Furth P A, Choe W T, Rex J H, Byrne J C, Baker C C
Laboratory of Tumor Virus Biology, National Cancer Institute, Bethesda, Maryland 20892.
Mol Cell Biol. 1994 Aug;14(8):5278-89. doi: 10.1128/mcb.14.8.5278-5289.1994.
Expression of bovine papillomavirus type 1 (BPV-1) late genes is limited to terminally differentiated keratinocytes in an infected epithelium. We have previously shown that although the BPV-1 late polyadenylation site is functional in nonpermissive cells, a 53-nucleotide (nt) fragment of the late 3' untranslated region acts posttranscriptionally to reduce polyadenylated cytoplasmic RNA levels. This 53-nt fragment does not appear to function by destabilizing polyadenylated cytoplasmic RNA (P. A. Furth and C. C. Baker, J. Virol. 65:5806-5812, 1991). In this study, we used site-directed mutagenesis and deletion analysis to demonstrate that the sequence AAG/GUAAGU, which is identical to the consensus 5' splice site sequence, was both necessary and sufficient for the inhibitory activity of the 53-nt fragment. Furthermore, base pairing between the 5' end of the U1 small nuclear RNA and this 5' splice site-like sequence was shown to be required for the inhibitory activity in vivo. We have also further mapped the human papillomavirus type 16 late 3' inhibitory element (I. M. Kennedy, J. K. Haddow, and J. B. Clements, J. Virol. 65:2093-2097, 1991) to a 51-nt region containing four overlapping sequence motifs with partial homology to 5' splice sites. Mutation of each of these motifs demonstrated that only one of these motifs is required for the inhibitory activity. However, the presence of the other motifs may contribute to the full inhibitory activity of the element. No BPV-1 or human papillomavirus type 16 mRNAs which are spliced by using the potential 5' splice sites present in the viral late 3' untranslated regions have been identified. This suggests that the primary function of these 5' splice site-like sequences is the inhibition of late gene expression. The most likely mechanism of action of these elements is reduction of polyadenylation efficiency, perhaps through interference with 3'-terminal exon definition.
1型牛乳头瘤病毒（BPV-1）晚期基因的表达仅限于感染上皮中终末分化的角质形成细胞。我们之前已经表明，尽管BPV-1晚期聚腺苷酸化位点在非允许细胞中具有功能，但晚期3'非翻译区的一个53个核苷酸（nt）的片段在转录后发挥作用，以降低聚腺苷酸化的细胞质RNA水平。这个53-nt片段似乎不是通过使聚腺苷酸化的细胞质RNA不稳定来发挥作用的（P.A.弗思和C.C.贝克，《病毒学杂志》65:5806 - 5812, 1991）。在本研究中，我们使用定点诱变和缺失分析来证明与共有5'剪接位点序列相同的序列AAG/GUAAGU对于53-nt片段的抑制活性是必需且充分的。此外，U1小核RNA的5'末端与这个5'剪接位点样序列之间的碱基配对在体内被证明是抑制活性所必需的。我们还进一步将16型人乳头瘤病毒晚期3'抑制元件（I.M.肯尼迪、J.K.哈多和J.B.克莱门茨，《病毒学杂志》65:2093 - 2097, 1991）定位到一个51-nt区域，该区域包含四个与5'剪接位点具有部分同源性的重叠序列基序。对这些基序中的每一个进行突变表明，这些基序中只有一个是抑制活性所必需的。然而，其他基序的存在可能有助于该元件的完全抑制活性。尚未鉴定出通过使用病毒晚期3'非翻译区中存在的潜在5'剪接位点进行剪接的BPV-1或16型人乳头瘤病毒mRNA。这表明这些5'剪接位点样序列的主要功能是抑制晚期基因表达。这些元件最可能的作用机制是降低聚腺苷酸化效率，可能是通过干扰3'末端外显子的界定。
