Study On The DNA Binding Activity Of Prrsv NSP10 And High Throughput Screening For Inhibitors

Porcine reproductive and respiratory syndrome virus(PRRSV)belongs to the genus Arterivirus of the family Arteriviridae. It has a single-strand positive-sense RNA genome whose size is 15 kb, and is composed of nine open reading frames. As a serious infectious virus, PRRSV can cause reproductive disorders in pregnant sows and respiratory disease in growing pigs. PRRSV has spread all over the world and brought serious loss to the pig industry.Up to now, there is no specific treatment method for the PRRS and vaccine doesn’t work well. So many scientists are studying the replication mechanism and the functions of proteins in order to figure out a better control strategy and specific drugs.PRRSV NSP10 is a protein with multiple functions, which is comprised of an N-terminal complex zinc binding domain(ZBD) and a C-terminal helicase SF1, owning binding activity and unwinding activity. ZBD consists of one α-helices with two antiparallel β-sheets. α-helices inserted in DNA major groove, so binding activity was obtained. The main work is as follows:1.Detecting the direction of helicase activity and the minimum length of 5’-ss DNA overhangUnwinding activities for nsp10 s generated in this study were tested by non-denaturing electrophoresis. The direction of helicase activity and the minimum length of 5’-ss DNA overhang was evaluated. The data suggest that the DNA duplex-unwinding activity strictly depends on the presence of 5’ single-stranded tails and it requires more than 5-base-long strand to efficiently unwind its substrate.2.The detection of non-specific binding ds DNA and ss DNA activity of nsp10Zinc-binding domain is a part of the nsp10 structure’s N-terminal domain. To investigate the nucleic-acid-binding activity of nsp10, we use the non-denaturing gel electrophoresis. The results suggested that the PRRSV nsp10 possessed both ss DNA- and dsDNA-binding activities and there is no need of ATP.3.Expression and purification of the ZBD mutant and the detection of its binding activity and unwinding activityCompared the zinc-binding domain of PRRSV with that of other members of the Arteriviridae family and using analysis result of the structure of EAV nsp10, 6 mutations were selected(C10、C25、H28、H32、C41、H43). To further explore the role of ZBD, all mutations were over-expressed and purified from E. coli. We tested the effect of six mutations soluble His-tagged proteins and found out that some mutations(C10、C25、H28 、 H32) could not be obtained. So we try to use other expression vectors p ET-28a-SUMO、Pgex6p-1, in order to get soluble proteins.To find the crucial amino acids associated with ZBD formation, Unwinding and binding activities for all mutant nsp10 s generated in this study were tested. These results suggested that C25 and H32 exhibited an activity defect.4.High throughput screening of compoundsHigh-throughput screening assay of the antiviral compounds was developed on the cellular level. Finally, we chose 5,000 cells per well as the optimized cell density, 0.05 MOI as the optimized infective dose, and 120 h post-inoculation as the endpoint. With the condition of S/B>5、Z′≥0.5 and CV<10%, The HTS assay was used to screen PRRSV inhibitors from the Library of Pharmacologically Active Compounds 1280. The inhibition rate of compounds identified to have>60% was obtained, in the secondary screening analysis. Considering the clinical application, two compounds were selected.5.Detecting the effect of antiviral compoundsAntiviral compounds A1895、D8555 are cheap and have a high rate of inhibition. The two compounds showed a good result in inhibition of PRRAV by western blot and indirect immunoinfluscent assay. A1895 is a DNA topoisomerase II inhibitor and D8555 is an anxiolytic drug acting on the peripheral benzodiazepine receptor.