MHV nucleocapsid synthesis in the presence of cycloheximide and accumulation of negative strand MHV RNA

We have found that genomic RNA synthesis is inhibited by cycloheximide in cells infected with mouse hepatitis virus, strain A59 (MHV-A59), in agreement with previously published results (Sawicki S.G. and Sawicki D.L. (1986) J. Virol. 57, 328–334). In the present study, the fate of the residual genomic RNA synthesized in the presence of cycloheximide was determined. Nearly all of the genomic RNA synthesized in the presence of drug was incorporated into nucleocapsid structures, suggesting that even in the absence of protein synthesis, genomic RNA synthesis and encapsidation are coupled in MHV-infected cells. Sufficient free nucleocapsid N protein was available for this purpose, since the pool of soluble N protein was determined to decay with a half-life of approximately one hour. Negative strand RNA is the template for the synthesis of both genomic and subgenomic positive strand RNA, and would be predicted to accumulate primarily during the early phases of the lytic cycle. In agreement with this prediction, negative strand RNA accumulated during the first 5–6 h of infection, with little additional accumulation occurring over the next 2.5 h. In marked contrast, positive strand RNA increased 5–6-fold over the same 2.5 h period. These results, taken in conjunction with published data, suggest that negative strand RNA is synthesized during the early period of the infectious cycle and is stable in infected cells and also suggest that treatment with cycloheximide at late times does not inhibit positive strand RNA synthesis indirectly by blocking the formation of negative strand templates.