Characterization of rare human papillomavirus type 11 mRNAs coding for regulatory and structural proteins, using the polymerase chain reaction.
Characterization of rare human papillomavirus type 11 mRNAs coding for regulatory and structural proteins, using the polymerase chain reaction.
复制标题
使用聚合酶链式反应表征编码调节蛋白和结构蛋白的罕见人乳头瘤病毒 11 型 mRNA。
DOI：
10.1016/0042-6822(89)90191-8
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发表时间：
1989
期刊：
影响因子：
3.7
通讯作者：
Broker,TR
中科院分区：
文献类型：
--
作者：
Rotenberg,MO;Chow,LT;Broker,TR
某些人乳头瘤病毒 (HPV) 类型会引起疣、发育不良以及肛门生殖器和口腔粘膜癌。由于 HPV 无法在培养细胞中繁殖、人类病变中病毒 mRNA 的缺乏以及源自不同启动子的选择性剪接转录本的复杂性，因此无法确定大多数 mRNA 种类和编码蛋白质的确切结构。我们采用了最近开发的聚合酶链式反应来扩增从无胸腺小鼠中有效感染的人包皮异种移植物中分离出的罕见 11 型 HPV mRNA 的 cDNA。寡核苷酸引物被设计为位于每个 mRNA 剪接位点的侧翼，这些剪接位点先前是通过电子显微镜分析克隆的 HPV-11 DNA 和从生殖器疣中分离的病毒 mRNA 之间形成的异源双链体而绘制的。通过直接测序 PCR 扩增的 cDNA 产物或将 cDNA 克隆到质粒载体后确定剪接点。我们为稀有 mRNA 的存在提供了第一个直接证据，这些 mRNA 具有编码被假设存在于 HPV 的调节蛋白的潜力。根据上游外显子的长度、使用的翻译框架以及翻译过程中内部重新启动的可能性，一对具有相同剪接点的 mRNA 可以编码病毒 DNA 拷贝数调节蛋白 E1-M、增强子抑制蛋白 E2-C 或两者。第二对 mRNA 也具有相同的剪接点，编码增强子调节蛋白 E2；两者中较长的一个也可以在其 5' 轴突中编码 E6 和 E7 蛋白之一或两者。最后，我们证明主要病毒颗粒衣壳蛋白Li的双重剪接晚期信息还包含前两个外显子中早期E1∧E4蛋白的整个编码区，Li蛋白的起始密码子精确地位于第三轴突的剪接受体处。这种晚期 mRNA 编码 E1∧E4 蛋白和衣壳蛋白的潜力可能有助于 E4 蛋白在病变中的优势。
Certain human papillomavirus (HPV) types cause warts, dysplasias, and carcinomas of the ano-genital and oral mucosa. Because of the inability to propagate HPVs in cultured cells, the paucity of viral mRNAs in human lesions, and the complexity of alternatively spliced transcripts derived from different promoters, it has not been possible to ascertain the exact structures of the majority of the mRNA species and the proteins encoded. We have adapted the recently developed polymerase chain reaction to amplify cDNAs of rare, type 11 HPV mRNAs isolated from a productively infected human foreskin xenograft in an athymic mouse. The oligonucleotide primers were designed to flank each of the mRNA splice sites previously mapped by electron microscopic analysis of heteroduplexes formed between cloned HPV-11 DNA and viral mRNAs isolated from genital warts. The splice junctions were determined by direct sequencing of the PCR-amplified cDNA products or after the cDNA was cloned into a plasmid vector. We provide the first direct evidence for the existence of rare mRNAs with the potential to encode regulatory proteins that have been hypothesized to exist for HPVs. Depending on the lengths of the upstream exons, the translation frame used and the possibility of internal reinitiation during translation, one pair of mRNAs with the same splice junction could encode the viral DNA copy number modulating protein E1-M, the enhancer repression protein E2-C, or both. A second pair of mRNAs, also with identical splice junctions, encode the enhancer-regulating protein E2; the longer of the two could also encode, in its 5′ axon, either or both of the E6 and E7 proteins. Finally, we demonstrate that the doubly spliced late message for the major virion capsid protein Li also contains the entire coding region for the early E1∧E4 protein in the first two exons, with the initiation codon for the Li protein located precisely at the splice acceptor of the third axon. The potential of this late mRNA to encode both the E1∧E4 protein and the capsid protein could contribute to the preponderance of the E4 protein in the lesion.