"Pretranscriptional capping" in the biosynthesis of cytoplasmic polyhedrosis virus mRNA

Abstract

The in vitro synthesis of cytoplasmic polyhedrosis virus (CPV) mRNA was previously shown to be dependent upon the presence of the methyl donor S-adenosylmethionine (AdoMet). We now find that the competitive inhibitor of methylation, S-adenosylhomocysteine (AdoHcy), also stimulates CPV mRNA synthesis efficiently, resulting in the synthesis of viral mRNAs containing 5'-terminal GpppA and ppA, rather than m(7)GpppAm as observed with Adomet. In addition to AdoHcy, other AdoMet analogues, including S-adenosylethionine and adenosine, also stimulate CPV mRNA synthesis but to a smaller extent than does AdoHcy or AdoMet. In order to study the relationship between cap formation and mRNA synthesis, nucleoside triphosphates were replaced in the RNA-synthesizing reaction mixture (containing AdoMet) by the corresponding beta,gamma-imido analogues, which are resistant to nucleotide phosphohydrolase, an enzyme involved in cap formation. Although mRNA synthesis occurred in the presence of UMP-pNHp or GMP-pNHp, none was observed when AMP-pNHp was substituted for ATP. Because the ATP molecule that becomes the 5'-terminal nucleotide of CPV mRNA must be cleaved at the beta-gamma position during cap formation, the results suggest that, in this viral transcription system, cap formation is prerequisite to mRNA synthesis-i.e., a "pretranscriptional" event.