Study On Function And Molecular Mechanism Of NS5B And Integrin β3in CSFV Proliferation

Classical Swine Fever (CSF) is a highly infectious and fatal pig disease, resulting inhuge economic loss to the swine industry. However, currently the main method to controlCSFV is to use vaccine and to rule out the sick livestock. Although those two mehods playedcritical role in preventing CSF, the frequently occurred chronic CSF and the CSF derivedfrom the CSFV mutation strains result in new chanlenge and problems to swine industry. So,study on the mechanism of the CSFV infection and its interactions with the host cell wouldoffer theoretical basis to define the mechanism of CSFV infection and finding effective waysto control it. This dissertation focused on the NS5B (RNA dependent RNA polymerase) ofCSFV and integrinβ3, the membrane-bound receptor to define their functions and themechnisms in CSFV infection and proliferation. Around this theme, we obtained some resultsas follows：（1）CSFV NS5B was successfully expressed in human293T cells and showed the RNApolymerase activity. NS5B primed the RNA synthesis without other CSFV proteins or theCSFV genome. The RNA product derived from NS5B could be recognized by RIG-I andemerged the inner immune responses. NS5B protein expressed in vitro could use theheterogenous synthetical RNA template to start the de novo initiated RNA synthesis.According to the predicted the2nd and3rd structure of CSFV NS5B, we designed8mutations of NS5B targeting the active sites. In vitro RNA synthesis assay demonstrate thatthe activities of NS5B mutations vary a lot from each other. The polymerase activity ofmutation K282A increased while NS5B mutations of K283R, R285K and I287Vdecreasedobviously compared to the wide type of CSFV NS5B protein. NS5B mutations of K283R,K283A, R285A and I287A basically lost all the polymerase activities. Furthermore, wedetected100chemically synthesized compounds to test the inhibitions to CSFV NS5B proteinand found2which are promising to be the inhibitors to CSFV NS5B, which offered thetheoretical reference to developing the antiviral drugs or the related antibody.（2）CSFV Core protein could enhance the polymerase activity of CSFV NS5B. The5BR assay uses transiently-expressed CSFV NS5B to produce RNAs that activate the RIG-Imediated signaling pathway to result in reporter protein production. Upon co-expression of the CSFV Core protein, we observed enhancement of the CSFV RdRp activity. The CSFVCore and NS5B proteins could co-immunoprecipitate with each other and co-localize incytoplasm. CSFV Core could enhance the CSFV NS5B activity in a virus species-specificmanner. Studies of truncated CSFV Core demonstrated that the first21residues of coreprotein are dispensable for the enhancement in CSFV NS5B activity. And the first53residuesare required for the expression of Core protein or responsible for the protein stability. PurifiedCore protein could enhance RNA synthesis by the purified NS5B in vitro, with the increasebeing in the synthesis of the de novo initiated RNA. These results demonstrate that the CSFVCore protein can regulate the mechanism of RNA synthesis by the CSFV RdRp. Furthermore,based on the knowledge that the core protein could reassembled in vitro, we got the CSFVlike VLPs using BMV RNA.（3）The crystals of CSFV NS5B was first obtained. Through aligning the amino acids ofNS5Bs from Flaviviridae, we define that there is a high similarity of amino acids betweenCSFV NS5B and BVDV NS5B. Compare the predicted3rd structure of CSFV NS5B with thereported NS5B protein structure from BCDV, HCV and WNV, we found the protein structuresare similar. According to the predicted2nd protein structure as well as the mass spectrometryanalysis, we established multiple constucts containing the different NS5B truncations, andobtained the high purity NS5B proteins from different constructs. After screening200differentcrystal conditions, we finally found the right crystal condition for CSFV NS5B protein and gotthe good shaped NS5B crystals. The crystal structure of CSFV NS5B remains unknown, wehope this result could fill up the blank.（4）Integrin β3is required in infection and proliferation of classical swine fever virus.Integrin β3is positively correlated to CSFV proliferation.Integrinβ3is a membrane-boundsignal mediator, expressed on a variety of cell surfaces and are known as receptors orco-receptors for many viruses. Here, we document that integrin β3is revealed to be requiredand has a receptor-like function in CSFV infection and proliferation and as such the integrinβ3could be a viable target for antiviral therapies to control CSFV infection.（5）Some microRNAs from Let7family which regulate the CSFV proliferation in cell,also regulate the integrinβ3. Conversely, the change of integrinβ3expression in cell couldaffect the expression of some microRNAs from Let7family.