Genetics of mouse hepatitis virus transcription: evidence that subgenomic negative strands are functional templates

(ts) mutantsbelonging tocomplementation group C were characterized andmappedbystandard genetic recombination techniques. Temperature shift experiments early ininfection suggested thatthegroup Callele can bedivided into twophenotypically distinct subgroups, designated ClandC2.Since previousdataindicated thatthegroup Clmutantsprobably contained an earlydefect whichaffects negative-strand synthesis, RNA synthesis was further examined byanalyzing replicative-form (RF)RNA.Full-length aswell as subgenomic-length RFRNAswere radiolabeled from3to12 hpostinfection (p.i.) andlabeled late ininfection after shift tothenonpermissive temperature (39.5°C). The relative percent molarratios ofeachmRNA andcorresponding RFRNA were roughly equivalent throughout infection. Temperature shift experiments at5.5or 6.0hp.i. resulted inan 83to92%reduction intheamount oftotal RF RNA at39.5°C. Radiolabeling timecourse experiments after temperature shift to39.5°C also demonstrated incorporation (6to9hp.i.) into bothsubgenomic-length andfull-length RFRNAs,suggesting thatpreviously transcribed negative strands were functional templates throughout infection. Todetermine if thereduction inRFRNAwas duetoa decrease inpositive- ornegative-strand RNA synthesis, ratesofmRNA synthesis werecalculated frombothfull-length andsubgenomic-length templates. TherateofmRNA synthesis after theshift was increased at39.5°C compared withthatat32°Cregardless ofthetemplate used;however, transcription ratescalculated fromsubgenomic-length templates were similar tothoseofotherviral and