FUNCTIONAL MAPPING OF THE HUMAN PAPILLOMAVIRUS TYPE-11 TRANSCRIPTIONAL ENHANCER AND ITS INTERACTION WITH THE TRANS-ACTING E2 PROTEINS
FUNCTIONAL MAPPING OF THE HUMAN PAPILLOMAVIRUS TYPE-11 TRANSCRIPTIONAL ENHANCER AND ITS INTERACTION WITH THE TRANS-ACTING E2 PROTEINS
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DOI：
10.1101/gad.2.1.54
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发表时间：
1988-01-01
影响因子：
10.5
通讯作者：
CHOW, LT
中科院分区：
文献类型：
--
作者：
HIROCHIKA, H;HIROCHIKA, R;CHOW, LT
乳头瘤病毒的转录增强子序列受到病毒E2开放阅读框编码的反式作用因子的调节。我们对 11 型人乳头瘤病毒 (HPV-11) 增强子进行了详细的功能和物理分析。在瞬时转染的猴 CV-1 细胞中使用氯霉素乙酰转移酶 (CAT) 测定，增强子区域定位于紧邻 E6 开放阅读框之前的 270 bp 区域，并且由两个功能组件组成。第一个是组成型增强子，包含与 SV40 和多瘤病毒增强子中发现的 GT-、Sph- 和 P-基序同源的序列；其他类似于 CTF (NF-1) 的识别序列，CTF 是一种刺激某些真核基因转录和腺病毒 DNA 复制的因子。第二个成分是诱导型增强子，具有对乳头瘤病毒编码的 E2 蛋白有反应的共有序列 ACCN6GGT。组成型增强子部分的串联拷贝充当不依赖于 E2 的增强子，而包含 E2 响应序列 (E2-RS) 的 HPV-11 DNA 限制性片段或合成寡核苷酸的多个拷贝在 HPV-1、HPV-11 或 1 型牛乳头瘤病毒 (BPV-1) 编码的 E2 蛋白存在时充当增强子。当 E2-RS 中引入突变或突变回文序列被取代时，诱导活性就会丧失。我们还在大肠杆菌中表达了 HPV-1、HPV-11 和 BPV-1 的 E2 蛋白，并在体外研究了它们与 E2 响应序列的物理相互作用。使用粗制大肠杆菌裂解物进行的过滤结合分析表明，E2 蛋白与 E2-RS 结合，但不与突变基序结合，其亲和力与拷贝数成正比。使用合成的 E2-RS 作为配体，通过序列特异性 DNA 亲和层析将这些 E2 蛋白纯化至接近均质。纯化的蛋白质在胰腺 DNase I 足迹分析中保护含有 E2-RS 和几个侧翼核苷酸的 DNA 片段。基于这些结果，我们得出结论，E2蛋白通过直接结合E2-RS并与其他转录因子相互作用来激活增强子，并且序列ACCN6GGT对于E2蛋白体外结合和体内RNA转录激活是必要且充分的。
The transcriptional enhancer sequences of the papillomaviruses are regulated by trans-acting factors encoded by the viral E2 open reading frame. We have performed detailed functional and physical analyses of the enhancer of the human papillomavirus type 11 (HPV-11). Using the chloramphenicol acetyltransferase (CAT) assay in transiently transfected monkey CV-1 cells, the enhancer region has been localized to a 270-bp tract immediately preceding the E6 open reading frame, and it consists of two functional components. The first is a constitutive enhancer containing sequences homologous to the GT-, Sph-, and P-motifs found in the SV40 and polyomavirus enhancers; others resemble the recognition sequence for CTF (NF-1), a factor which stimulates transcription of certain eukaryotic genes and replication of adenovirus DNA. The second component is an inducible enhancer with a consensus sequence ACCN6GGT responsive to the E2 protein encoded by papillomaviruses. Tandem copies of portions of the constitutive enhancer function as an E2-independent enhancer, whereas multiple copies of HPV-11 DNA restriction fragments or synthetic oligonucleotides containing the E2-responsive sequence (E2-RS) act as an enhancer in the presence of the E2 protein encoded by HPV-1, HPV-11, or bovine papillomavirus type 1 (BPV-1). The inducible activity is lost when mutations are introduced into the E2-RS or when a mutant palindromic sequence is substituted. We have also expressed the E2 proteins of HPV-1, HPV-11, and BPV-1 in Escherichia coli and studied their physical interactions with the E2-responsive sequence in vitro. Filter-binding analyses with crude Escherichia coli lysates show that the E2 proteins bind to the E2-RS, but not to mutated motifs, with an affinity proportional to the copy number. These E2 proteins have been purified to near-homogeneity by sequence-specific DNA affinity chromatography using the synthetic E2-RS as a ligand. The purified proteins protect a DNA segment containing the E2-RS and several flanking nucleotides in pancreatic DNase I footprinting analyses. Based on these results, we conclude that E2 proteins activate the enhancer by binding directly to the E2-RS and interacting with other transcriptional factors and that the sequence ACCN6GGT is both necessary and sufficient for the E2 protein binding in vitro and for activation of RNA transcription in vivo.