BINDING-SITE FOR S-ADENOSYL-L-METHIONINE IN A CENTRAL REGION OF MAMMALIAN REOVIRUS LAMBDA-2 PROTEIN - EVIDENCE FOR ACTIVITIES IN MESSENGER-RNA CAP METHYLATION

One or more proteins in mammalian reovirus core particles mediate two RNA methylation activities, (guanosine-7-N)-methyltransferase and (gua nosine-2'-O)-methyltransferase, that contribute to forming the 5' cap 1 structure on viral mRNA, We used UV irradiation to identify core pro teins that bind S-adenosyl-L-methionine (SAM), the methyl-group donor for both methyltransferases. A [methyl-H-3]SAM-binding site was observ ed among the reovirus lambda proteins; was shown to be specific by com petition with low levels of S-adenosyl-L-homocysteine, the product of methyl-group transfer from SAM; and was subsequently localized to prot ein lambda 2, lambda 2 mediates the guanylyltransferase reaction in ca p formation and was previously proposed to mediate one or both methyla tion reactions as well. SAM binding was demonstrated for both lambda 2 in cores and lambda 2 expressed in insect cells from a recombinant ba culovirus. Using three different methods to cleave lambda 2, a binding site for SAM was tentatively localized to a central region of lambda 2, between residues 792 and 1100, which includes a smaller region with sequence similarity to the SAM-binding pocket of other methyltransfer ases. Alanine substitutions at positions 827 and 829 within this predi cted binding region greatly reduced the capacity of baculovirus-expres sed lambda 2 protein to undergo UV cross-linking to SARI but had no ef fects on either the guanylyltransferase activity of this protein or it s conformation as judged by partial proteolysis, suggesting that one o r both of these residues is essential for SAM binding. Based on these findings, we propose that the two methyltransferase activities involve d in mRNA capping by reovirus cores utilize a single SAM-binding pocke t within a central region of lambda 2.