Detailed kinetics of adenovirus type-5 steady-state transcripts during early infection

The appearance and steady-state accumulation of specific viral RNAs during the early phase of adenovirus type 5 (Ad5) infection was examined. HeLa cells were synchronously infected and harvested at 30 min intervals throughout the first 12 h of infection. Total cytoplasmic RNA was extracted from infected cells and analyzed by hybridization-selection and translation to identify the viral mRNAs from each early region on the basis of the protein products they encode. The same RNA samples were used for S-1 nuclease and Northern blot analyses to quantitatively compare the levels of individual viral RNAs that accumulate within each early transcription region (E1A, E1B, L1, E2A, E3 and E4). The salient features of this analysis show that RNA accumulation occurs first from E1A followed by E2A, E3 and E4, E1B and lastly, L1. Although the profile of RNA accumulation was unique for each early region, overlapping RNAs within E1A, E3, and E4, respectively, remained generally parallel to one another throughout early infection, in contrast to RNAs from E1B and L1, respectively. Since both the appearance and quantitative accumulation of specific early viral mRNAs were examined at many time points, a number of subtleties associated with the complex dynamics of early Ad5 gene expression were revealed. In particular, the L1 region was shown to transcribe from the major late promoter two early RNAs of 3.81 Kb and 3.5 Kb, either or both of which encode the 52,55 kDa proteins; the auxiliary i leader sequence was found on the 3.81 Kb RNA but not on the 3.5 Kb RNA.