Encephalomyocarditis virus replication complexes preferentially utilizing nucleoside diphosphates as substrates for viral RNA synthesis

Replication complexes (RC) of the encephalomyocarditis (EMC) virus were shown previously to contain components that exhibit marked preference for nucleoside diphosphates over nucleoside triphosphates (NTP) as substrates for viral RNA synthesis [Koonin and Agol (1983), Virology 129 , 309–318]. These NDP‐preferring components have now been found to posses the following properties. When RC preparations were fractionated by sucrose density gradient centrifugation, the fractions containing NDP‐preferring components exhibited a considerably higher nucleotide kinase activity as compared to either the fractions containing NTP‐preferring components or corresponding fractions from mock‐infected cells. When NDP‐preferring RC were incubated with ADP and three other NTP, very low concentrations of endogenously generated ATP ensured a greater rate of RNA synthesis than did much higher concentrations of exogenous ATP. When an equimolar mixture of differently labelled UDP and UTP was used as a substrate for NDP‐preferring RC, the label from UDP predominated in the newly synthesized RNA, even though the UDP‐derived UTP constituted a minor portion of the total UTP pool. When labelled UDP was diluted with unlabelled uridine nucleotides, unlabelled UTP proved to be far less efficient than unlabelled UDP in diminishing the specific radioactivity of UMP incorporated into RNA by NDP‐preferring RC. These data are interpreted in the sense that the NTP generated by the built‐in nucleotide kinase system are not freed into the external milieu but rather form a separate pool preferentially used for synthesis of viral RNA by NDP‐preferring RC. It is suggested that this functional compartmentation of NTP may be significant for the replication of viral RNA in vivo.