Reaction in alphavirus mRNA capping : Formation of a covalent complex of nonstructural protein nsPl with 7 - methyl - GMP ( mRNA guanylyltransferase / covalent catalysis / Semliki Forest virus / Sindbis virus )

After the start of transcription, the 5' ends of eukaryotic mRNA molecules are modified by the addition of a guanylyl residue to form a cap structure, G(5')ppp(5')N. The guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) reaction responsible for cap formation usually proceeds via a covalent enzyme-GMP intermediate. We have studied the alphavirus-specific guanylyltransferase by incubating lysates from Semliki Forest and Sindbis virusinfected cells with [k-32P]GTP, using vaccinia virus and mock-infected cells as controls. One additional 32P-labeled protein was detected in alphavirus-infected cells but only in the presence of S-adenosylmethionine. This protein was identified as the nonstructural protein nsPl. The properties of the covalent enzyme-guanylate complex were studied with Semliki Forest virus nsPl expressed in recombinant baculovirus-infected cells. S-Adenosylmethionine and divalent cations were required for the complex formation. The reaction was specific for guanylate nucleotides (GTP, dGTP) and was inhibited by pyrophosphate. nsPl could be labeled with S-adenosyl [methyl-3H]methionine but only under conditions in which the nsPl-guanylate complex was formed. 7-Methyl-GMP was released from the nsPlguanylate complex by treatment with acid or acidic hydroxylamine. Similar treatment of vaccinia virus capping enzyme released GMP. These findings suggest that in the capping of alphavirus mRNAs the guanine is methylated before linkage to the mRNA molecule. The genome of alphaviruses consists of a single-stranded RNA molecule of positive polarity. Its translation in infected cells yields a polyprotein, which is processed into four nonstructural proteins (nsPl-nsP4) that form a membrane-associated replication complex. In addition to the genomic 42S RNA (11.5 kb), a subgenomic 26S RNA, which codes for the virus structural proteins, is produced during infection (1, 2). The 5' end of both mRNAs is blocked by a cap 0 structure (3, 4). Since cap formation of cellular mRNAs is a nuclear function and alphaviruses replicate exclusively in the cytoplasm of the infected cells (5), these viruses are expected to devote a substantial part of their coding capacity for producing their own capping enzymes. The capping of mRNA has been studied in mammalian and yeast cells and in several viral systems (reviewed in ref. 6). The capping enzymes of vaccinia virus have been characterized in great detail (7-9). Cap formation in these systems proceeds by a common mechanism: Step 1: pppN1pN2pN3 * > ppN,pN2pN3*** + P Step 2: GTP + enzyme > enzyme-GMP + PPi Step 3: enzyme-GMP + ppN,pN2pN3... GpppN1pN2pN3