However, other insect viruses are known to contain RNAi suppressors that aid in their replication via suppression of the RNAi response. The B2 protein from the insect-pathogenic FHV is a potent viral suppressor of RNA silencing (VSR) that binds to dsRNA as a dimer in a sequence-independent manner and can bind a range of dsRNA

sizes [11, 12]. The Selleck Staurosporine generic and promiscuous nature of dsRNA binding by B2, evidenced by its ability to inhibit RNAi in plants, nematodes, and insects, makes it an excellent candidate to study the effects of RNAi suppression in mosquitoes [13–16]. This report describes the production of a recombinant SINV that expresses a heterologous VSR protein and use of the virus to directly study the effects of RNAi on mosquito infection. A TE/3’2J virus was engineered to express the B2 protein, with the hypothesis that expression of B2 during SINV infection would inhibit the RNAi EGFR inhibitor response in infected mosquito cells and that this inhibition will lead to increased virus replication within the mosquito. The VSR was functional in mosquito cells and affected the replication of TE/3’2J virus in Ae. aegypti cell culture. Mosquito ACY-1215 ic50 infection experiments show that not only are rates of infection and dissemination of SINV in Ae. aegypti increased if RNAi is inhibited, but that the B2-expressing

virus became highly pathogenic in the mosquito, significantly shortening the mosquitoes’ lifespan. These

studies highlight the necessity for RNAi from both the standpoint of mosquito survival and arbovirus persistence. Results Inhibition of RNAi by a SINV-expressed VSR After rescue of infectious virus from cDNA-derived RNA, expression of V5 epitope-tagged B2 protein from the second subgenomic promoter was verified by immunoblot analysis of total protein from infected Aag2 cells. Using a commercial antibody against the V5 epitope, we observed a single band of approximately 12 kilodaltons (kDa) in B2-infected cells (Figure 1), in agreement with the predicted size of V5-tagged B2 protein (12.4 kDa). No bands were detected in cells infected with TE/3’2J, TE/3’2J/GFP, or mock-infected cells. Figure 1 Detection of V5-B2 Mannose-binding protein-associated serine protease protein in mosquito cell culture. V5-B2 protein was detected by immunoblot using anti-V5 antibody in total protein from Aag2 cell culture. Molecular weights are indicated on the left side of each panel. Lane 1, protein molecular weight marker. Lane 2, TE/3’2J/B2-infected cells. Lane 3, TE/3’2J/GFP-infected cells. Lane 4, TE/3’2J-infected cells. Lane 5, Mock-infected cells. To determine the ability of SINV-expressed B2 protein to inhibit the mosquito RNAi response, an in vitro dicing assay was performed. A synthetic 500 bp biotinylated dsRNA derived from the bacterial β-galactosidase gene was introduced into Aag2 cell lysates produced from cells mock-infected or infected with GFP- or B2-expressing virus.