Characterization of cDNAs of spliced HPV-11 E2 mRNA and other HPV mRNAs recovered via retrovirus-mediated gene transfer.
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, Cell 37, 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 E1 ORF in the first exon, 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 E1 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.
Rotenberg
M O
MO
Biochemistry Department, University of Rochester School of Medicine, New York 14642.
Chiang
C M
CM
Ho
M L
ML
Broker
T R
TR
Chow
L T
LT
D001483:Base Sequence; D002460:Cell Line; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics*; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics; D027383:Papillomaviridae / Q000235:genetics*; D010957:Plasmids; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D014162:Transfection
D004279:DNA, Viral; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; D012367:RNA, Viral