Suppression of RNA silencing by Flock house virus B2 protein is mediated through its interaction with the PAZ domain of Dicer.
RNA silencing is a conserved pathway that functions as an antiviral mechanism. The majority of viruses encode silencing suppressors that interfere with siRNA- and miRNA-guided silencing pathways. The insect flock house virus B2 protein (FHVB2) functions as an RNAi silencing suppressor that inhibits siRNA biogenesis. Here, we describe the generation of a GFP silent sensor line (Sf21) and a GFP sensor line expressing FHVB2 to study RNAi suppression mechanisms. Overexpression of FHVB2 resulted in suppression of GFP-RNAi and resumption of GFP expression. Protein fractionation studies with FHVB2-transfected cells showed that FHVB2 associates with a high-molecular-weight complex of Dicer and dsRNA/siRNAs. Yeast two-hybrid and pulldown assays revealed an interaction between FHVB2 and Drosophila Dicer proteins that appeared to involve PAZ domains. To map the FHVB2 domains interacting with Dicer, we used a 17-residue C-terminal deletion mutant. RNAi suppression was reversed in cells transfected with the FHVB2 mutant as revealed by loss of GFP. Additional yeast two-hybrid and in vitro pulldown assays confirmed that the C-terminal region of FHVB2 was involved in the interaction with the PAZ domains of Dicers. These results thus reveal a novel interaction between FHVB2 and Dicer that leads to suppression of siRNA biogenesis.
Singh
Gatikrushna
G
International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067, India.
Popli
Sonam
S
Hari
Yukti
Y
Malhotra
Pawan
P
Mukherjee
Sunil
S
Bhatnagar
Raj K
RK
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D030381:Nodaviridae / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D029721:Drosophila Proteins; D019476:Insect Proteins; D034741:RNA, Small Interfering; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D043244:Ribonuclease III