Viperin Protein Restricts Porcine Reproductive And Respiratory Syndrome Virus Replication

Porcine reproductive and respiratory syndrome virus (PRRSV) is classified in the order Nidovirales, family Arteriviridae, and genus Arterivirus. The genome is approximately 15 kb in length, and encodes non-structural protein nsp 1-12 and structural protein GP2a, GP3, GP4, GP5, GP5a, M, N. This disease has many clinical manifestations but the two most prevalent are severe reproductive failure in sows and gilts characterized by late-term abortions, an increased number of stillborn, mummified and weak born pigs and respiratory problems in pigs of all ages associated with a non-specific lymph mononuclear interstitial pneumonitis. PRRSV suppressed the production of IFNs and exogenous IFNs strongly inhibited the replication of PRRSV. Viperein is an interferon stimulated gene and has significantly antiviral effect in the replication of many of kind viruses. In this study, we amplified the swine and monkey Viperin genes from the Marc-145 and PK-15 cells and prepared the swine Viperin protein polyclonal antibodies. This research is the first to show that Viperin inhibited the replication of PRRSV and revealed its antiviral mechanism. And we constructed the recombinant sViperin adenoviral expression vector and demonstrated that the sViperin inhibited the PRRSV replication in Marc-145 cells and PAMSs.Cloning, prokaryotic expression of Viperin gene and preparation of anti- Viperin ployclonal antibodyViperin is an IFN-stimulated gene (ISG), to study the anti-PRRSactivity; we extracted and cloned the monkey and swine Viperin genes from Marc-145 and PK-15 cells. Marc-145 and PK-15 cells were seeded onto 24-well plates for 24 h and treaded with IFN-a. The cells were harvested to extract the total RNA, and the Viperin genes of swine and monkey were amplified by RT-PCR. Then the gene was cloned into the pEASY-blunt simple plasmid for sequencing. The antigenicity and hydrophobic of swine Viperin were analyzed by DNAstar software, and the fragment with good immunogenicity were amplified to clone into the prokaryotic expression vector pET-28a+. E coli BL21 containing recombinant plasmids pET-sVIP were induced by IPTG,and SDS-PAGE analysis demonstrated that recombinant protein were expressed in the form of inclusion bodies in E.Coli. Then inclusion bodies protein were purified, the BALB/c mice with 9 days old were immunized two times with purified recombinant sViperin protein emulsified with equal amounts Freund' s complete adjuvant. Two weeks after final immunization, the serums were collected and the antibody specificity was identified by Western-blot, IFA assay. The result showed that the recombinant protein could react with the sViperin protein expressed by eukaryotic expression vector pVAX-sVIP. Preparation of polyclonal antibody established the foundation for the antiviral activity research of Viperin.Monkey Viperin restricts porcine reproductive and respiratory syndrome virus replicationThe first line of defense against viral infection is the innate immunity response,interferon production play a key role in innate immunity. Various antiviral proteins were induced by IFNs. Viperin is an endoplasmic reticulum related protein that is induced by IFNs, and it inhibited the replication of many kinds of virus, but the more detailed mechanics has yet to be studied. In ours study, we explored the anti-PRRSV activity of mViperin in Marc-145 cells, the result demonstrated that mViperin gene were upregulated by IFN-a and suppressed the replication of PRRSV in dose- and time-dependent manner.The expression of IFN-a were strong inhibited in Marc-145 cells infected with PRRSV BB0907, and mViperin were induced by PRRSV via IFN-independent manner. Over expression of mViperin suppressed the replication of PRRSV in a dose-dependent manner,and knockdown of mViperin induced with IFN-a enhances PRRSV replication in Marc-145 cells. And then, our results demonstrated that over expression of mViperin suppresses PRRSV replication by blocking the early steps of PRRSV entry but not inhibiting assembly and release. The results demonstrated that over-expressed mViperin co-localized with PRRSV GP5 and N protein in cytoplasm by confocal microscopy experiment. The experiments result of Co-immunoprecipitation assays showed that mViperin could interact with PRRSV N protein in Marc-145 cells. It was found that the 13-16 amino acids of mViperin were essential for inhibiting PRRSV replication and the N-termini of mViperin determines the distribution of mViperin protein in the cells.Construction and identification of recombinant adenovirus expressing swine Viperin proteinAbove experiments have proved that monkey Viperin play a key role in inhibiting the PRRSV replication. To further study the antiviral activity of swine Viperin in Marc-145 and PAMS cells, we constructed recombinant adenovirus vector to express the swine Viperin protein. The sViperin gene were amplified by PCR from the pEASY-sVIP and cloned into the adenovirus shuttle vector pTrack-CMV. The recombinant plasmid pTrack-CMV-sViperin was indentified by enzyme digestion and sequencing analysis. The pTrack-CMV-sViperin were linearized by pme-I and homologous recombined with pAdEasy-1 backbone vector in E.coli BJ5183.The recombinant adenoviral genome DNA were extracted and digested by pac-1, then the DNA were transfected into the HEK-293A cells for packaging whole virus particle. The CPE and GFP fluorescence of cells were observed, the cell were harvested and adenoviral expressed recombinant sViperin protein were indentified by RT-PCR and western-blot. The result showed that the sViperin were correctly expressed as expected, and the protein has good reactivity with mouse anti-Viperin serum, thus laying a foundation for further antiviral activity study.Swine Viperin restricts porcine reproductive and respiratory syndrome virus replicationWe constructed the expression plasmid pCI-sVIP and Marc-145 cells were transfected with pCI-sVIP to study the sViperin anti-PRRSV activity. The result showed that over expressed mViperin protein inhibited the replication of PRRSV in dose-dependent manner.And then the experiments demonstrated over expressed sViperin blocked an early step of PRRSV entry but does not affected virus assembly or release. Confocal microscopy experiment demonstrated that over expressed sViperin co-localizes with PRRSV GP5 and N protein in cytoplasm. The experiment results of Co-immunoprecipitation assays experiments showed that mViperin could interact with PRRSV N protein in Marc-145 cells to inhibit the replication of PRRSV protein. To further identified the anti-PRRSV activity of sViperin in PAMSs cells, the PAMSs cells were infected by recombinant adenovirus expressed sViperin protein, and the result showed over expressed sViperin protein inhibited the replication of PRRSV in PAMSs cells, and sVipern expressed with adenovirus also partly blocked the PRRSV entry.