Interaction between Bombyx mori Cytoplasmic Polyhedrosis Virus NSP8 and BmAgo2 Inhibits RNA Interference and Enhances Virus Proliferation.

Abstract

Some insect viruses encode suppressors of RNA interference (RNAi) to counteract the antiviral RNAi pathway. However, it is unknown whether Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) encodes an RNAi suppressor. In this study, the presence of viral small interfering RNA (vsiRNA) in BmN cells infected with BmCPV was confirmed by small RNA sequencing. The Dual-Luciferase reporter test demonstrated that BmCPV infection may prevent firefly luciferase (Luc) gene silencing caused by particular short RNA. It was also established that the inhibition relied on the nonstructural protein NSP8, which suggests that NSP8 was a possible RNAi suppressor. In cultured BmN cells, the expressions of viral structural protein 1 (vp1) and NSP9 were triggered by overexpression of nsp8, suggesting that BmCPV proliferation was enhanced by NSP8. A pulldown assay was conducted with BmCPV genomic double-stranded RNA (dsRNA) labeled with biotin. The mass spectral detection of NSP8 in the pulldown complex suggests that NSP8 is capable of direct binding to BmCPV genomic dsRNA. The colocalization of NSP8 and B. mori Argonaute 2 (BmAgo2) was detected by an immunofluorescence assay, leading to the hypothesis that NSP8 interacts with BmAgo2. Coimmunoprecipitation further supported the present investigation. Moreover, vasa intronic protein, a component of RNA-induced silencing complex (RISC), could be detected in the coprecipitation complex of NSP8 by mass spectrum analysis. NSP8 and the mRNA decapping protein (Dcp2) were also discovered to colocalize to processing bodies (P bodies) for RNAi-mediated gene silencing in Saccharomyces cerevisiae. These findings revealed that by interacting with BmAgo2 and suppressing RNAi, NSP8 promoted BmCPV growth. IMPORTANCE It has been reported that the RNAi pathway is inhibited by binding RNAi suppressors encoded by some insect-specific viruses belonging to Dicistroviridae, Nodaviridae, or Birnaviridae to dsRNAs to protect dsRNAs from being cut by Dicer-2. However, it is unknown whether BmCPV, belonging to Spinareoviridae, encodes an RNAi suppressor. In this study, we found that nonstructural protein NSP8 encoded by BmCPV inhibits small interfering RNA (siRNA)-induced RNAi and that NSP8, as an RNAi suppressor, can bind to viral dsRNAs and interact with BmAgo2. Moreover, vasa intronic protein, a component of RISC, was found to interact with NSP8. Heterologously expressed NSP8 and Dcp2 were colocalized to P bodies in yeast. These results indicated that NSP8 promoted BmCPV proliferation by binding itself to BmCPV genomic dsRNAs and interacting with BmAgo2 through suppression of siRNA-induced RNAi. Our findings deepen our understanding of the game between BmCPV and silkworm in regulating viral infection.