The Crucial Sites In Prrsv Nsp10 And Fragment-Based Inhibitor Screen

Porcine reproductive and respiratory syndrome virus(PRRSV) is a single-stranded positive sense RNA virus, which is classified in the order Nidovirales, family Arteriviridae, genus Arterivirus. Since it emerged in 1990, PRRSV has rapidly been one of the most important diseases of swine. It is characterized by quality drop of boar semen, high mortality of neonatal piglets, other age’s swine respiratory disease and the sow abortion, stillbirth, premature or weak young. The virus and the syndrome continue to evolve with clinical variations of the disease, such as swine mortality and abortion syndrome or atypical PRRS, thus inducing large number of swine death and abortion. Vaccination is a predominant strategy for the prevention and control of PRRS. However, neither of modified live-attenuated vaccines and killed virus vaccines, which are two types of commercially available PRRS vaccines, is able to protect completely against PRRSV infection. Besides, PRRSV infection impairs and delays both neutralizing antibody and T cell responses, which is considered to be the reason for the poor effect of the vaccine immunization. Therefore, it is essential to explore the effective method for the control of PRRSV.The Nsp10 helicase of arterivirus participates in multiple viral processes, including subgenomic RNA synthesis, the early steps of mRNA transcription, genome replication, and virion biogenesis. A comparison between the genomic sequences of attenuated and highly pathogenic PRRSV suggests that mutations in Nsp10 may be involved in the virulence of virus. Since the functional properties of Nsp10 were reported in 2012, there was little report about it in these ten years. Therefore, it is meaningful to find the key active site of Nsp10 and inhibitors towards it for uncovering the unwinding mechanism of Nsp10 and developing the anti-PRRSV drugs, respectively.In this study, primarily the soluble Nsp10 with high activity was obtained through the Escherichia coli expression system and Nickel column affinity chromatography purification. The optimum reaction systems for detecting the ATPase and unwinding activity of NSP10 were established using a Kinase-Glo Plus Luminescent Kinase Assay kit and a fluorescence resonance energy transfer(FRET) method, respectively. Also, a series of mutants were constructed. The key active site in Nsp10 conserved motif Ⅰand Ⅱ was studied through comparing the ATPase and unwinding activity of wild type protein and mutants. And subsequently a high throughout screening system was established to find inhibitors towards unwinding actiivity through the screening of a library of SPECS Company, which has 4536 kinds of small molecular fragments. Then the anti-viral activity of these inhibitors was tested in cells. In addition, we tried to unlock the structure of Nsp10 protein. The main results are as follows: 1. The prokaryotic expression, affinity chromatography purification and activity testing of Nsp10 proteinThe soluable Nsp10 protein can be obtained through transforming the constructed expression plasmid pET30a-Nsp10 into Rosetta E.coli and inducing for 16 hours at 16 。C. Through the Nickel column affinity chromatography purification, the relatively pure Nsp10 protein was gained and identified by SDS-PAGE and Western blot. Then, the ATPase activity was tested using the Kinase-Glo Plus Luminescent Kinase Assay Kit and the reaction system was optimized to that 0.6μM Nsp10 protein, 2mM MgCl2, 0.2mM ATP and pH7.9 reacted for 30 min at 37 。C. And it is found that the soluable Nsp10 protein was chelated with some metal ions. Meanwhile, the optimum reaction system for detecting unwinding activity of Nsp10 was established using a fluorescence resonance energy transfer(FRET) method. It is that 2mM MgCl2, 80μM Nsp10 protein, 400 nM double-strand fluorogenic substrates, 4μM capture strand, 0.2mM ATP and pH7.9 reacted for 90 min at 37 。C. 2. Study on the key active site in Nsp10 conserved motif Ⅰand ⅡThrough site-directed mutation by over-lap PCR, a series of mutants were obtained. And the ATPase and unwinding activity were detected. The single mutation at residue K155 was associated with an 88.1% decrease in ATPase activity, whilst the E226 A mutation reduced ATPase activity by 8.0%. All other mutations failed to affect Nsp10 ATPase activity. K155 was demonstrated to be the key active site of ATPase activity. The unwinding ability of K155 A, D225 A, E226 A, A228 D and A228 V was reduced relative to the WT Nsp10. The D225 A and A227 S mutations reduced helicase activity by 77.0% and 90.3 %, respectively. The ATPase and unwinding activity of D225 and E226 were both reduced significantly. The helicase activity disappeared when Ala227 was substituted by Ser, while changing the same residue to Val clearly increased helicase activity. D225 and E226 were proved to have a close association with both ATPase and unwinding activity. A227 were demonstrated to be the key active site of unwinding activity. 3. High throughout screening for unwinding inhibitors and the anti-viral activity testing of these inhibitors in cellsA high throughout screening system was established to find inhibitors towards unwinding activity. Finally, 18 small molecular fragments with an inhibition ratio to the Nsp10 unwinding activity exceeded 50% were found through the screening of a library of SPECS company, which has 4536 kinds of small molecular fragments. Then the anti-viral activity of these inhibitors was tested in cells. However these small molecular fragments were found to have no inhibitory effects towards the replication and infection of PRRSV on Marc-145 cells. 4. Research on Nsp10 protein structureThe soluable Nsp10 protein obtained through Nickel column affinity chromatography purification was disposed with gel filtration chromatography to reach the purity of over 95%. The relatively absolute Nsp10 protein was concentrated using the Millipore ultrafiltration device. Once the concentration reached 5mg/ml or higher, then the protein was applied to screen the suitable condition for protein crystallization. At last, we found that the cylinder-shaped protein crystal with high quality can grow in buffer with pH 7.5 that contain 2.2M NaCl and 1.0M HEPES. But the best resolution ratio was just 3.6?. We still can not uncover the structure of Nsp10 protein.