Transcriptional complexity of vaccinia virus in vivo and in vitro

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Abstract
The transcriptional complexity of vaccinia virus in vivo and in vitro was measured by using DNA:RNA hybridization with RNA in excess. In vivo, early or prereplicative RNA saturated at 25% or 1/2 of the viral genome. Late or postreplicative RNA from infected [human cervical carcinoma] HeLa cells saturated at 52% or essentially the entire genome. This well-regulated transcriptional pattern of the virus in vivo was not maintained in vitro. In a number of experiments a range of saturation values from 40-50% was obtained for in vitro synthesized RNA. The complexity of polyadenylated and non-polyadenylated RNA, and purified 8-12S RNA released from the virus, was indistinguishable from purified high-MW virion-associated RNA with a sedimentation value of > 20S and equivalent to total in vitro synthesized RNA. No additional hybrid formation was observed in experiments in which total in vitro RNA and late in vivo RNA from infected HeLa cells were combined, suggesting that the virus does not transcribe in vitro DNA sequenes that are not also transcribed during productive infection. About 15% complementary RNA was detected when radiolabeled total in vitro RNA was allowed to reanneal with late in vivo RNA, while as much as 8% of the in vitro synthesized RNA was complementary.