Hummel M, Lim H B, Laimins L A
Department of Microbiology-Immunology, Northwestern University, Chicago, Illinois 60611, USA.
J Virol. 1995 Jun;69(6):3381-8. doi: 10.1128/JVI.69.6.3381-3388.1995.
Expression of the human papillomavirus (HPV) capsid genes, L1 and L2, as well as amplification of viral DNA and virion assembly occur in the terminally differentiated layers of infected stratified squamous epithelium in vivo. These processes can be duplicated in the laboratory through the use of organotypic or raft cultures. When CIN612 cells, which contain episomal copies of the high-risk HPV type 31b, are allowed to differentiate in raft cultures, the expression of transcripts encoding the early genes E1--E4 and E5 is induced. These transcripts are initiated at the differentiation-dependent P742 promoter located in the middle of the E7 open reading frame. Exposure of raft cultures to activators of protein kinase C, such as phorbol esters, results in the further induction of late gene expression as well as virion assembly. In this study, we have investigated the mechanism by which activators of protein kinase C induce late gene expression. The major L1 transcript was found to be encoded by a bicistronic E1--E4, L1 RNA which initiated at the differentiation-dependent promoter P742. Additional low-level expression of L1-containing RNAs was also observed from the early-region promoter, P97. The major L2 transcripts were found to be encoded by E1--E4, E5, L2, L1 RNAs which were also initiated in the early region, probably at the differentiation-specific promoter P742. While early and late RNAs were found to be expressed from the same promoter, they differed in utilization of splicing and polyadenylation sites. Raft cultures treated with activators of protein kinase C induced expression of late genes, but no change in the abundance of early RNAs initiated at the P742 promoter was observed. Thus, the increase in late gene expression was likely due to changes in RNA processing or stabilization rather than an increase in the rate of transcription from P742. Regulation of HPV late gene expression therefore occurs at two levels: differentiation-dependent induction of the P742 promoter, which can be mimicked in vitro by growth in raft cultures, and posttranscriptional changes that can be induced by activation of protein kinase C. These posttranscriptional changes may occur through inactivation or down-regulation of splicing factors which inhibit use of the late region polyadenylation site, resulting in increased stability of late region transcripts.
人乳头瘤病毒（HPV）衣壳基因L1和L2的表达，以及病毒DNA的扩增和病毒体组装，在体内感染的复层鳞状上皮终末分化层中发生。这些过程可通过使用器官型或筏式培养在实验室中重现。当含有高危型HPV 31b游离型拷贝的CIN612细胞在筏式培养中分化时，编码早期基因E1 - E4和E5的转录物的表达被诱导。这些转录物起始于位于E7开放阅读框中间的分化依赖性P742启动子。将筏式培养物暴露于蛋白激酶C的激活剂，如佛波酯，导致晚期基因表达以及病毒体组装的进一步诱导。在本研究中，我们研究了蛋白激酶C激活剂诱导晚期基因表达的机制。发现主要的L1转录物由双顺反子E1 - E4、L1 RNA编码，其起始于分化依赖性启动子P742。还从早期区域启动子P97观察到含L1 RNA的额外低水平表达。发现主要的L2转录物由E1 - E4、E5、L2、L1 RNA编码，这些RNA也起始于早期区域，可能在分化特异性启动子P742。虽然发现早期和晚期RNA从相同启动子表达，但它们在剪接和聚腺苷酸化位点的利用上有所不同。用蛋白激酶C激活剂处理的筏式培养物诱导晚期基因表达，但未观察到在P742启动子起始的早期RNA丰度的变化。因此，晚期基因表达的增加可能是由于RNA加工或稳定性的变化，而不是P742转录速率的增加。因此，HPV晚期基因表达的调控发生在两个水平：P742启动子的分化依赖性诱导，这可通过筏式培养中的生长在体外模拟，以及可由蛋白激酶C激活诱导的转录后变化。这些转录后变化可能通过抑制晚期区域聚腺苷酸化位点使用的剪接因子的失活或下调而发生，导致晚期区域转录物稳定性增加。
