Identification of a Novel Regulatory Region within the Interdomain Loop of Mouse Hepatitis Virus (MHV) Protease nsp5

Indianapolis, IN

Human coronaviruses are responsible for upper and lower respiratory infections including the common cold and more severe diseases such as severe acute respiratory syndrome (SARS). Mouse hepatitis virus (MHV) is a coronavirus that remains an important and safe model to study coronavirus biology. During MHV replication, the positive-sense single-strand RNA (+ssRNA) genome is translated by host cell ribosomes into long polyproteins encoding the nonstructural protein (nsp) machinery required for replication. Production of mature replication proteins requires proteolytic processing of the polyprotein by 3 viral encoded proteases. Coronavirus protease nsp5 mediates 11 proteolytic cleavages of the polyprotein, is essential for virus replication, and remains a primary target for design of coronavirus inhibitors. Connecting two functional domains of nsp5 is a long 15 amino acid interdomain loop (IDL) structure of unknown function. In this study, we employed site-directed mutagenesis and functional replication assays to evaluate individual residue positions throughout the length of this loop. Four amino acid positions within a novel horseshoe-like motif either failed to tolerate mutagenesis or resulted in temperature-sensitivity of the mutant. These studies define, for the first time, a key functional role of the coronavirus nsp5 IDL and highlight a new putative target for active inhibitor design efforts.