Study Of SARS-CoV M Protein By RNA Interference, And The Role Of Three Conserved Histidines In SFV Infection

SectionⅠStudy of SARS-CoV M protein by RNA interferenceSevere acute respiratory syndrome-associated coronavirus(SARS-CoV) belongs to group 2 coronavirus.It is an enveloped virus,with a single-stranded plus-sense RNA genome of about 30 kb in length.It mainly encodes six structural proteins,which are spike (S),envelope(E),membrane(M),nucleocapsid(N) and the newly defined Orf3a and Orf7a.Among them,M protein is the most abundant viral membrane glycoprotein and plays key role in viral assembly and budding.In this study,the subcellular distribution of M protein was established in mammalian cells where it showed "dot" expression.Small interfering RNA(siRNA) targeting to the two terminal sequences were designed, transcribed in vitro,and then introduced into the cells expressing the M protein.The results demonstrated that three candidate siRNAs specifically degraded M mRNA and significantly inhibited M protein expression,which provides a new target for the development of anti-SARS drugs.1,Localization of SARS-CoV membrane protein in cultured cellsUsing EGFP as report gene,the plasmid pEGFP-M,which encodes M-EGFP recombinant protein,were co-transfected with Golgi/pDsRed-N1,a specific Golgi marker, into the HEK 293T cells.After 48 hours,the transfected cells were fixed by paraformaldehyde,and examincd by confocal fluorescence microscopy.The cellular distribution of M-EGFP recombinant protein and Golgi-Red protein were analyzed and overlapped by the use of image overlap software,which showed that they co-localized in Golgi apparatus.2.Silencing of SARS-CoV membrane protein by siRNA in cultured cellsWe adopted T7 transcription in vitro to acquire five siRNAs,three of which were targeting to M gene,and the other two were positive and negative control.Then the five siRNAs and pEGFP-M werc co-transfected into HEK 293T cells,respectively.After 48 hours,grecn fluorescence was observed using fluorescence microscope;Samples were counted and analyzed with CellQuest software,using non-transfected HEK 293T cells as control.The values were calculated as the percentage of the cell population that exceeded the fluorescence intensity of the control cells and the mean fluorescence intensity of this population;Total RNA from the transfected or non-transfected cells was extracted and semi-quantitative RT-PCR and Real-time PCR were performed using SARS-CoV M and GAPDH gene specific primers after being reverse transcribed into cDNA.The expression of M-EGFP recombinant protein was detected by Western blot.The results demonstrated that compared with the cells transfected with pEGFP-M alone,the cells co-transfected scramble siRNA gave no significant reduction of EGFP expression,while the EGFP-siRNA gave an about 1.9- and 4.3-fold reduction in percentage of fluorescence cell population and mean fluorescence intensity respectively.Percentage of fluorescence cell population and mean fluorescence intensity reduced about 1.5- and 5.0-fold for M-siRNA1, 1.9- and 3.7-fold for M-siRNA2 and 2.2- and 5.0-fold for M-siRNA3.Compared with the cells transfected with pEGFP-M alone,the transcript levels of SARS-CoV M gene were decreased about 45-,56- and 52-fold in cells co-transfected with SARS-CoV M-siRNA1, M-siRNA2 and M-siRNA3 respectively.Correspondingly,SARS-CoV M transcript levels were not significantly changed in cells co-transfected with scramble siRNA,while GAPDH transcript level was not affected by the five siRNAs.M-EGFP recombinant protein was expressed at high level in cells transfected with pEGFP-M alone,similar to that co-transfected with scramble siRNA.The recombinant protein expression levels were obviously decreased in cells co-transfected with EGFP-siRNA,M-siRNA1,M-siRNA2 and M-siRNA3 for their weak bands,while the siRNAs had no effect on the expression of GAPDH.These results demonstrated that siRNA can inhibit the target protein expression effectively and specifically through down-regulating the transcript level of target gene.PartⅡthe Role of three conserved Histidines in SFV infectionSFV(Semliki Forest Virus),belonging to enveloped alphavirus,infects cells by low pH-dependent membrane fusion.Its envelope protein E1 is classⅡfusion protein,which is composed of three domains(DⅠ,DⅡ,DⅢ) enriched inβ-structure.A series of conformational changes occur during membrane fusion,e.g.E1/E2 dissociation,fusion peptide exposure and E1 HT formation.However,it is not clear what triggers membrane fusion.Prior studies showed that some residues on E2 were related with E1/E2 dissociation. But as for E1,its ectodomain could also insert membrane and form HT after acid treatment, indicating that E1 has its own pH sense system.So,what controls E1's low pH-dependent conformational change? What is the mechanism? SFV fuses at the threshold of about pH 6.2,which is close to the pKa of Histidine(～6-7),suggesting that its proton state change is associated with E1's conformational change.Therefore,in the study,mutagenesis was performed to mutate His(H3,H125 and H331) into Alanine,three SFV mutants were constructed and investigated for their respective role in viral infection and fusion.1.Construction and initial phenotype identification of SFV mutantsFirst,different E1 sequences from alphavirus were aligned and analyzed for their sequence conservation.We found that three His(H3,H125 and H331) in SFV E1 were highly conserved,and located at key position in E1 crystal structure.H3 is close to DⅠ-DⅢadapter,H125 is near to the hinge region of DⅠ/DⅡ,and H331 is located at the interface of DⅢand DⅠ.They all moved during the fusion.Therefore,mutated SFV infectious clones (His to Ala) were constructed by mutagenesis.Infectious RNA was transcribed in vitro, and electroporated into BHK cells.The infectivity of wt and mutant SFV was compared by spread assay.Results showed that the secondary infectivity of H125A and H331A mutants was similar with that of wt,while that of H3A was much lower.However,both H3A E1 and E2 proteins were expressed and transported to cell surface.The results of growth dynamics for the three mutants demonstrated that H125A and H331A mutants produced equal amount of virus efficiently as wt,but the virus titer produced by H3A mutant was about two logs lower than wt,and mutated virus assembled normally.Therefore,the decrease of H3A mutant infectivity was not due to the expression and location of E1 and E2,and also the virus assembly.2.Fusion activities of SFV mutantsFusion-Infection Assay(FIA) and cell-cell fusion assay were performed to test the fusion activities,which occur either between virus and plasma membrane or between cell and cell mediated by SFV envelope proteins.Results of FIA demonstrated that H331A mutant had the similar fusion activity with wt,while H125A and H3A mutants showed pH shift in their fusion,especially for H3A mutant,which needed lower pH to trigger fusion. The fusion threshold of H125A and H3A mutants was pH5.75 and pH5.5(wt～pH6.25), with the maximal fusion at pH5.5 and pH4.5(wt～5.75),respectively.The results of cell-cell fusion assay were similar.H331A mutant was identical to wt,the average fusion index of H125A mutant,with the threshold of pH6,was about 20%lowed than wt,but the size and formation rate between them showed no significant difference.As for H3A mutant, it needed pH5 or lower pH to trigger cell fusion.The fusion index was still much lower than wt even at pH4.5,just 4 or 5 nuclei were seen in the syncytium(～30 nuclei for wt), and about 80%of cells was single nucleus.Therefore,the fusion activity of H3A mutant was much lower than wt,which was the main reason why this mutant decreased in its infectivity.Next,the major steps during the fusion were tested for H3A mutant.3.Tests of H3A mutant fusion processFirst,trypsin-digestion at 0℃was performed to test the stability of H3A E1/E2 heterdimer.Similar with wt,H3A E1/E2 was stable at neutral pH,but dissociated at low pH.Secondly,virus-liposome binding assay was used to test the dissociation of E1/E2 and subsequent E1's membrane insertion.At low pH,H3A mutant was able to associate with complete liposome and cofloated to the upper fractions of sucrose gradient just as wt, indicated that H3A E1/E2 dissociated at low pH and monomeric E1 was able to insert into lipid bilayer.Finally,SDS-sensitivity,trypsin-digestion and co-IP with E1 acid-specific antibody experiments were performed to test whether H3A mutant was able to form correct conformational HT.Although wt SFV could form effectively correct E1 HT at pH6 and lower pH,H3A mutant could not until pH5 or even lower and less efficiently.The HT formed by H3A mutant was also SDS-and trypsin-resistant,but its acid-specific epitope was exposed less efficiently.Thus,H3A mutation does not affect E1/E2 dissociation and monomeric E1's membrane insertion,but the subsequent trimerization was blocked and no correct conformational E1 HT was formed,which resulted in defected membrane fusion and virus infection.4.Screening and identification of H3A revertantsAlthough H3A mutant spreaded less efficiently than wt,it can generate some viable revertants to compensate for the defect.To analyze and understand how H3A mutant regulates low pH-dependent fusion,single revertant was isolated and purified by agarose overlay.After amplification,viral RNA extraction,E1 gene amplification and sequencing, ten independent revertants were obtained.They were all single mutation in addition to the introduced H3A.Based on their positions in E1*(E1 ectodomain) HT,these revertants were divided into two groups:the first group is located on DⅠ/DⅢadapter,and contains five revertants but just two amino acids mutation,that is D284 and A286;the second group is located in DⅠand also contains five revertants,including A3T,P8A,V10A and V140A. The positions these revertants located are the regions where changed a lot during hairpin-like E1 HT formation.Thus,the study of revertants will provide more structural information for the phenotype of H3A mutant.Three SFV mutants were constructed by mutagenesis,analyzed and compared with wt in viral infection and membrane fusion.Data here demonstrated that H331A did not affect SFV infection and fusion,H125A showed no effect on infection and a small pH shift on fusion,while H3A decreased the fusion threshold and infection by affecting correct HT formation.Ten H3A revertants were isolated and the second-site mutations were all located key region for DⅢfold,suggesting that they rescued the H3A fusion and infection by conformational modification for H3A HT.All these data suggested that H3 protonation was directly associated with E1 HT formation,which plays important role in the regulation of low pH-dependent membrane fusion.