyrate aminotransferase associated with the midgut and ensuring introduction of virus and dsRNA into the hemocoel [7]. A significant increase in SINV titers was observed at both four and seven days post infectious bloodmeal in mosquitoes ingesting TE/3’2J/B2 virus. The RNAi response is continuously inhibited by B2 protein as it is produced in infected mosquito cells. dsRNA intermediates or secondary structure of the virus genome will not be recognized by the RNAi machinery, allowing virus replication to continue unabated. Our data indicate that SINV becomes pathogenic to mosquitoes when RNAi is suppressed during virus infection.

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