The RNA-dependent RNA polymerase (Rd. Rp) of SARS coronavirus (SARS-CoV) is essential for viral replication and a potential target for anti-SARS drugs. We report here the cloning, expression, and purification of the N-terminal GST-fused SARS-CoV Rd. Rp and its polymerase catalytic domain in Escherichia coli. During purification, the full-length GST-Rd. Rp was found to cleave into three main fragments: an N-terminal p12 fragment, a middle p30 fragment, and a C-terminal p64 fragment comprising the catalytic domain, presumably due to bacterial proteases. Biochemical assays show that the full-length GST-Rd. Rp has Rd. Rp activity and the p64 and p12 fragments form a complex that exhibits comparable Rd. Rp activity, whereas the GST-p64 protein has no activity, suggesting that the p12 domain is required for polymerase activity possibly via involvement in template-primer binding. Nonnucleoside HIV-1 RT inhibitors are shown to have no evident inhibitory effect on SARS-CoV Rd. Rp activity. This work provides a basis for biochemical and structural studies of SARS-CoV Rd. Rp and for development of anti-SARS drugs. © 2005 Elsevier Inc.
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