Functional Analysis Of Papain-like Proteases (PLP) Of Porcine Epidemic Diarrhea Virus (PEDV)

Coronaviruses are a large class of viruses infecting people and animals. The host has evolved an important defensive mechanism against virus invasion, which is regarded as antiviral innate immune response. Due to the diversity in varieties, pathogenicity, and the immunity characteristics between human and animal coronaviruses, its regulation mechanism of host antiviral innate immune response to coronaviruses is still unclear. Previously, we found that the papain like protease (PLPs) of the human coronavirus NL63and SARS both have deubiquitinase (DUB) activity, and is one of host interferon antagonists that encoded by the coronaviruses. In order to confirm whether all coronavirus PLPs have the DUB activity and interferon antagonist activity, animal coronavirus of Porcine epidemic diarrhea virus (PEDV) was selected. This study showed that the host cells infected by PEDV did not induce the evident production of type ⅠIFN, indicating negative regulation of IFN expression by PEDV infection. PEDV also suppresses the activation pathway of nuclear transcription factor IRF3. Thus, PEDV is speculated to negatively regulate host antiviral interferon expressions pathway via inhibiting IRF3pathway. PEDV non-structural protein, nsp3, harbours two core domain of papain-like proteases (PLPs), PLP1and PLP2, and PEDV PLP1does not have the DUB activity and IFN antagonistic activity; whereas PLP2has strong DUB activity. PLP2also exhibits DUB activity for both K48-and K63-linked polyubiquitin modification, which indicates that PLP2DUB activity does not have substrate specificity to K48-and K63-linked polyubiquitin. However, the DUB activity of PLP2was significantly suppressed when PLP2protease catalytic active sites of C1729, H1888and D1901were mutated, suggesting that the PLP2protease catalytic active sites are simultaneously the key sites that is required for the DUB activity. Additionally, PLP2obviously suppresses IFN expression stimulated by Sendai virus, indicating that PLP2is a PEDV encoding interferon antagonist, and its IFN antagonist function is closely related to its protease activity. PLP2inhibits IFN production, and activation of NF-κB and IRF3signaling pathways inducted by RIG-Ⅰ and STING, the key regulators of IFN signaling pathway. Mechanically, PLP2interacts with RIG-Ⅰ (the key pattern recognition receptor) and STING (the important regulatory protein), and PLP2negatively regulates the host antiviral innate immune response through deubiquitinating the ubiquitinated modification of RIG-Ⅰ and STING. Overall, these studies reveal a new regulation mechanisms for PEDV for negative modulation of the host antiviral innate immune responses and thereof the pathogensis, which will provide some clues for the development of new immune control measures.