Identification and characterization of human rhinovirus-14 3C protease deamidation isoform

A purified recombinant human rhinovirus-14 3C protease preparation containe d only similar to 50% active enzyme as titrated using specifically designed irreversible 3C protease inhibitors. Analysis of the purified 3C protein b y isoelectric focusing showed differently charged 3C isoforms that had isoe lectric points (pI) of 8.3 (55%) and 9.0 (45%), with the latter one being c onsistent with the predicted pi of the human rhinovirus-14 3C protein. Furt her analysis indicated that the pi 8.3 protein was the deamidated form of 3 C, and it displayed similar to 10-fold reduced cleavage activity relative t o the original 3C protease sample. Peptide mapping followed by sequence ana lysis revealed that a single asparagine, Asn-164, was deamidated to asparti c acid in the pi 8.3 isoform, Converting Asn-164 to Asp by site-directed mu tagenesis resulted in a mutated 3C protease with extremely low activity, as seen with the pi 8.3 isoform, indicating a role of Asn-164 in substrate re cognition and binding, In addition, the deamidated 3C protease was found to be present in vivo, and its abundance was related to the viral replication cycle, Moreover, mutant virus carrying Asp-164 showed reduced viability in infected cells. Taken together, our data suggest that 3C protein deamidati on plays a role in the regulation of its enzymatic activity.