Doorbar J, Parton A, Hartley K, Banks L, Crook T, Stanley M, Crawford L
Department of Pathology, University of Cambridge, United Kingdom.
Virology. 1990 Sep;178(1):254-62. doi: 10.1016/0042-6822(90)90401-c.
The W12 cell line was derived from a low grade cervical lesion, and is unique among HPV16-containing cell lines in carrying its HPV16 genome as a multicopy episome. As such it is thought to be more representative of a premalignant HPV16-induced tumor than the cervical cancers from which other cell lines have been derived. Using the polymerase chain reaction (PCR), we report here the identification and cloning of a number of novel cDNA species, which appear to be characteristic of the W12 cell line. Two species were identified with E6* coding capacity (E6I and E6III). The smaller of these (1009 bp) was predicted to encode a novel E6III polypeptide containing C-terminal amino acids derived from an out of frame region of the E2/E4 ORFs. The larger species (1480 bp) contained, in addition to the E6I ORF, an intact E7 ORF and probably represents the transcript for E7 expression, as the E7 protein was readily detectable in the W12 cell line. Both species appeared to be transcribed from the p97 promoter which has been shown to be active in other cell lines. A putative E2 repressor cDNA (891 bp), an E1/E4 message (883 bp), and two novel late cDNA species (1757 and 2031 bp) were also detected, allowing the identification of a splice acceptor immediately in front of the L1 open reading frame (nt 5637) and a splice donor at nt 3631. Although the 1757-base species has the capacity to encode a full-length L1 protein, both messages use a splice donor at nt 1301, and are thus not analogous to late species previously identified in HPV11. Of the six cDNAs cloned, only the 1480-bp E7 message has been observed in other HPV16-containing cell lines. The presence of L1 transcripts, and an E2 repressor mRNA, although unexpected, may reflect the different origins of the W12 cell line.
W12细胞系源自低度宫颈病变，在含HPV16的细胞系中独具特色，其HPV16基因组以多拷贝附加体形式存在。因此，与其他细胞系所源自的宫颈癌相比，它被认为更能代表HPV16诱导的癌前肿瘤。我们在此报告，利用聚合酶链反应（PCR）鉴定并克隆了一些新的cDNA种类，它们似乎是W12细胞系所特有的。鉴定出两种具有E6编码能力的种类（E6I和E6III）。其中较小的一种（1009 bp）预计编码一种新的E6III多肽，其C末端氨基酸源自E2/E4开放阅读框的移码区域。较大的种类（1480 bp）除了含有E6*I开放阅读框外，还含有完整的E7开放阅读框，可能代表E7表达的转录本，因为在W12细胞系中很容易检测到E7蛋白。这两种种类似乎都从已证明在其他细胞系中有活性的p97启动子转录而来。还检测到一个假定的E2抑制因子cDNA（891 bp）、一个E1/E4信使（883 bp）以及两种新的晚期cDNA种类（1757和2031 bp），从而确定了紧邻L1开放阅读框（第5637位核苷酸）前方的一个剪接受体和第3631位核苷酸处的一个剪接供体。尽管1757碱基的种类有能力编码全长L1蛋白，但这两种信使都在第1301位核苷酸处使用一个剪接供体，因此与先前在HPV11中鉴定出的晚期种类不同。在克隆的六个cDNA中，只有1480 bp的E7信使在其他含HPV16的细胞系中被观察到。L1转录本和E2抑制因子mRNA的存在虽然出乎意料，但可能反映了W12细胞系的不同起源。
