Human rhinovirus-14 protease 3C (3Cpro) binds specifically to the 5'-noncoding region of the viral RNA. Evidence that 3Cpro has different domains for the RNA binding and proteolytic activities

Abstract

Protease 3C (3Cpro) encoded by human rhinovirus type 14 was purified from recombinant Escherichia coli and shown to bind specifically to the 5'-terminal 126 nucleotides of the viral RNA (126 RNA) in addition to efficiently cleaving a synthetic peptide in trans. The binding of 3Cpro to the viral RNA may be required for the initiation of plus strand viral RNA synthesis, suggesting a second non-proteolytic function for 3Cpro. Single amino acid substitutions were generated in 3Cpro at residues that are highly conserved among picornaviruses or that lie within the putative catalytic triad. Conservative changes at Asp-85 (D85E and D85N) destroyed the ability of 3Cpro 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 inactive mutants that bound to the 126 RNA. These results suggest that the highly conserved Asp-85 is essential for specific binding to the 126 RNA, but is unlikely to function in proteolysis as the acidic member of the catalytic triad. Moreover, 3Cpro appears to have different domains for the RNA binding and proteolytic activities.