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 (3CLpro) 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 3CLpro from PEDV. Analysis of the PEDV 3CLpro structure and comparison to other coronaviral 3CLpro’s from the same alpha-coronavirus phylogeny shows that the overall structures and active site architectures across 3CLpro’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) 3CLpro, (R)-16, to have inhibitor activity against PEDV 3CLpro, despite that SARS-3CLpro and PEDV 3CLpro share only 45.4% sequence identity. Structural comparison reveals that the majority of residues involved in (R)-16 binding to SARS-3CLpro are conserved in PEDV-3CLpro; 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 3CLpro architecture and contributes to the broader structural knowledge of coronaviral 3CLpro’s.

Porcine Epidemic Diarrhea Virus and the Host Innate Immune Response

“…Of these reported immune evasion strategies employed by CoVs, the cleavage of innate immune adaptors is a particularly effective manner to disrupt antiviral responses. Nsp5 is essential for the life cycle of PEDV and other CoVs [210,211]. It is a potential target for the development of anti-coronaviral therapeutics.…”

Broad-spectrum inhibition of coronavirus main and papain-like proteases by HCV drugs

“…The S2 subsite is filled with the 6,6-dimethyl-3-azabicyclo[3.1.0]hexane group of boceprevir and is perhaps the greatest determinant of substrate and inhibitor specificity for coronavirus 3CLpro enzymes. This subsite is formed by the non-conserved S2 loop (16), which is a demonstrably important effector of specificity, both within this binding pocket and elsewhere in the enzyme (17). For coronavirus 3CLpros, there is no defined pocket or S3 subsite for a P3 group on a substrate or inhibitor.…”

Porcine Epidemic Diarrhea Virus 3C-Like Protease-Mediated Nucleocapsid Processing: Possible Link to Viral Cell Culture Adaptability

“…3B). As a negative control, 3Cpro-FLAG harboring the C144A protease active-site mutation did not release the GFP-based cleavage product, indicating the specificity of the assay (34,35).…”

“…The general substrate specificity of 3Cpro is determined primarily by the residues in the P1, P2, and P1= sites. The P1 position contains a conserved glutamine (Q), while the P2 position usually contains a hydrophobic residue, preferably leucine (L), and the P1= position contains a small aliphatic residue such as serine, glycine, or alanine (12,29,34,35,40). For the PEDV N cleavage site that we identified, the P1 and P2 positions strictly conform to the CoV 3Cpro substrate specificity.…”