A dsRNA-binding protein of a complex invertebrate DNA virus suppresses the Drosophila RNAi response.
Bottom Line: Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R.Using biochemical assays, we reveal that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs).We demonstrate that 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex.
Affiliation: Department of Medical Microbiology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.Show MeSH
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Mentions: The IIV-6 340R gene encodes a 23-kDa protein that contains a 70-aa dsRBD flanked by a 30-aa N-terminal sequence and a 73-aa C-terminal sequence. Alignment of the the dsRBD of 340R to the dsRBDs of DCV 1A and cellular proteins from different model organisms shows that conserved amino acids are present throughout the motif (Figure 2A). Homology modeling suggests that the dsRBD of 340R adopts the expected αβββα topology, in which two α helices are packed along a three-stranded antiparallel β sheet, and that the dsRBD is preceded by an N-terminal helical structure (Figure 2B). Based on these analyses, we selected for site-directed mutagenesis four highly conserved residues (L35Y, F63A and AA92LL) and two residues within a region expected to interact with dsRNA (K86A and K89A) (Figure 2A and B and Supplementary Figure S2). In addition, we generated a C-terminally truncated version of 340R, consisting of the N-terminal 100-aa that contains the complete dsRBD (dsRBD100).
Affiliation: Department of Medical Microbiology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.