Process of Cap Formation of Messenger RNA by Vaccinia Virus Particles Carrying an Organized Enzyme System

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Abstract
Vaccinia virus mRNA carry the cap structure m7G5''pppAm- or m7G5''pppGm- at the 5''-terminus, which is synthesized by a series of RNA polymerase and capping enzymes contained in the virus particle. The process of the cap formation at the 5''-terminus of mRNA was studied using an in vitro system under similar conditions to those of vaccinia virus multiplication in its host cell. After adding a methyl-group donor, methyl-3H-S-adenosylmethionine, the oligonucleotides, which were the de novo synthesized 5''-terminal part of mRNA, were isolated from the RNA-synthesizing virion at appropriate time intervals and analyzed. The 5''-5''-confronting nucleotides with 2''-O-methylation; G5''pppAm and G5''pppGm were found with the completed cap structure m7G5''pppAm and m7G5''pppGm. The confronting nucleotides with only 7-methyl guanine as a methylated component, m7G5''pppA and m7G5''pppG, were not detected at any incubation time. Methylation at the 2''-position of the 5''-terminal purine nucleoside of mRNA thus must precede methylation of the 7-position of the blocking guanosine. This result is different from that obtained using the enzymes isolated from vaccinia virus and from the results obtained using other kinds of virus particles which carry RNA polymerase and capping enzymes. These differences may be due to the specific organization of a series of capping enzymes and RNA polymerase in each virus particle.