The surface region of the bifunctional vaccinia RNA modifying protein VP39 that interfaces with Poly(A) polymerase is remote from the RNA binding cleft used for its mRNA 5' cap methylation function.

VP39 is a single-domain, bifunctional viral protein, which acts at both ends of nascent mRNA. At the 5' end, it acts as a cap-specific 2'-O-methyltransferase. At the 3' end, it acts as a poly(A) polymerase processivity factor, requiring its direct association with poly(A) polymerase. Although crystallographic and biochemical data show the catalytic center and associated binding sites for VP39's methyltransferase function to be juxtaposed around a superficial cleft on the protein surface, surface regions required for VP39's mRNA 3' end modifying functions are not known. Here, we identify a surface region that interfaces directly with poly(A) polymerase, taking three independent approaches: (i) development of a direct in vitro dimerization assay, which is applied to numerous VP39 point mutants; (ii) identification of sites within VP39 that become protected from protease cleavage upon dimerization and further mutagenesis based upon these data; (iii) site-specific photo-cross-linking of VP39 to VP55. We find that the dimerization interface lies on a surface region remote from the methyltransferase cleft and contains a 3-5-residue "hot-spot," which is very sensitive to amino acid substitutions. Various other sites within VP39 consistently became hypersensitive to protease cleavage upon interaction with VP55, indicating the occurrence of extensive conformational changes.