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X-Ray Structure and Inhibition of 3C-like Protease from Porcine Epidemic Diarrhea Virus.

ABSTRACT: Porcine epidemic diarrhea virus (PEDV) is a coronavirus that infects pigs and can have mortality rates approaching 100% in piglets, causing serious economic impact. The 3C-like protease (3CL(pro)) is essential for the coronaviral life cycle and is an appealing target for the development of therapeutics. We report the expression, purification, crystallization and 2.10 Å X-ray structure of 3CL(pro) from PEDV. Analysis of the PEDV 3CL(pro) structure and comparison to other coronaviral 3CL(pro)'s from the same alpha-coronavirus phylogeny shows that the overall structures and active site architectures across 3CL(pro)'s are conserved, with the exception of a loop that comprises the protease S2 pocket. We found a known inhibitor of severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL(pro), (R)-16, to have inhibitor activity against PEDV 3CL(pro), despite that SARS-3CL(pro) and PEDV 3CL(pro) share only 45.4% sequence identity. Structural comparison reveals that the majority of residues involved in (R)-16 binding to SARS-3CL(pro) are conserved in PEDV-3CL(pro); however, the sequence variation and positional difference in the loop forming the S2 pocket may account for large observed difference in IC50 values. This work advances our understanding of the subtle, but important, differences in coronaviral 3CL(pro) architecture and contributes to the broader structural knowledge of coronaviral 3CL(pro)'s.

SUBMITTER: St John SE

PROVIDER: S-EPMC4865815 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledPorcine epidemic diarrhea virus (PEDV) is an enteropathogenic coronavirus causing lethal watery diarrhea in piglets. Since 2010, a PEDV variant has spread rapidly in China, and it emerged in the United States in 2013, posing significant economic and public health concerns. The ability to circumvent the interferon (IFN) antiviral response, as suggested for PEDV, promotes viral survival and regulates pathogenesis of PEDV infections, but the underlying mechanisms remain obscure. Here, we show that PEDV-encoded 3C-like protease, nsp5, is an IFN antagonist that proteolytically cleaves the nuclear transcription factor kappa B (NF-κB) essential modulator (NEMO), an essential adaptor bridging interferon-regulatory factor and NF-κB activation. NEMO is cleaved at glutamine 231 (Q231) by PEDV, and this cleavage impaired the ability of NEMO to activate downstream IFN production and to act as a signaling adaptor of the RIG-I/MDA5 pathway. Mutations specifically disrupting the cysteine protease activity of PEDV nsp5 abrogated NEMO cleavage and the inhibition of IFN induction. Structural analysis suggests that several key residues outside the catalytic sites of PEDV nsp5 probably impact NEMO cleavage by modulating potential interactions of nsp5 with their substrates. These data show that PEDV nsp5 disrupts type I IFN signaling by cleaving NEMO. Previously, we and others demonstrated that NEMO is also cleaved by 3C or 3C-like proteinases of picornavirus and artertivirus. Thus, NEMO probably represents a prime target for 3C or 3C-like proteinases of different viruses.ImportanceThe continued emergence and reemergence of porcine epidemic diarrhea virus (PEDV) underscore the importance of studying how this virus manipulates the immune responses of its hosts. During coevolution with its hosts, PEDV has acquired mechanisms to subvert host innate immune responses for its survival advantage. At least two proteins encoded by PEDV have been identified as interferon (IFN) antagonists, papain-like protease (PLP) and N protein. Here, we report that the PEDV nsp5 gene, which encodes the 3C-like protease of PEDV, is another IFN antagonist. Mechanistically, the cysteine protease activity of PEDV nsp5 mediates proteolysis of NEMO, the key adaptor for IFN synthesis, and NEMO is cleaved at glutamine 231 (Q231). The new molecular details and determinants impacting NEMO scission by PEDV nsp5 delineated in this study are fundamental to our understanding of critical virus-host interactions that determine PEDV pathogenesis.

Project description:Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality rates in newborn piglets, leading to massive losses to the swine industry worldwide during recent epidemics. Intense research efforts are now focusing on defining viral characteristics that confer a growth advantage, pathogenicity, or cell adaptability in order to better understand the PEDV life cycle and identify suitable targets for antiviral or vaccine development. Here, we report a unique phenomenon of PEDV nucleocapsid (N) cleavage by the PEDV-encoded 3C-like protease (3Cpro) during infection. The identification of the 3Cpro cleavage site at the C terminus of N supported previous observations that PEDV 3Cpro showed a substrate requirement slightly different from that of severe acute respiratory syndrome coronavirus (SARS-CoV) 3Cpro and revealed a greater flexibility in its substrate recognition site. This cleavage motif is present in the majority of cell culture-adapted PEDV strains but is missing in emerging field isolates. Remarkably, reverse-genetics-derived cell culture-adapted PEDVAVCT12 harboring uncleavable N displayed growth retardation in Vero E6-APN cells compared to the wild-type virus. These observations altogether shed new light on the investigation and characterization of the PEDV nucleocapsid protein and its possible link to cell culture adaptation.ImportanceRecurrent PEDV outbreaks have resulted in enormous economic losses to swine industries worldwide. To gain the upper hand in combating this disease, it is necessary to understand how this virus replicates and evades host immunity. Characterization of viral proteins provides important clues to mechanisms by which viruses survive and spread. Here, we characterized an intriguing phenomenon in which the nucleocapsids of some PEDV strains are proteolytically processed by the virally encoded main protease. Growth retardation in recombinant PEDV carrying uncleavable N suggests a replication advantage provided by the cleavage event, at least in the cell culture system. These findings may direct us to a more complete understanding of PEDV replication and pathogenicity.

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X-Ray Structure and Inhibition of 3C-like Protease from Porcine Epidemic Diarrhea Virus.

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