Study On The Relationship Between The Structure And The Function Of The Endodomain Of Mouse Coronavirus S Protein

Coronaviruses are enveloped RNA viruses, with a single-stranded, positive-sense RNA genome of28to32kb, the largest known in all RNA viruses, which belong to the Coronaviridae family in the Nidovirales order. They are medically important viruses that cause principally respiratory or enteric infections in humans and a broad range of animals. Mouse hepatitis virus (MHV) is one of Betacoronaviruses, the same as SARS-CoV, which causes neurovirulence and hepatitis in rodents including mouse and rat. Mouse hepatitis virus (MHV) produces a series of subgenomic RNAs for viral protein expression. The structure proteins contain S, E, M and N.The S protein of MHV is a180kDa N-glycosylated protein, a typical type I transmembrane protein, mediating receptor binding and membrane fusion. S protein of coronavirus can be divided into three functional parts including N-terminal part (S1), extracellular part of S2and C-terminal part of S2. S1containing receptor binding domain is responsible for viral tropism. Extracellular part of S2containing a fusion peptide and two heptad repeats (HR1and HR2) domains is involved in membrane fusion. There is a unique cysteine-rich motif in S endodomains of most coronaviruses. Previous experiments showed that a13-residues deletion in the motif of recombinant MHVs reduced the formation of syncytia dramatically but remained relatively high replication in L2cells. Based on the previous results, in the study our objectives are to investigate the feasibility of recombinant MHV as a viral vector to express foreign proteins, and to analyze the function of the cystein-rich motif in virus replication.Firstly, we investigated the influence of the deletion mutation of the cysteine-rich motif on the expression of foreign proteins by recombinant MHV. Secondly, the function of the cysteine-rich motif on the virus replication was analyzed.Previously, a13-residue of deletion in MHV spike (S) protein endodomain was found to reduce the formation of syncytia dramatically while slightly influence virus replication. In this study, effects of the S mutation on MHV infectivity and foreign protein expression were further examined in murine L2, NIH/3T3and Neuro-2a cells. The replacement of MHV gene2a/Hemaglutinin-Esterase with a membrane-anchored protein hook (HK) and gene4with EGFP did not change the adaptability and cytopathology of recombinants in these cells. However, the cytopathic effect of recombinants with partial S deletion was significantly reduced in these cells. The replication and foreign protein expression of the S-mutated recombinants were found more efficient in L2than in Neuro-2a and in NIH/3T3. Meanwhile HK as well as EGFP expressed by recombinants were observed to locate appropriately in infected cells and the distribution pattern was similar to that in cells transfected with eukaryotic expression vector. These results suggest that partial deletion in S endodomain be useful for improvement of MHV as a viral vector for protein expression through attenuating toxicity and maintaining foreign protein expression.In this study, we investigated the functional importance of the cysteine-rich region in virus replication and the role of posttranslational palmitoylation of the conserved cysteines in the domain using reverse genetics.7-10highly conserved cysteine residues are located adjacent to the carboxy side of the hydrophobic domain of all coronavirus S proteins. We hypothesized that conservation of the cysteines residues should be significant for viral replication and the palmitoylation may facilitate protein-lipid membrane interaction. First, the effect of replacement of cysteines on virus growth and the level of palmitoylation on S proteins with different number of cysteines were analyzed. The ability of S protein anchoring membrane and the lipid raft association were also investigated. The data strongly indicate that the cysteine-rich region is important for coronavirus replication since mutant viruses with only above three cysteine residues could grow on L2cells. The level of palmitoylation was detected by shift of electrophoresis mobility after treatment with hydroxylamine. The results also show that the palmitoylation can influence the lipid raft association of S protein and may be important for the S protein transportation to the cell membrane after infection.