Mosquito and Drosophila entomobirnaviruses suppress dsRNA- and siRNA-induced RNAi.
Bottom Line: We found that the Culex RNAi machinery processes CYV double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNAs).VP3 was found to bind long dsRNA as well as siRNAs and interfered with Dicer-2-mediated cleavage of long dsRNA into siRNAs.Slicing of target RNAs by pre-assembled RNA-induced silencing complexes was not affected by VP3.
Affiliation: Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Radboud Institute for Molecular Life Sciences, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.Show MeSH
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Mentions: VSRs of different viruses may target different aspects of the RNAi machinery, such as Dcr-2-mediated cleavage of dsRNA and slicing of target RNAs by AGO2. To characterize the VSR activity of CYV and DXV VP3 in more detail, we performed a series of biochemical assays using maltose-binding protein (MBP)-tagged recombinant proteins purified from Escherichia coli. We first tested whether the recombinant VP3 proteins interfere with dicing of dsRNA, the initiation phase of the RNAi pathway. We incubated radioactively-labeled 126-nt dsRNA in D. melanogaster S2, A. albopictus U4.4, C. quinquefasciatus Hsu and C. tarsalis CT cell extracts and monitored its processing into 21-nt siRNAs on denaturing polyacrylamide gels. The dsRNA was efficiently processed into siRNAs in extracts from all cell types (Figure 6A, lanes 10, 15, 20 and 25). Processing of the dsRNA was, however, inhibited in a dose-dependent manner by the VP3 proteins of both CYV (Figure 6A, lanes 6–8, 11–13, 16–18 and 21–23) and DXV (Figure 6A, lanes 3–5), and in the presence of DCV 1A (Figure 6A, lane 1), a VSR that is known to interact with dsRNA (2). As expected, MBP alone did not inhibit dsRNA processing (Figure 6A, lanes 9, 14, 19 and 24). These data indicate that the entomobirnavirus VP3 proteins interfere with siRNA production by Dcr-2. Importantly, inhibition of dsRNA cleavage into siRNAs was also observed in extracts from Drosophila S2 and Culex CT cells infected with CYV. The dsRNA was processed in extracts from mock-infected cells (Figure 6B, lanes 5 and 10), but no dsRNA processing was observed in extracts from CYV-infected cells (Figure 6B, lanes 1 and 6). Titration of CYV-infected cell extracts into mock-infected cell extracts abolished dsRNA processing (Figure 6B, lanes 2–4 and 7–9), which confirms the presence of a Dcr-2 inhibitor in CYV-infected cells.
Affiliation: Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Radboud Institute for Molecular Life Sciences, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.