LOCALIZATION OF BINDING-SITE FOR ENCEPHALOMYOCARDITIS VIRUS-RNA POLYMERASE IN THE 3'-NONCODING REGION OF THE VIRAL-RNA

We previously showed that encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3D(pol)) binds specifically to 3'-terminal segments of EMC virus RNA. This binding, which depends on both the 3'-noncoding region (3'-NCR) and 3'-poly (A) tail [together denoted 3'-NCR(A)], may be an important step in the initiation of virus replication. In this paper, the 3'-NCR and 3'-poly(A) were separately transcribed then mixed, but no complex with 3D(pol) was obtained, showing that covalent attachment of the 3'-poly(A) to the 3'-NCR is essential for complex formation. Mutational and deletion analyses localized a critical determinant of 3D(pol) binding to a U-rich sequence located 38-49 nucleotides upstream of the 3'-poly(A). Similar analyses led to the identification of a sequence of A residues between positions +10 and +15 of the 3'-poly(A) which are also critical for 3D(pol) binding. As U-rich and A-rich regions are important for 3D(pol) binding, a speculative model is proposed in which 3D(pol) induces and stabilizes the base-pairing of the 3'-poly(A) with the adjacent U-rich sequence to form an unusual pseudoknot structure to which 3D(pol) binds with high affinity.