The Interaction Of Rotavirus NSP2 And Dynein Intermediate Chain Mediates The Formation Of Rotavirus Viroplasms

Rotavirus(RV)is one of the causative agents that cause severe dehydrated gastroenteritis in children,piglets and many other young animals.Despite more than a decade of efforts to vaccinate against RV in health systems and veterinary medicine,it continues to threaten the health of children in low-income countries,and to inflict serious economic losses on the pig industry.Therefore,in-depth understanding of RV replication characteristics is urgently needed to develop effective prevention and control methods against RV.The main site of RV replication is viroplasm,and the viroplasms formation is particularly important for effective RV replication.It is reported that RV viroplasms formation is associated with the stability of microtubule and that microtubule-dependent dynein transport is involved in multiple viral replications.But it is still not clear whether microtubule-dependent dynein transport affects RV replication and its viroplasms formation.In this study,the inhibitor of cytoplasmic dynein transport Dynapyrazole-A was used to study the infection of RV on MA-104 cells.And it was found that microtubule-dependent dynein transport was involved in RV infection.By laser confocal imaging technique in living cells,two viroplasms was observed to move along microtubule and finally fuse into a single viroplasm,in MA-104 cells infected with RV.Then,by controlling the time of adding Dynapyrazole-A and using hyperimmune serum that could neutralize RV,the effect of Dynapyrazole-A on RV replication stage was studied in particular(excluding the effect of Dynapyrazole-A on the invasion and release of RV): on the one hand,the result of laser confocal microscope showed that Dynapyrazole-A inhibited the number and size of the early viroplasms,as well as the size of the late viroplasms,while increasing the amount of the late viroplasms(previous studies have shown that RV viroplasms mainly increased their number in the early formation stage,and in the late formation stage viroplasms increased in size and decreased of number through fusion).It suggesting that microtubule-dependent dynein transport was involved not only in the formation of early viroplasms,but also in the fusion of late viroplasms.The result by electron microscopy showed that Dynapyrazole-A inhibited the production of subviral particles surounding viroplasms.Interestingly,Dynapyrazole-A did not affect the expression of viral proteins Nsp2,Nsp5 and VP6 in the RV replication stage.These results suggest that microtubule-dependent dynein transport promotes the RV viroplasms formation and the production of subviral particles surrounding viroplasms.But the expression of RV proteins has nothing to do with these biological processes.In addition,dynein intermediate chain(DIC)was observed to co-locate with RV viroplasms in MA-104 cells infected with RV by laser confocal Airyscan detector.By co-immunoprecipitation test,it was found that DIC directly interacted with viroplasm nucleoprotein non-structural protein 2(NSP2),but not with the other viroplasm nucleoprotein non-structural protein 5(NSP5).And the WD40 repeat domain of DIC directly interacted with the 161?212aa region of NSP2.Moreover,si RNA was used to interfere with the expression of DIC in MA-104 cells,and it was found that silencing the expression of DIC inhibited RV infection,viroplasms formation and the production of subviral particles surrounding viroplasms,which were similar to the results of the dynein transport inhibitor Dynapyrazole-A.These results suggested that DIC participated in RV viroplasms formation by its direct interaction with NSP2,and promoted the production of subviral particles surounding viroplasms.To sum up,dynein mediates the RV viroplasms formation through the direct interaction of DIC with NSP2,and promotes the production of subviral particles surounding viroplasms.Our study reveals the internal molecular mechanism of intracellular transport utilized by RV viroplams,which influences on virioplasm fusion and even the virioplasms formation.The identified interaction region between DIC and NSP2 provides a new insight for the development of prevention and treatment against RV in the future.