The Molecular Mechanism Of Porcine Bocavirus NP1 Protein Regulation The Type Ⅰ Interferon Immune Response

The innate immune response is the first-line defense against viral infection in host. As the major effectors that cytokines against virus infection, type I IFNs, containing IFN-α and IFN-β, are widely expressed in host to possess strong antiviral and immunomodulatory properties. Many viruses have evolved various countermeasures to inhibit the IFN production to subvert host immune response. It is a hot spot to research how viruses regulate host IFN antiviral system. Porcine bocavirus(PBo V), a newly emergent Parvovirus, has been reported worldwide in postweaning multisystemic wasting syndrome(PMWS), diarrheic, respiratory or asymptomatic swine. Compared to healthy pigs, PBo V has higher co-infection prevalence in PMWS piglets with porcine circovirus type 2(PCV2), porcine reproductive and respiratory syndrome(PRRSV), while similar situation has been observed for PBo V and porcine epidemic diarrhea virus(PEDV) in diarrheic pigs. Hence PBo V was suggested to play an important role in triggering immunologic suppression to facilitate infection of co-infection pathogens. PBo V contains three potential open reading frames(ORFs) encoding two nonstructural proteins(NS1 and NP1) and two structural proteins(VP1 and VP2), which the functions were rarely reported. And whether these proteins play role in host immune system modulation is still unknown. The present study tries to discover PBo V encoding protein that has immune regulation function. With this effort, we identify NP1 is a novel IFN-I antagonist and could also induce inflammation. We further demonstrate the detail mechanisms of PBo V antagonize host immune defense. This research will provide an explanation of innate immune suppression for the pathogenesis of PBo V and help us to understand the role of PBo V in co-infection. The specific studies include: 1. PBo V NP1 negative regulates type I IFN productionViruses have evolved various survival strategies to combat the antiviral effects of innate immune. Members in the Parvoviridae family infect a wide range of host, however, their evasions of the innate immune system have rarely been reported. In this study, we found that PBo V non-structural protein NP1 inhibited both IFN-β promoter activity and type I IFN production. We screened several components of the RLR signaling pathway and found that NP1 impaired IRF3-mediated IFN-β production and target at or down of IRF3. Detailed analysis demonstrated NP1 had no effect on the expression, phosphorylation and nuclear translocation of IRF3, but blocked the association of IRF3 with IFN-β promoter in turn suppress type I IFN production. 2. PBo V NP1 negative regulates type I IFN signaling pathwayTo subvert host antiviral immune responses, many viruses have evolved countermeasures to inhibit IFN-β signaling pathway. In this study, we screened the structural and non-structural proteins encoded by PBo V and found that the non-structural protein NP1 significantly suppressed IFN-stimulated response element(ISRE) activity and subsequent IFN stimulated gene(ISG) expression. However, NP1 affected neither the activation and translocation of STAT1/STAT2, nor the formation of the heterotrimeric transcription factor complex ISGF3(STAT1/STAT2/ IRF9). Detailed analysis demonstrated that PBo V NP1 interacted with the ISGF3 and blocked its combination with ISRE. 3. Molecular mechanism of PBo V NP1 blocking IRF3 and IRF9 DNA binding activityAlthough some pleiotropic virus proteins both target IFN production and signaling pathway, it was rarely seen that virus proteins utilized broad-spectrum mechanisms to regulate IFN system by targeting IRFs. In this study, we found PBo V NP1 both interacted with IRF3 and IRF9. Further research demonstrated that NP1 combined with DNA binding domain(DBD) of IRF3 and IRF9. Detailed analysis revealed that NP1 directly interacted with the DNA binding sites on DBD of IRF3 and IRF9. In summary, these results indicate that PBo V NP1 interferes with both the production and signaling of type I IFN by a similar mechanism resulting in a broad-spectrum ability to inhibit innate immune responses. 4. PBo V NP1 induces NF-κB activationFor optimal survival, some IFN-antagonistic viruses induce the activation of NF-κB to facilitate the inflammatory response and the production of immunosuppressive cytokines. In this study, we observed that PBo V NP1 induced the activation of NF-κB and promoted several pro-inflammatory cytokines expression. Further research demonstrated NP1 induced the degradation of IκBα and enhanced phosphorylation and translocation of p65. 5. Differential contributions of PBo V NP1 N- and C-terminal regions to its nuclear localization and immune regulationPBo V NP1 participated in IFN antiviral immune regulation, however, its functional relative domain still remained unknown. In this study, we showed that the N-terminal region of PBo V NP1 contains two classical nuclear localization signals(c NLSs) and a non-classical NLS. The N-terminal region also inhibited the promoter activity of IFN-β and IFN-stimulated response element activity the same as full-length NP1 protein, but the PBo V NP1 C-terminal region did not. We also demonstrated that the C-terminal region(aa 168-218) is responsible for the induction of NF-κB, although the c NLS region of NP1 enhanced this activation. These data suggested that PBo V NP1 contains two functionally independent domains in its N- and C-terminal regions, and the N-terminal region of PBo V NP1 is critical for its nuclear localization and IFN-related promoter inhibition, and the C-terminal region is critical for its induction of NF-κB.6. Screen the interaction host protein of PBo V NP1 by phage display libraryNP1 is a protein unique to the genus Bocavirus and its function is not fully understood. In this study, we purified PBo V NP1 protein by prokaryotic expression system to screen and identify host proteins that interact with the NP1 protein by means of T7-phage display system. The selected positive clones were identified by DNA sequence and analyzed with BLAST program in Gen Bank. After BLAST in all positive clones, two protein(ZNF APA-1 and PTDSS1) were found to interact with the NP1 protein.