DNA sequence requirements for the activation of a CATAAA polyadenylation signal within the hepatitis B virus X...

Truncated hepatitis B virus transcripts terminating downstream of a cryptic CAUAAA polyadenylation signal within the HBx open reading frame have previously been identified in tissue samples from two patients with hepatocellular carcinoma (Hilger et al., 1991, J. Virol. 65, 4284-4291). In this study an HBx expression plasmid was systematically deleted in order to elucidate the DNA sequence context which is required for the conversion of the usually inactive CAUAAA motif into a functional polyadenylation signal. Deletions were made progressively on a stretch of viral DNA which, seen on the transcript level, started downstream of the established UAUAAA polyadenylation signal and proceeded to the cryptic CAUAAA motif. The plasmid constructs obtained were used to transfect cells of the HepG2 line. The analysis of newly synthesized RNA via an RNase protection assay revealed termination downstream of the CAUAAA motif following the removal of GU-rich auxiliary sequences downstream of the poly(A) addition site of the UAUAAA signal. Similar results were obtained when an anchored oligo(dT) primer which recognizes selectively truncated RNA was used for the differential, RT/PCR-mediated amplification of 3'-ends. Thus it could be documented in two ways that inactivation or removal of the UAUAAA signal rendered the CAUAAA motif functional as a poly(A) signal. On the basis of the results obtained, we suggest that chromosomally integrated viral DNA on which the TATAAA motif is removed may constitute a template for truncated as well as for virus/cell hybrid transcripts. We also suggest the use of anchored oligo(dT) primers for the rapid identification of truncated transcripts in tissue samples of HCC patients.