HUMAN RHINOVIRUS-14 PROTEASE-3C (3C(PRO)) BINDS SPECIFICALLY TO THE 5'-NONCODING REGION OF THE VIRAL-RNA - EVIDENCE THAT 3C(PRO) HAS DIFFERENT DOMAINS FOR THE RNA-BINDING AND PROTEOLYTIC ACTIVITIES

Protease 3C (3C(pro)) encoded by human rhinovirus type 14 was purified from recombinant Escherichia coli and shown to bind specifically to t he 5'-terminal 126 nucleotides of the viral RNA (126 RNA) in addition to efficiently cleaving a synthetic peptide in trans. The binding of 3 C(pro) to the viral RNA may be required for the initiation of plus str and viral RNA synthesis, suggesting a second non-proteolytic function for 3C(pro). Single amino acid substitutions were generated in 3C(pro) at residues that are highly conserved among picornaviruses or that li e within the putative catalytic triad. Conservative changes at Asp-85 (D85E and D85N) destroyed the ability of 3C(pro) to bind specifically to the 126 RNA, whereas the D85N mutation resulted in almost wild-type levels of proteolytic activity. Conversely, substitutions at His-40, Glu-71, or Cys-146 (H40D, E71A, or C146S) gave proteolytically inactiv e mutants that bound to the 126 RNA. These results suggest that the hi ghly conserved Asp-85 is essential for specific binding to the 126 RNA , but is unlikely to function in proteolysis as the acidic member of t he catalytic triad. Moreover, 3C(pro) appears to have different domain s for the RNA binding and proteolytic activities.