The Clone Expression Of Human Parvovirus B19 Genome NS1,VP1,VP2 And Its Function On Type â…  Interferon Signaling

Background Human parvovirus B19 is non-enveloped, single-strand DNA, only 23nm in diameter and it was firstly discovered in 1975. Transmission of infection occurs via the respiratory route, through blood-derived products administered parenterally, and vertically from mother to fetus and is widespread in the world year round. Transmission mainly occurs in spring. Once infected, the host firstly produces IgM to defense the invasion, this kind of immunoglobulin could last 3 months. About 2 weeks after the infection, the host produces IgG and that can persist for life. The genome of B19 virus contains three main open reading frames, which are NS1, VP1 and VP2. Our previous study mainly focused on the prevalence of B19 virus in Chinese blood donors, but the study on how the B19 virus influence on the host innate immune especially the type I interferon signaling is still lacking.Objective To make sure that NS1, VP1 and VP2 can express in Hela cell line. To test the influence of this expression on the endogenous IFN production of the host and the type I IFN signaling.Methods We used gene cloning method to clone NS1, VP1 and VP2 to the pcDNA3.1-3tag vector and we constructed the high expression pcDNA3.1-3tag-NSl, pcDNA3.1-3tag-VP1 and pcDNA3.1-3tag-VP2 plasmids. We had the Hela cells transfected with the plasmids and tested whether NS1, VP1 and VP2 could be highly expressed on mRNA level and protein level through the methods of RT-PCR and Western Blot. After making sure the high expression on protein level, we examined the influence on the production of endogenous IFN and ISGs expression by RT-PCR; we examined the influence on the phosphorylation of STAT1 through Western Blot; we examined the activation of ISRE by dual luciferase reporter gene assay.Results We succeed to construct pcDNA3.1-3tag-NS1, pcDNA3.1-3tag-VP1 and pcDNA3.1-3tag-VP2 plasmids. All the three plasmids could be highly expressed on mRNA level but only NS1 could be highly expressed on protein level. The production of endogenous IFN can be increased after the NS1 transfection, while NS1 can suppress the type I IFN signaling on phosphorylation of STAT1, activation of ISRE and ISGs expression levels after the IFN stimulation.Conclusion NS 1 can activate host innate immune and stimulate host cells to produce typeâ…  IFN. NS1 can suppress the type â…  IFN signaling on phosphorylation of STAT1, activation of ISRE and ISGs expression levels.