Research On The Interacting Cellular Proteins Of Porcine Rotavirus Structural Protein VP6

Porcine rotaviruses(PoRV)are the major cause of acute viral gastroenteritis in piglets.PoRV infection is transmissive by fecal-oral route,and these diseases are endemic.PoRV group A(RVA)spread widely in China now,and the prevalence rates in pigs vary from 3.3% to 67.3%.G9 genotypes of PoRV strains re-emerge in recent years.PoRV infection alone or mixed infection with other pathogens,such as porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV)and porcine circovirus(PCV),impact productivity in swine herds.This becomes an important factor to hinder pig industry development,and it causes huge economic losses.Therefore,it is extremely urgent and important that we start to have an insight into PoRV replication process in vivo,and study the interaction mechanisms of viral and cellular proteins,for controlling these diseases prevalence effectively,and reduce the hazard.Rotavirus A pig/China/NMTL/2008/G9P[23](abbreviated as NMTL strain)was isolated in our lab.We selected PoRV NMTL strain as object of this study,not only because it had high pathogenicity for piglets,but also there are potential risks for G9P[23] genotype strains of RVA transmission from porcine to human,which pose a great threaten to the public health,and should be given full consideration.PoRV VP6 is the major structural protein,and locates in the intermediate layer of virion.It involves in the transcriptive activity of double-layered particles(DLPs),which were released into the cytoplasm after virus uncoating.Recent researches suggest that VP6 may associate with plenty of cellular factors,and these interactions play essential roles during RV infection,but the interaction partners of VP6 and their fuctions are still unclear.In this study,we amplified VP6 gene of the PoRV NMTL strain by reverse transcriptionpolymerase chain reaction(RT-PCR),and the VP6 PCR products were ligated into the glutathione S-transferase(GST)fusion vector p GEX-6P-1.After that,it was transformed into DH5? competent cells.The resulting recombinant plasmid p GEX-PoRV-VP6 was verified by DNA sequencing.GST and the GST-VP6 fusion protein were expressed in the Escherichia coli BL21(DE3)strain by induction with 1 m M isopropyl-?-D-thiogalactopyranoside.By GST pull-down assay and matrix-assisted laser desorption/ionization dual time of flight(MALDI-TOF/TOF)mass spectrometry(MS),we discovered three interactive proteins of PoRV VP6 in MA104 cells,and they are beta-actin,TPM1 and RPS16.These interactions were further confirmed by Co-immunoprecipitation(Co-IP)assay.The interaction with beta-actin was further studied.Cellular beta-actin protein expression was knocked down by si RNA interference,and then MA104 cells were infected with PoRV.PoRV VP6 protein expression in the cell lysates were detected bywestern blotting analysis,and the culture supernatants of differently treated groups were collected to perform infectivity assays.Virus particles releasing from MA104 cells to media were measured at different time points post PoRV infection(2h/4h/6h/8h/10h/12h)using q RT-PCR analysis,to determine the time necessary for a single lifecycle of PoRV infection.On the basis of electron microscopy observation and expression kinetics of PoRV VP6,define PoRV replication stages.Cyto D were added to MA104 cells at different time points after virus inoculation(0h/2h/4h/6h),and releasing viruses were detected at 8hpi by q RT-PCR,to make sure whether actin polymerization have an effect on PoRV propagation and virus release.Co-localization of beta-actin and PoRV VP6 were detected in PoRV infected cells by double immunofluorescence,and transport pathways of PoRV VP6 were observed by immuno-electron microscope.PoRV DLPs were purified by cesium chloride(Cs Cl)density-gradient centrifugation,in order to determine the roles PoRV DLPs played in actin polymerization by actin-binding proteins(ABPs)spin-down assay.Our study discovered beta-actin,tropomyosin1(TPM1)and 40 S ribosomal protein S16(RPS16)are interactive proteins of VP6 in PoRV infection.These three proteins mentioned above exist extensively in various histocytes,and they are highly conservative during species evolution.PoRV utilizing these common cellular proteins is beneficial to virus adaptation and infectivity,furthermore make it possible for RV interspecies transmission.PoRV VP6 synthesis and virions release were significantly reduced when beta-actin was knocked down by si RNA in PoRV infected cells.MA104 cells were transfected with si ACTB for72 h,and then were infected with the PoRV NMTL strain for another 48 h.Cell lysates were collected for western blot analysis,and PoRV VP6 protein expression was significantly reduced compared with that of the control groups.The culture supernatants were collected to infect MA104 cells.After 18 hours post infection,infectivity rates of different groups were examined with the Operetta TM High Content Screening System.We found that there were less PoRV-infected cells in the si ACTB treatment group than the others(Untreated,Mock and si FAM groups).These results suggest that the interaction between PoRV VP6 and beta-actin might efficiently mediate a productive virus infection,and plays essential roles in DLPs delivery,m RNA transcription initiation to synthesize viral proteins,and TLPs maturation and release.By double immunofluorescence assay,we found that PoRV VP6 extensively co-localized with beta-actin throughout the cell cytoplasm in the infected cells.By immunoelectron microscopy observation,we found VP6 localized on the actin microfilaments at the early stage of PoRV infection,and then they distributed in the ribosome,mitochondria,endoplasmic reticulum(ER)and nucleus.These findings suggest that depending on the actin network,DLPs can be delivered to specified sites for replication in PoRV infected cells.It revealed the pathways of VP6 travelling in the infected cells and cellular components that involved in PoRV replication.ABPs spin-down assays confirmed that PoRV VP6 bound to actin filaments,but PoRV DLPs didn't have actin polymerization enhancement activity in vitro.Rely on this interaction,PoRV DLPs hijack cellular actin network,and DLPs can be delivered to specified organelles in thecytoplasm like cargoes for replication.But during PoRV infection in vivo,whether PoRV VP6 recruit other cellular proteins to co-stimulate actin polymerization,such as TPM1,it needs further verified.During the PoRV infection,there are various interactions between virus and host cells,which establish a complex network with proteins.They play critical roles in virus replication and innate immune response.These findings in this study reveal the ways that PoRV VP6 and its interactive proteins involving in virus replication process.It will provide a scientific reference for us to understand PoRV pathogenic mechanisms,control and prevent diarrheal diseases.