Studies On Receptor Protein Of SARS Coronavirus Replicase Nsp8 And Nsp9

Severe acute respiratory syndrome(SARS) is a new infectious disease of human caused by a novel coronavirus which leads to atypical pneumonia with the manifestation of febrile illness, headaches ,myalgia and cough followed by progressive acute respiratory distress and respiratory failure. This infectious disease spreads widely, transmits rapidly and causes acute pathological changes, and leads to a high mortality rate up to 10%. To date, SARS has been controlled using conventional measures such as rapid detection, infection control, isolation, quarantine, contact tracing, etc. Clearly, these measures cannot be sustained indefinitely or repeatedly, and there is an urgent need to elucidate the natural history and pathogenesis of SARS-CoV infection, as well as to develop improved diagnostic tests and specific antiviral drugs and vaccines.As invasion into the body, this viral agent can express numbers of replicases (nonstructural protein),including the RNA dependent RNA polymerase, according to the viral genomic RNA. And these replicases involves in transcription of subgenomic mRNA, synthesis structural protein mRNA and viral genomic RNA through the discontinuous transcription model. The SARS-CoV replicase gene has been shown, or is predicted to encode multiple enzymatic functions which include a papain-like proteinase activity, a 3C-like serine proteinase activity, an RNA-dependent RNA polymerase activity, a superfamily 1-like helicase activity , 3'-5'exoribonuclease activity , endoribonuclease activity , ribose 2'-O-methyl transferase activity. These replicases play important roles in viral transcription, translational and post-translational regulatory mechanisms, whose molecular details remain to be determined. According to the structures and functions of replicases, a lot of drug anti-SARS-CoV has been designed and developed to prevent transmission of the disease. Now, the functions of nsp1, nsp2, nsp4, nsp6, nsp7, nsp8, nsp9, nsp10 and nsp11 are still unkown, but they can be detected in the SARS-CoV-infected cells as early as 6h postinfection. Nsp8 and nsp9 detected with immunofluorescence microscopy show punctate perinuclear and distribiued cytoplasmic foci,which locate at endoplasmic reticulum and small vesicle.Dual-labeling studies demonstrate collocation of nsp8 with nsp2 and nsp3 in cytoplasmic complexes and also with LC3, a potein marker for autophagic vacuoles. Nsp9 has a RNA binding activity,and can interact with nsp8 to change the behavior of nsp8. In this study, the gene of full length 594bp of replicase nsp8 was cloned from SARS-CoV BJ01 strain by RT-PCR and inserted into the vector pGEX-6p-1 to get the construct pNSP8E. The recombinant strain BL21(DE3)pNSP8E expressed souble 22kDa protein of nsp8 induced with IPTG. The receptor protein of nsp8 was captured by bait protein GST-nsp8 from the A549 cell lysate in the pull-down experiment. The result of peptide mass fingerprint(PMF) of target protein showed the potential receptor protein was zinc finger protein 324(ZNF324). Subsequently, the gene sequence of partial ZNF324 was cloned from A549 cell, and the protein of ZNF324 was harvested in prokaryotic expression system. The reaction of nsp8 to ZNF324 in vitro will be proved by pull-down experiment. The nsp8 gene was cloned into the eukaryotic expression vector pcDNA3.1 to construct pNSP8, which was transfected into A549 cell to establish the stable transfection cell strain. Laser scanning confocal microscropy detected distributed cytoplasmic fluorescence foci of nsp8 with IgG-anti-nsp8(FITC),and also demonstrate colocalization of nsp8 and ZNF324 in cytoplasmic complex. The reaction of nsp8 to ZNF324 in vivo will be proved by cosedimentation assays. The gene of full length 339bp of replicase nsp9 was cloned from SARS-CoV BJ01 strain by RT-PCR and inserted into the vector pGEX-6p-1 to get the construct pNSP9E. The recombinant strain BL21(DE3)pNSP9E expressed souble 12kDa protein of nsp9 induced with IPTG. Two receptor proteins of nsp9 were captured by bait protein GST-nsp9 from the A549 cell lysate in the pull-down experiment. One potential receptor protein of nsp9 analyzed by PMF was T cell associated kelch repeat protein(TA-KRP). The gene of TA-KRP was cloned from A549 cell, and the protein of TA-KRP was harvested in prokaryotic expression system. The reaction of nsp9 to TA-KRP in vitro will be proved by pull-down experiment. The nsp9 gene was cloned into the eukaryotic expression vector pcDNA3.1 to construct pNSP9, which was transfected into A549 cell to establish the stable transfection cell strain. Laser scanning confocal microscropy detected distributed cytoplasmic fluorescence foci of nsp9 with IgG-anti-nsp9(FITC),and also demonstrate colocalization of nsp9 and TA-KRP in cytoplasmic complex. N-terminal sequenceing of peptide showed the other potential receptor protein of nsp9 was Beckwith-Wiedemann syndrome chromosome region 2 associated zinc-finger protein 2(BAZ2,ZNF215). The gene of BAZ2 was cloned from A549 cell, and the protein of BAZ2 was harvested in prokaryotic expression system. The reaction of nsp9 to BAZ2 in vitro was proved by pull-down experiment. And also nsp9 and BAZ2 colocalized with each other in cytoplasmic complex. The reaction of nsp9 to TA-KRP and to BAZ2 in vivo will be proved by cosedimentation assays.The information of receptor proteins of nsp8 and nsp9 showed that these receptor proteins are zinc finger protein. Zinc finger protein play important roles in regulation of gene transcription and translation, cytogenesis and embryogeny with its special domain to bind the target structure. Studies on the receptor protein of nsp8 and nsp9 will help elucidate their functions in viral genomic RAN replication, subgenomic RNA synthesis and viral particle assembly. The structure of nsp8and nsp9 and their receptor protein such as BAZ2 will provide important tools to design and develop inhibitor and diagnosis methods to inhibit viral replication and prevalence.