The Formation Mechanism Of Viroplasm Induced By Rice Gall Dwarf Virus Infection In Its Insect Vector Cells

Rice gall dwarf virus (RGDV) is a member of the genus Phytoreovirus in the family Reoviridae, which is effectively multiplication in the host plant and vectors. RGDV transmitted by leafhopper Recilia dorsalis and Nephotettix cincticeps in a persistent-propagative manner. The viral genome of RGDV encodes six structural proteins (P1, P2, P3, P5, P6 and P8) and six nonstructural proteins (Pns4, Pns7, Pns9, Pns10, Pns11 and Pns12). In this paper, we focus on the fuctions of nonstructural proteins of Pns7, Pns9 and Pns12 and the structural proteins of P8 in the cells.The results showed that Pns7 and Pns9 localise in the cytoplasm, and Pns12 localise in the cytoplasm and nucleus when they expressed in Sf9 cell respectively. Co-expression the nonstructural proteins:Pns7 and Pns9 distributed in the cytoplasm. However, Pns7 and Pns12 have no co-localzilation in Sf9. While Pns9 could recruit Pns12 from nucleus to cytoplasm, suggesting these two proteins interacts in Sf9 cells RNA interference (RNAi) induced by dsRNA synthesized in vitro was used to knockout Pns7, Pns9 and Pns12 of RGDV in the cultured cells derived from R. Dorsalis. The results showed that the expression of the three nonstructural proteins were decreased obviously. Viroplasms formed were inhibited in size and number These result suggest that the nonstructural proteins Pns7, Pns9 and Pns12 of RGDV participate in the formation of viroplasm. These three proteins interacts with each other and one of the proteins is important for the formation of viroplasm and the replication of virus particles.The structural protein P8 is a component of viral outer capsid protein, but the function of this protein in viral infectionis still unknown in insect vector. Here, RNAi induced by dsRNA synthesized in vitro was used to investigate the role of RGDV P8 protein in viral infection in the cultured cells derived from R. dorsalis. Immunofluorescence assay of RGDV infection in cultured cells after the treatment of dsRNAs from P8 gene (dsP8), showed that dsP8 not only knocked down the expression of P8, but also reduced the accumulation of RGDV in the insect vector cells. Then viral dsRNA genome and proteins were detected by SDS-PAGE and Western blot, the results indicated that the treatment of dsP8 significantly decreased the synthesis of viral dsRNA genome and the expression of P8 protein in insect vector cells. Thus, the results suggested that structural protein P8 of RGDV may play a role in viral assembly and multiplication in insect vector cells. This provide an important theoretical for controling the spreading of rice gall dwarf disease by using RNAi Technology.