EVIDENCE THAT THE RNA METHYLATION AND POLY(A) POLYMERASE STIMULATORY ACTIVITIES OF VACCINIA VIRUS PROTEIN VP39 DO NOT IMPINGE UPON ONE ANOTHER

Vaccinia protein VP39 has two RNA modifying activities. In monomeric f orm, it acts as an mRNA cap-specific 2'-O-methyltransferase, specifica lly modifying the ribose moiety of the first transcribed nucleotide of m(7)G-capped mRNA. In association with VP55, the catalytic subunit of the vaccinia poly(A) polymerese, VP39 facilitates the rapid elongatio n of poly(A) tails that are already greater than similar to 35 nt in l ength. Introducing new assays, we provide evidence that substrates for each of VP39's two activities do not detectably modulate the converse reaction and that VP39's 2'-O-methyltransferase activity is not signi ficantly affected by its association with VP55. In an electrophoretic mobility shift assay, VP39 interacted with a short (5 nucleotide) RNA only when the latter was m7G-capped. Complexes with longer (22 nucleot ide) RNAs were more stable (i.e., cap-independent) but were further st abilized by the presence of an m(7)G cap. An additional complex was ob served at elevated RNA:protein molar ratios, indicating the presence o f two RNA binding sites per VP39 molecule. Interaction at one of these sites was stabilized by the cap structure. Additional experiments ind icated that RNA molecules undergoing poly(A) tail elongation by the VP 55-VP39 heterodimer are not favored as cap-methylation substrates. (C) 1998 Academic Press.