Schutz T, Kairat A, Schröder C H
Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Virology. 1996 Sep 15;223(2):401-5. doi: 10.1006/viro.1996.0495.
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.
此前在两名肝细胞癌患者的组织样本中已鉴定出截短的乙型肝炎病毒转录本，这些转录本在HBx开放阅读框内一个隐蔽的CAUAAA多聚腺苷酸化信号下游终止（Hilger等人，1991年，《病毒学杂志》65卷，4284 - 4291页）。在本研究中，对一个HBx表达质粒进行了系统删除，以阐明将通常无活性的CAUAAA基序转化为功能性多聚腺苷酸化信号所需的DNA序列背景。在一段病毒DNA上逐步进行删除，从转录水平看，这段病毒DNA始于已确定的UAUAAA多聚腺苷酸化信号下游，并延伸至隐蔽的CAUAAA基序。将获得的质粒构建体用于转染HepG2细胞系的细胞。通过核糖核酸酶保护试验对新合成的RNA进行分析，结果显示在去除UAUAAA信号的多聚腺苷酸添加位点下游富含GU的辅助序列后，CAUAAA基序下游出现了终止。当使用选择性识别截短RNA的锚定寡聚（dT）引物进行3'端的差异RT/PCR介导扩增时，也获得了类似结果。因此，可以通过两种方式证明，UAUAAA信号的失活或去除使CAUAAA基序具有了作为多聚（A）信号的功能。根据所获得的结果，我们认为去除TATAAA基序的染色体整合病毒DNA可能构成截短转录本以及病毒/细胞杂交转录本的模板。我们还建议使用锚定寡聚（dT）引物来快速鉴定肝癌患者组织样本中的截短转录本。
