FIG. 5.

Real-time PCR analysis of icTS-LA6. Cells were infected in triplicate with wt-icMHV, icTS-LA6, and mock at an MOI of 1 PFU/cell and maintained at the permissive temperature of 32°C and the standard temperature of 37°C. Cells were harvested in TRIzol reagent at 8 h p.i., total RNA was isolated, and 5 μg of RNA was used for reverse transcription using random hexamer primers to generate cDNA. Viral cDNAs were normalized to the housekeeping gene GAPDH. (A) Dilutions were prepared to normalize the total cDNA of all infections to approximate levels using the housekeeping gene GAPDH. All four normalized samples amplified at similar cycle threshold values indicating equivalent starting template concentrations. (B) Subgenomic mRNAs were detected using primers to the leader and the first 122 nt of the N gene. Cells infected with wt-icMHV and maintained at 37°C generated the most subgenomic mRNAs, while cells infected with wt-icMHV and icTS-LA6 and maintained at the permissive temperature were reduced by ∼2.5 logs. icTS-LA6 infections initiated and maintained at 37°C generated extremely reduced concentrations of subgenomic mRNAs. (C) Genomic mRNAs were detected using primers to 122 nt of ORF1a. Cells infected with wt-icMHV and maintained at 37°C generated the most genomic mRNAs, while cells infected with wt-icMHV and icTS-LA6 and maintained at the permissive temperature were reduced by 2 to 2.5 logs. icTS-LA6 infections initiated and maintained at 37°C generated extremely reduced concentrations of genomic RNAs. (D) Comparisons of the reductions of subgenomic and genomic mRNAs for icTS-LA6 and wt-icMHV suggest that RNA synthesis is equivalent in both viruses at the permissive temperature. Black, subgenomic; white, genomic.