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

Abstract

SUMMARYVaccinia virus mRNAs carry the cap structure mTGS'pppAm -or mTGS'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 RNAsynthesizing virion at appropriate time intervals, and were analysed. The 5'-5' confronting nucleotides with 2'-O-methylation, GS'pppAm and GS'pppGm, were found with the completed cap structure, mTGS'pppAm and mTG5'pppGm. The confronting nucleotides with only 7-methyl guanine as a methylated component, m7GS'pppA and mTGS'pppG, were not detected at any incubation time, and it was concluded that methylation at the T-position of the 5'-terminal purine nucleoside of mRNA precedes methylation of the 7-position of the blocking guanosine. This result is different from that obtained using the enzymes isolated from vaccinia virus (Moss et al., 1976) and also 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.

Vaccinia virus RNA polymerase associated with nuclei of infected HeLa cells

“…However, one distinguishing feature of vaccinia mRNA is that, to date, no identified viral mRNA requires splicing as a processing step in its formation (6,42). Vaccinia DNA-dependent RNA polymerase (22,25,26,36), polyadenylated polymerase (28), and the enzymes involved in mRNA capping (10,39) are all coded for by the virus genome and are all components of the cores. The localization of viral RNA synthesis in the cytoplasm and the presence therein of the viral enzymes involved in RNA synthesis suggest that the cytoplasm is the major site of viral RNA synthesis.…”