Robert P. Ricciardi

Adenovirus type 12 E1A gene represses accumulation of MHC class I mRNAs at the level of transcription

The levels of the class I antigens and mRNAs of the major histocompatibility complex (MHC) are greatly diminished in cells transformed by adenovirus type 12 (Ad12). Although the Ad12-transforming gene, E1A, is responsible for reduced class I expression, the site at which E1A blocks accumulation of class I transcripts is not known. In this study, we demonstrate by nuclear run-on assays that in Ad12-transformed mouse cells, E1A acts by reducing the rate of transcription of class I genes.

Transactivation of BKV and SV40 early promoters by BKV and SV40 T-antigens.

The early promoters of BKV and SV40 plasmids were transactivated in both BKV and SV40-transformed cells which failed to support replication of these plasmids. This suggests that the T-antigen of either virus can transactivate BKV and SV40 early promoters by either increasing the availability of cellular transcription factors or by directly interacting with specific sequences which comprise the transcriptional control region of the early promoters. We also observed that removal of 8-bp on the early side of T-antigen binding site I of BKV does not alter viral-plasmid replication.

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.

How reliable is amino acid sequence homology in predicting similarity of structure and function of c-myc and Ad12 E1A oncogenic proteins

SummaryAdenovirus E1A and c-myc genes are known to be capable of transforming primary rat cells when they occur in combination with either polyoma middle-T or T24 Harvey-ras 1 genes. There was a low level of amino acid sequence homology between the nuclear adenovirus-12 (Ad12) E1A protein product (289 amino acids) and the c-myc protein based on optimal alignment and percentage identity. In contrast to others [Ralston R, Bishop JM (1983) Nature 306:803–806], we concluded that this low level of amino acid sequence homology was not significant, since rabies glycoprotein (RGP), which has no transforming function and localizes to the cell surface, had a similar low level of amino acid sequence homology to the c-myc protein. Furthermore, dot-matrix analysis, when used to test the overall level of amino acid sequence homology, showed no significant homology between c-myc and Ad12 E1A, E1B, or RGP. Thus, low levels of amino acid sequence homology between two proteins may not be sufficient to predict structural and functional similarities between them reliably, even if the two proteins appear to share a common function.