FASEB J. Integrated DNA Technologies
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Published as doi: 10.1096/fj.08-125120.
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(The FASEB Journal. 2009;23:1845-1857.)
© 2009 FASEB

Suppression of RNA silencing by Flock house virus B2 protein is mediated through its interaction with the PAZ domain of Dicer

Gatikrushna Singh, Sonam Popli, Yukti Hari, Pawan Malhotra1, Sunil Mukherjee1 and Raj K. Bhatnagar1

International Centre for Genetic Engineering and Biotechnology, New Delhi, India

1Correspondence: International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067, India. E-mail: P.M., pawanm{at}icgeb.res.in; S.M., sunilm{at}icgeb.res.in; R.K.B., raj{at}icgeb.res.in

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, G., Popli, S., Hari, Y., Malhotra, P., Mukherjee, S., Bhatnagar, R. K. Suppression of RNA silencing by Flock house virus B2 protein is mediated through its interaction with the PAZ domain of Dicer.


Key Words: viral suppressors • RNAi sensor line






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