Detection of novel splicing patterns in a HPV16-containing keratinocyte cell line.
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 (E6*I and E6*III). The smaller of these (1009 bp) was predicted to encode a novel E6*III 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 E6*I 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.
Doorbar
J
J
Department of Pathology, University of Cambridge, United Kingdom.
Parton
A
A
Hartley
K
K
Banks
L
L
Crook
T
T
Stanley
M
M
Crawford
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000032:analysis; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014412:Tumor Virus Infections / Q000235:genetics
D004279:DNA, Viral; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger