Characterization of cDNAs of spliced HPV-11 E2 mRNA and other HPV mRNAs recovered via retrovirus-mediated gene transfer.
Characterization of cDNAs of spliced HPV-11 E2 mRNA and other HPV mRNAs recovered via retrovirus-mediated gene transfer.
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通过逆转录病毒介导的基因转移回收的剪接 HPV-11 E2 mRNA 和其他 HPV mRNA 的 cDNA 的表征。
DOI：
10.1016/0042-6822(89)90189-x
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发表时间：
1989
期刊：
影响因子：
3.7
通讯作者：
Chow,LT
中科院分区：
文献类型：
--
作者：
Rotenberg,MO;Chiang,CM;Ho,ML;Broker,TR;Chow,LT
人乳头瘤病毒（hpv）与皮肤或粘膜上皮的过度增生有关。这些病毒不能在任何细胞培养系统中繁殖。由于从人类病变中恢复的HPV mrna的cDNA拷贝的克隆仅取得了非常有限的成功，因此HPV mrna的表征一直存在问题。利用Moloney小鼠白血病病毒载体系统（C. L. Cepko， B. E. Roberts, and R. C. Mulligan, 1984,Cell37, 1053-1062），我们恢复了人乳头瘤病毒11型剪接的E2 mrna和其他11型和18型mrna的cdna，并确定了DNA序列中开放阅读框（orf）的利用。与先前描述的剪接位点相同的信息的cDNA拷贝的恢复强烈表明，新表征的剪接供体和受体也是真实的。HPV-11 E2 cDNA包含完整的E6和E7 ORF，以及位于第一个轴突的El ORF的开始部分，然后在E2 ORF起始密码子上游100个核苷酸的nt 2622处从nt 847拼接到第二个外显子。El ORF的起始密码子之后是4个额外的帧内AUG三联体和一个位于E2蛋白起始密码子上游30个核苷酸的帧内终止密码子。在几种细胞系的氯霉素乙酰转移酶（CAT）实验中，证实了E2 cDNA的真实性。该剪接受体位点的基因组DNA突变消除了其活性，表明剪接对E2蛋白的表达至关重要。我们得出结论，hpv - 11e2蛋白的翻译需要内部起始。
Human papillomaviruses (HPVs) are associated with hyperproliferations of cutaneous or mucosal epithelium. These viruses cannot be propagated in any cell culture system. Because cloning cDNA copies of HPV mRNAs recovered from human lesions has met with only very limited success, the characterization of HPV mRNAs has been problematic. Using the Moloney murine leukemia virus vector system (C. L. Cepko, B. E. Roberts, and R. C. Mulligan, 1984,Cell37, 1053–1062), we have recovered cDNAs of spliced E2 mRNAs of human papillomavirus type 11 and additional mRNAs of type 11 and type 18 and determined the utilization of open reading frames (ORFs) in the DNA sequences. The recovery of cDNA copies of messages with splice sites identical to those previously described strongly suggests that the newly characterized splice donors and acceptors are also authentic. The HPV-11 E2 cDNA contains the intact E6 and E7 ORFs and the beginning of the El ORF in the first axon, which is then spliced from nt 847 to the second exon at nt 2622, 100 nucleotides upstream from the initiation codon for the E2 ORF. The initiation codon in the El ORF is followed by four additional in-frame AUG triplets and an in-frame termination codon positioned 30 nucleotides upstream from the initiation codon for the E2 protein. The authenticity of this putative E2 cDNA was shown by its ability to provide enhancer transactivating activity in chloramphenicol acetyltransferase (CAT) assays in several cell lines. A mutation in the genomic DNA at this splice acceptor site eliminates its activity, demonstrating that the splice is essential for the expression of the E2 protein. We conclude that the translation of the HPV-11 E2 protein requires internal initiation.