A unique class of compound, guanosine-nucleoside tetraphosphate G(5')pppp(5')N, synthesized during the in vitro transcription of cytoplasmic polyhedrosis virus of Bombyx mori. Structural determination and mechanism of formation

Abstract

Two structurally different classes of oligonucleotides accumulate in vitro in cytoplasmic polyhedrosis virus (CPV) transcription mixtures in molar excess as compared to the completed RNA products. The first class consists of oligonucleotides which correspond to the 5'-terminal sequence of the virus mRNAs (referred to as initiator oligonucleotides). The major species of initiator oligonucleotides are (p)ppApG and (p)ppApGpN together with smaller amounts of homologous capped structures (Furuichi, Y. (1981) J. Biol. Chem. 256, 483-493). In addition to initiator oligonucleotides, CPV transcription mixtures yielded a second new class of compounds which were radiolabeled by [alpha-32P]GTP and resistant to phosphatase digestion. Their structures were identified as G(5')pppp(5')A, G(5')pppp(5')C, G(5')pppp(5')G, and G(5')pppp(5')U. With the exception of G(5')pppp(5')G, these compounds have not been observed previously. The mechanism of synthesis of these unique compounds was elucidated as pppG + pppN leads to GppppN + PPi. The reaction resembles, in principle, a guanylylation reaction which occurs during cap formation in CPV and other eukaryotic mRNA syntheses. It is likely that these compounds are formed in a similar way by a condensation reaction involving a viral guanylyltransferase-pG intermediate complex and ribonucleoside triphosphate. When the amounts of G(5')pppp(5')N were measured, it was found that G(5')pppp(5')N reached maximum concentrations (0.4 to 0.7 microM) shortly after the onset of RNA synthesis (1 h) and these levels were maintained or diminished gradually. By contrast, mRNA and (p)ppApG were continuously synthesized. The relative molar ratios of total G(5')pppp(5')N and (p)ppApG versus mRNA were comparable (74:24:1 and 30:27:1 during 1 to 4 h transcription, respectively). The results imply that these unusual compounds G(5')pppp(5')N as well as initiator oligonucleotides may be produced reiteratively during initiation when RNA chain elongation and capping are uncoupled.