The Formation Mechanism Of Viroplasm Induced By Southern Rice Black-streaked Dwarf Virus Infection Insect Cells

Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus, in the family Reoviridae, is transmitted by the white-backed planthopper (WBPH, Sogatella furcifera Horvath) in a persistent-propagative manner.In the recent years, SRBSDV has caused severe damage to rice production, and has spread rapidly throughout southern China and northern Vietnam as well as western Japan. The study of SRBSDV mainly focused on the researches of classification, genome, protein functions as well as the interaction between host and vector, however, the mechanism of replication of virus and spread in WBPH is still unknown. Generally, plant reoviruses are thought to replicate and assemble within cytoplasmic, nonmembranous structure called viroplasm which plays an important role in virus replication. Therefore, studying on the formation mechanism of viroplasm induced by fijivirus has significant impact on breaking down transmission route in its vector.This paper studied the formation mechanism of viroplasm induced by SRBSDV infection in insect vector. The baculovirus system was used to express three nonstructural proteins P5, P6 and P9-1. Recombinant baculovirus vectors containing P5, P6 and P9-1 were introduced into DH10Bac for transposition into the bacmid. Sf9 cells were transfected with recombinant bacmids in the presence of Cellfectin. As seen with immunofluorescence microscopy, P5 was aggregated to form filament-like structures in the cytoplasm, P6 was distributed diffusely throughout the cytoplasm, and P9-1 was aggregated to form granular inclusions in the cytoplasm when expressed alone in the Sf9 cells. At the same time, coexpression experiments demonstrated that P6 and P5 were associated with the large circular structures. Furthermore, P6 and P9-1 were colocalized on granular inclusions in the cytoplasm. By contrast, P5 was not observed in association with the inclusions formed by P9-1. At the same time, the results of yeast two-hybrid analysis also showed that P6 interacted directly with P5 and P9-1, while P5 did not interact with P9-1. The previous studies had proved that P9-1 was essential protein of viroplasm matrix. Taken together, these results suggested that viral nonstructural proteins P5, P6 and P9-1 are collectively required for the formation of the viroplasm induced by SRBSDV infection.In conclusion, we use the immunofluorescence to study the structure and function of nonstructural proteins P5, P6 and P9-1 in the Sf9 cells. We firstly proved that nonstructural proteins P5, P6 and P9-1 participated in the formation of viroplasm. It provides scientific basis for studying the replication and spread mechanism of SRBSDV and pays the way for developing a new method to control the disease.