Identification And Studies On The Tubule-forming Proteins Of Two Kinds Of Reovirues

In recent years, the epidemic of diseases induced by several reoviruses including SRBSDV and RRSV has caused great losses to rice production in our country. Many studies have been carried out on the etiology, epidemiology, serology, taxonomic status, genome structure and function and host-virus interaction of SRBSDV and RRSV. However, our knowledge on the mechenism by which these two viruses infect their hosts and how they replicate and spread remains poor. Therefore, identifying the viral proteins that are invoved in viral infection and spread is of great importance to the development of prevention measures.The ORFs of SRBSDV-P7-1 and RRSV-P7 were amplified by RT-PCR and then inserted into the baculovirus expression vector pDEST8, resulting pDEST8- SRBSDV-P7-1 and pDEST8 -RRSV-P7 respectively. The plasmids were transformed into E coli DH10Bac to obtain the shuttle vector Bacmid- SRBSDV-P7-1 and Bacmid- RRSV-P7. The Bacmid plasmids were then used to transfect sf9 cells. The expression of the proteins was observed by Immunofluorescence staining and detected by Western blot. Also, the structure of the proteins was observed by electron microscopy after their purification. The results showed that SRBSDV-P7-1 and RRSV-P7 were successfully expressed in sf9 cells. They formed tubular structures in sf9 cells, which can extend from the surface of the infected cell to reach neighboring cells. The tubules are approximately 85 nm in diameter. These results indicated that SRBSDV-P7-1and RRSV-P7 are tubular proteins.In order to study the domain of tubules, Computer analysis of the primary sequence of the SRBSDV-P7-1and RRSV-P7 was conducted. One potential transmembrane region was found in each of the two proteins. The importance of the transmembrane region in tubule formation of the two proteins was studied by deletion exprements. Mutant SRBSDV-P7-1 and RRSV-P7 that lack the N terminal, C terminal or the transmembrane reion respectively were created and cloned into the shuttle vector Bacmid- P7, creating Bacmid- P7-1-ΔTM1, Bacmid-P7-1-ΔTM2, Bacmid-P7-1-ΔTM3, Bacmid-P7-1-ΔN, Bacmid-P7-1-ΔC , Bacmid-P7-ΔTM, Bacmid-P7-ΔN and Bacmid-P7-ΔC respectively. After transfection and Immunofluorescence staining, the accumulation patterns of these mutant proteins was observed under a confocal microscopy. The results showed that the formation the ability to form tubules was lost in mutant SRBSDV-P7-1and RRSV-P7 that lack the transmembrane and C or N terminal regions. This indicated that both of the transmembrane and the conserved terminal regions of these two proteins are involved in tuble formation. In conclusion,we expressed wildtype- and mutant forms of SRBSDV-P7-1 and RRSV-P7 by Bac-to-Bac baculovirus Expression System; The expression of the proteins was detected by Western Blot; the accumulation patterns of the proteins was observed using a confocal microscopy after immunofluorescence staining and the structures of them was observed using Electron microscopy after purification; The function of SRBSDV-P7-1 and RRSV-P7 was investigated in this study; the domains that were responsiable for tubule formation of SRBSDV-P7-1 and RRSV-P7 were sdudied. This will be helpful for further researches of the mechenisms underlying the infection and spread of SRBSDV-P7-1 and RRSV-P7 in their hosts and for the development of prevention measures for the diseases induced by the two viruses.