Functional Study Of The Conserved Region LLRKxGxKG In Non-structural Protein 1 Of Coronavirus GroupⅡ

Coronavirus can cause severe disease in livestock animals and lead thereby to high economic losses. In humans, almost 10%-30% mild respiratory tract disease (common cold) is caused by coronavirus infections. In 2002-2003, the appearance of severe acute respiratory syndrome (SARS) in our country,caused by a formerly unknown coronavirus (SARS-CoV), exemplified the potential of coronaviruss to seriously affect human health. Therefore, it is of the utmost importance to reveal the pathogenic mechanism of coronavirus.But SARS-CoV related study is severely restricted for bio-security reasons. Under this circumstances, mouse hepatitis virus A59 (MHV A59) is the most convenient and valuable type strain for study. That's because first, both MHV and SARS-CoV belong to groupⅡcoronavirus. And they are homologous to each other. So the study of MHV can give us important indications for SARS. Second, MHV is harmless to human beings. And there is well developed animal model for MHV study. So it is easy to carry out the research.Some reports showed that in MHV and SARS-CoV, non-structural protein 1 (NSP1) is an important pathogenic factor and it can suppress the production of IFN-β. Sequence alignment revealed that there is a highly conserved region (LLRKxGxKG) in MHV and SARS-CoV NSP1. It may be an important functional domain, and plays a key role in some identical functions between MHV NSP1 and SARS-CoV NSP1. It deserves a further study. For this goal, we constructed MHV NSP1 mutant gene with LLRKxGxKG region deletion, and also rescued mutant MHV. By detecting the changes of IFN-βregulation function, replication capacity, virulence and tissue tropism, we hope to characterize the function of the conserved LLRKxGxKG region in NSP1 of coronavirus groupⅡ.1. In vitro functional study of MHV NSP1 and conserved region LLRKxGxKGIn order to reveal the effect of MHV NSP1 and the LLRKxGxKG region on IFN-βproduction, we constructed the eukaryotic plasmids pcDNA3.1-NSP1 which can express MHV NSP1 and pcDNA3.1-NSP1-mu which can express the LLRKxGxKG deleted NSP1. Then they were transfected into L929 cells. After stimulation, the expression level of IFN-βin the culture supernatant was measured by ELISA. Results showed that MHV NSP1 can reduce IFN-βexpression by 12.8%, and the mutant one by 18.4%. While the LLRKxGxKG region seems to have no effect on this.To further investigate the effect of MHV NSP1 on IFN signal pathways, two reporter plasmids (-110-IFNβ)-CAT and ISRE-Luc, respectively, were used to co-transfect with the eukaryotic expressing plasmids. The activity of IFN-βpromoter and Interferon Stimulated Response Element (ISRE) were determined by CAT ELISA assay and Luciferase assay. Results showed that MHV NSP1 can inhibit the activity of IFN-βpromoter and ISRE dramatically (P<0.01). But it makes no difference whether the LLRKxGxKG region was deleted or not.In former experiments, we also found by accident that without stimulation the inhibiting capacity decreased dramatically after the deletion of the LLRKxGxKG region. And someone else found that SARS-CoV NSP1 can suppress host gene expression. We speculate that MHV NSP1 may have the same function. So in another set of experiments, some other reporter plasmids (pRLuc-CMV,pGL3-basic and pGL3-control) were used to co-transfect with the eukaryotic expressing plasmid. The expressing levels of reporter genes were examined. Results showed that MHV NSP1 can suppress reporter genes expression dramatically (P<0.01). And the suppression decreased dramatically (P<0.01) after the deletion of the LLRKxGxKG region. Our results suggest that MHV NSP1 can suppress host gene expression, and the conserved region LLRKxGxKG plays an important role in this process.2. Rescue of MHV mutant with LLRKxGxKG region deletedTo deeply understand the relationship between NSP1 conserved region LLRKxGxKG and viral pathogenicity, we used the MHV reverse genetic system to rescue MHV mutant with this region deleted.The E. coli guanine-phosphoribosyl transferase gene (gpt) was used as both a positive and a negative selection marker. By vaccinia virus-mediated homologous recombination, we replaced NSP1 gene in MHV genome by the mutant one. After GPT selection and plaque purification, we got the vaccinia virus which contains the mutant MHV genome. Then the genomic vMHV-inf-1 DNA was prepared from this virus stocks and used as a template to transcribe, in vitro, a capped RNA corresponding to the MHV genome with bacteriophage T7 RNA polymerase. When this RNA was transfected into BHK-21 cells by lipofection, cytopathic effects indicative of mouse hepatitis virus infection developed throughout the culture after 24-48 hours. A virus, designated MHV-NSP1⊿ 27, was rescued from the culture supernatant, and then was plaque purified and identified. And viral growth and peak titers of MHV-NSP1⊿ 27 in 17CL-1cells were almost the same with that of wide type MHV (MHV wt).3. Pathogenicity analysis of MHV mutant with LLRKxGxKG region deletedOur study revealed MHV-NSP1⊿ 27 is trongly attenuated in vivo. Infections with a high dose (5×106pfu, intraperitoneal) of wide type MHV may result in 87.5% fatality. While for MHV-NSP1⊿ 27 the fatility was 37.5%. And mice infected intracranialy with 2×104pfu of MHV wt succumbed to infection. Whereas mice infected with 2×104pfu of MHV-NSP1⊿ 27 all survived. Both MHV-NSP1⊿ 27 and MHV wt could replicate in spleen and liver, whereby MHV-NSP1⊿ 27 titers were consistently lower than MHV wt. Furthermore, MHV-NSP1⊿2 7 was rapidly cleared and not detectable after day 2 p.i.. And liver enzyme values of mice infected with MHV-NSP1⊿ 27 were much lower than that infected with MHV wt. Finaly, MHV-NSP1⊿ 27 can elicit strong immune response and protect mice from homologous viral infections. So it is a good candidate of live attenuated vaccine.The most remarkable finding of this study is the level of attenuation of the nsp1 mutant MHV. This finding reveals that the conserved region LLRKxGxKG is an important pathogenic site. And MHV mutant reserved good immunogenicity,so it provides good evidence for the study of SARS-CoV pathogenic mechanism and attenuated vaccine. But it needs a further study on the details of LLRKxGxKG region's function.And it's the first time that the recombinant coronavirus was successfully rescued using vaccinia virus vector reverse genetic system in China. This provides us a convenient platform for coronavirus research. And it can be used to some other fields. The use of vaccinia virus vectors also enlight us on the development of reverse genetics of other viruses.