Localization Of Viroplasm Assembly Related Domains On Rotavirus NSP2 And Its Effect On Replication

Rotavirus?RV?is a pathogen that causes severe dehydrating diarrhea in infants and young animals.Currently,there has no effective drug to treat RV,vaccination is an important component of prevention strategies.Viroplasm can be observed in the cytoplasm during the early stage of rotavirus infection,and then the packaging and replication of rotavirus were carried out.Viroplasm-like structures?VLS?formation requires the interaction of two nonstructural proteins,NSP2 and NSP5.NSP2 is essential for the viroplasm formation and rotaviruses replication,however,the effect of its key domains on viroplasm assembly and virus replication remain largely unknown.The study of the influence of key sites or domains of NSP2 on the formation of viroplasm by reverse genetics system can provide reference for the research and development of new RV drugs and vaccines.In this study,recombinant plasmid p LVX-IRES-Puro-T7 RNAP with helper plasmids were co-transfected into 293T cells.Lentivirus was harvested and infected BHK-21 cells.T7 RNAP and puromycin were introduced into BHK-21 by lentivirus infection and the cells were screened with puromycin to obtain BHK-21 cells expressing T7 RNAP stably.The open reading frame of NSP2 of rotavirus strain SA11was expressed in prokaryotic cells and guniea pigs were immunized by the purified recombinant protein with the aid of ISA206.The serum was separated,then the anti-NSP2 polyclonal antibody was prepared.To predict sites on NSP2 that may be associated with viroplasm replication by read articles.The recombinant mutant plasmids p T7-NSP2-H225,p T7-NSP2-H225A,p T7-NSP2-784-820 and p T7-NSP2-K188AH221AH225A were constructed according to the mutant sites.The recombinant viruses were rescued by cotransfected of BHK-T7 cells with 14 plasmids reverse genetic system.RT-PCR was used to detect the expression of NSP2 and NSP5 protein in recombinant viruses.Meanwhile,quantitative real-time PCR was used to quantify the difference of VP6 gene expression of mutant viruses.Since viroplasm appear at the early stage of viral infection,indirect immunofluorescence was used to detect the formation of viroplasm after the recombination virus infected cells 4 and 8h.TCID50 of recombinant virus was determined by Reed-Muench method to detect the difference of viral titer of mutant virus.The BHK-21 cell lines stable expressing T7 RNAP were successfully obtained,and RT-PCR shown that the T7 RNAP could be stably expressed in different generations.The NSP2 polyclonal antibody was successfully prepared,and reached a titer of higher than 1:8000.The recombinant viruses rescued by reverse genetics system were named as wt r SA11,mu r SA11-NSP2-H225,mu r SA11-NSP2-H225A,mu r SA11-NSP2-784-820 and mu r SA11-NSP2-K188AH221AH225A.The total RNA of the rescued recombinant RV were extracted,and amplified with NSP2 and NSP5 specific primers after reverse transcription to obtain the target band.In the real-time PCR,compared with wt r SA11,the VP6 expression of mu r SA11-NSP2-H225,mu r SA11-NSP2-H225A,mu r SA11-NSP2-784-820 and mu r SA11-NSP2-K188AH221AH225A were 79.00%,39.78%,46.65%and 53.58%,respectively.In T-test,there was no significant difference between mu r SA11-NSP2-H225 and wt r SA11,mu r SA11-NSP2-H225A,mu r SA11-NSP2-784-820 and mu r SA11-K188AH221AH225A compared with wt r SA11 have significant difference.In the early stage of virus packaging and replication,the indirect immunofluorescence assay was used to detect NSP2 expression and viroplasm formation.After 4 h and 8 h of inoculation with recombinant viruses,showed that the green fluorescence of mutant viruses were less than that of wild-type,and the formation of viroplasm and replication of mutant viruses were delayed.The TCID₅₀was detected the difference in viral titer of mutant viruses,the value of wt r SA11,mu r SA11-NSP2-H225,mu r SA11-NSP2-H225A,mu r SA11-NSP2-784-820 and mu r SA11-NSP2-K188AH221AH225A were 10^-3.86,10^-3.8,10^-3.42,10^-2.61and 10^-1.61,respectively.The difference in viral titer indicated that their mutation sites had different effects on virus replication.Above of all,compared with wt r SA11,mutant viruses mu r SA11-NSP2-H225A,mu r SA11-NSP2-784-820 and mu r SA11-NSP2-K188AH221AH225A showed delayed viroplasm formation,delayed viral replication,reduced relative expression of VP6,and decreased viral titer,indicating the importance of this site in viral packaging replication.The conclusion is of great significance to elucidate the effect of NSP2 protein on the packaging and replication of rotavirus and provide a new method for the development of novel drugs and vaccines against rotavirus.