Human coronavirus OC43 3CL protease and the potential of ML188 as a broad-spectrum lead compound: homology modelling and molecular dynamic studies.

BACKGROUND: The coronavirus 3 chymotrypsin-like protease (3CL(pro)) is a validated target in the design of potential anticoronavirus inhibitors. The high degree of homology within the protease's active site and substrate conservation supports the identification of broad spectrum lead compounds. A previous study identified the compound ML188, also termed 16R, as an inhibitor of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) 3CL(pro). This study will detail the generation of a homology model of the 3CL(pro) of the human coronavirus OC43 and determine the potential of 16R to form a broad-spectrum lead compound. MODELLER was used to generate a suitable three-dimensional model of the OC43 3CL(pro) and the Prime module of SchrÓ§dinger predicted the binding conformation and free energy of binding of 16R within the 3CL(pro) active site. Molecular dynamics further confirmed ligand stability and hydrogen bonding networks. RESULTS: A high quality homology model of the OC43 3CL(pro) was successfully generated in an active conformation. Further studies reproduced the binding pose of 16R within the active site of the generated model, where its free energy of binding was shown to equal that of the 3CL(pro) of SARS-CoV, a receptor it is experimentally proven to inhibit. The stability of the ligand was subsequently confirmed by molecular dynamics. CONCLUSION: The lead compound 16R may represent a broad-spectrum inhibitor of the 3CL(pro) of OC43 and potentially other coronaviruses. This study provides an atomistic structure of the 3CL(pro) of OC43 and supports further experimental validation of the inhibitory effects of 16R. These findings further confirm that the 3CL(pro) of coronaviruses can be inhibited by broad spectrum lead compounds.

The acetyl-esterase activity of the hemagglutinin-esterase protein of human coronavirus OC43 strongly enhances the production of infectious virus.

Most betacoronaviruses possess an hemagglutinin-esterase (HE) protein, which appears to play a role in binding to or release from the target cell. Since this HE protein possesses an acetyl-esterase activity that removes acetyl groups from O-acetylated sialic acid, a role as a receptor-destroying enzyme has been postulated. However, the precise function of HE and of its enzymatic activity remains poorly understood. Making use of neutralizing antibody and of molecular clones of recombinant human coronavirus OC43 (HCoV-OC43), our results suggest that the HE protein of this HCoV could be associated with infection of target cells and, most notably, is important in the production of infectious viral particles. Indeed, after transfecting BHK-21 cells with various cDNA infectious clones of HCoV-OC43, either lacking the HE protein or bearing an HE protein with a nonfunctional acetyl-esterase enzymatic activity, we were reproducibly unable to detect recombinant infectious viruses compared to the reference infectious HCoV-OC43 clone pBAC-OC43(FL). Complementation experiments, using BHK-21 cells expressing wild-type HE, either transiently or in a stable ectopic expression, demonstrate that this protein plays a very significant role in the production of infectious recombinant coronaviral particles that can subsequently more efficiently infect susceptible epithelial and neuronal cells. Even though the S protein is the main viral factor influencing coronavirus infection of susceptible cells, our results taken together indicate that a functionally active HE protein enhances the infectious properties of HCoV-OC43 and contributes to efficient virus dissemination in cell culture.