THE SURFACE REGION OF THE BIFUNCTIONAL VACCINIA RNA MODIFYING PROTEINVP39 THAT INTERFACES WITH POLY(A) POLYMERASE IS REMOTE FROM THE RNA-BINDING CLEFT USED FOR ITS MESSENGER-RNA 5'-CAP METHYLATION FUNCTION

VP39 is a single-domain, bifunctional viral protein, which acts at bot h ends of nascent mRNA, At the 3' end, it acts as a cap-specific 2'-O- methyltransferase, At the 3' end, it acts as a poly(A) polymerase proc essivity factor, requiring its direct association with poly(A) polymer ase, Although crystallographic and biochemical data show the catalytic center and associated binding sites for VP39's methyltransferase func tion to be juxtaposed around a superficial cleft on the protein surfac e, surface regions required for VP39's mRNA 3' end, modifying function s are not known. Here, we identify a surface region that interfaces di rectly with poly(A) polymerase, taking three independent approaches: ( i) development of a direct in vitro dimerization assay, which is appli ed to numerous VP39 point mutants; (ii) identification of sites within VP39 that become protected from protease cleavage upon dimerization a nd further mutagenesis based upon these data; (iii) site-specific phot o-crosslinking of VP39 to VP55. We find that the dimerization interfac e 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 beca me hypersensitive to protease cleavage upon interaction with VP55, ind icating the occurrence of extensive conformational changes.