Subgenomic negative-strand RNA function during mouse hepatitis virus infection

Mouse hepatitis virus (MHV)-infected cells contain full-length and subgenom ic-length positive- and negative-strand RNAs. The origin and function of th e subgenomic negative-strand RNAs is controversial. In this report we demon strate that the synthesis and molar ratios of subgenomic negative strands a re similar in alternative host cells, suggesting that these RNAs function a s important mediators of positive-strand synthesis. Using kinetic labeling experiments, we show that the full-length and subgenomic-length replicative form RNAs rapidly accumulate and then saturate with label, suggesting that the subgenomic-length negative strands are the principal mediators of posi tive-strand synthesis. Using cycloheximide, which preferentially inhibits n egative-strand and to a lesser extent positive-strand synthesis, we demonst rate that cycloheximide treatment equally inhibits full-length and subgenom ic-length negative-strand synthesis. Importantly, following treatment, prev iously transcribed negative strands remain in transcriptionally active comp lexes even in the absence of new negative-strand synthesis. These findings indicate that the subgenomic-length negative strands are the principal temp lates of positive-strand synthesis during MHV infection.