Maturation Mechanism of Severe Acute Respiratory Syndrome (SARS) Coronavirus 3C-like Proteinase

The 3C-like proteinase (3CL(pro)) of the severe acute respiratory syndrome (SARS) coronavirus plays a vital role in virus maturation and is proposed to be a key target for drug design against SARS. Various in vitro studies revealed that only the dimer of the matured 3CL(pro) is active. However, as the internally encoded 3CL(pro) gets matured from the replicase polyprotein by autolytic cleavage at both the N-terminal and the C-terminal flanking sites, it is unclear whether the polyprotein also needs to dimerize first for its auto-cleavage reaction. We constructed a large protein containing the cyan fluorescent protein (C), the N-terminal flanking substrate peptide of SARS 3CL(pro) (XX), SARS 3CL(pro) (3CLP), and the yellow fluorescent protein (Y) to study the autoprocessing of 3CL(pro) using fluorescence resonance energy transfer. In contrast to the matured 3CL(pro), the polyprotein, as well as the one-step digested product, 3CLP-Y-His, were shown to be monomeric in gel filtration and analytic ultracentrifuge analysis. However, dimers can still be induced and detected when incubating these large proteins with a substrate analog compound in both chemical cross-linking experiments and analytic ultracentrifuge analysis. We also measured enzyme activity under different enzyme concentrations and found a clear tendency of substrate-induced dimer formation. Based on these discoveries, we conclude that substrate-induced dimerization is essential for the activity of SARS-3CL(pro) in the polyprotein, and a modified model for the 3CL(pro) maturation process was proposed. As many viral proteases undergo a similar maturation process, this model might be generally applicable.