FIG. 4.

RNA synthesis from rExoN1 and rExoN3 mutant viruses. (A) Kinetics of viral RNA synthesis during multicycle growth. DBT-9 cells were mock infected or infected with the indicated viruses at an MOI of 0.1 PFU/cell. Viral RNA was metabolically labeled with [³H]uridine in the presence of ActD at 4-h intervals that began at 4, 8, 12, 16, 20, 24, 28, 32, 36, and 40 hpi. Total RNA was precipitated with 5% TCA, and incorporation of radiolabel was quantitated by scintillation counting. Mean values and standard deviations from duplicate samples obtained from duplicate infection series are indicated. (B) Kinetics of viral RNA synthesis during single-cycle growth. DBT-9 cells were mock infected or infected with the indicated viruses at an MOI of 3 PFU/cell. Viral RNA was metabolically labeled with [³H]uridine in the presence of ActD at 2-h intervals that began at 3, 5, 7, 9, 11, 13, and 15 hpi. (C) Electrophoretic analysis of RNA replication and transcription products. DBT-9 cells were mock infected (m) or infected with the indicated viruses at an MOI of 3 PFU/cell. Viral RNA was metabolically labeled with [³H]uridine in the presence of ActD from 11 to 13 hpi. Intracellular RNA was isolated, denatured, and resolved by electrophoresis in 1% agarose gels. Labeled RNA species were visualized by fluorography. Genomic RNA (R1) and sg mRNAs (R2 to R7) are indicated. The apparent band migrating between RNA3 and RNA4 in lanes 1, 3, 4, and 5 is most likely due to a physical effect of abundant 28S rRNA. The asterisk indicates that lane 1 received the same RNA sample as lane 5 but fivefold less. Note that all samples were electrophoresed on the same gel, but the image was cropped to remove extraneous lanes between lanes 4 and 5. (D) RT-PCR of MHV RNA2 (top) and cellular β-actin mRNA (bottom). The same RNA samples analyzed in panel C were subjected to RT-PCR, and the products were resolved in 1.2% (top) and 1.5% (bottom) agarose gels in the presence of ethidium bromide. The VUSS3 RNA sample was diluted fourfold prior to reverse transcription. Sizes (in kbp) of bands from a 100-bp DNA ladder are indicated. The predicted sizes of the RNA2-specific and actin-specific amplicons are 437 and 348 bp, respectively. Abbreviations: m, mock; con, negative control PCR for β-actin using water as a template. Images in panels C and D were prepared using Adobe Photoshop CS2.