Localization of binding site for encephalomyocarditis virus RNA polymerase in the 3′-noncoding region of the viral RNA

Abstract

We previously showed that encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3Dpol) 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 3Dpol was obtained, showing that covalent atttachment of the 3′-poly(A) to the 3′-NCR is essential for complex formation. Mutational and deletion analyses localized a critical determinant of 3Dpol 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 3Dpol binding. As U-rich and A-rich regions are important for 3Dpol binding, a speculative model is proposed in which 3Dpol 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 3Dpol binds with high affinity.