Deletions or mutations of the retinoblastoma gene, RB1, are common features of many tumors and tumor cell lines. Recently, the RB1 gene product, p105-RB, has been shown to form stable protein/protein complexes with the oncoproteins of two DNA tumor viruses, the adenovirus E1A proteins and the simian virus 40 (SV40) large T antigen. Neither of these viruses is thought to be associated with human cancer, but they can cause tumors in rodents. Binding between the RB anti-oncoprotein and the adenovirus or SV40 oncoprotein can be recapitulated in vitro with coimmunoprecipitation mixing assays. These assays have been used to demonstrate that the E7 oncoprotein of the human papilloma virus type-16 can form similar complexes with p105-RB. Human papilloma virus-16 is found associated with approximately 50 percent of cervical carcinomas. These results suggest that these three DNA viruses may utilize similar mechanisms in transformation and implicate RB binding as a possible step in human papilloma virus-associated carcinogenesis.

The E7 proteins encoded by the human papillomaviruses (HPVs) associated with anogenital lesions share significant amino acid sequence homology. The E7 proteins of these different HPVs were assessed for their ability to form complexes with the retinoblastoma tumor suppressor gene product (p105‐RB). Similar to the E7 protein of HPV‐16, the E7 proteins of HPV‐18, HBV‐6b and HPV‐11 were found to associate with p105‐RB in vitro. The E7 proteins of HPV types associated with a high risk of malignant progression (HPV‐16 and HPV‐18) formed complexes with p105‐RB with equal affinities. The E7 proteins encoded by HPV types 6b and 11, which are associated with clinical lesions with a lower risk for progression, bound to p105‐RB with lower affinities. The E7 protein of the bovine papillomavirus type 1 (BPV‐1), which does not share structural similarity in the amino terminal region with the HPV E7 proteins, was unable to form a detectable complex with p105‐RB. The amino acid sequences of the HPV‐16 E7 protein involved in complex formation with p105‐RB in vitro have been mapped. Only a portion of the sequences that are conserved between the HPV E7 proteins and AdE1A were necessary for association with p105‐RB. Furthermore, the HPV‐16 E7‐p105‐RB complex was detected in an HPV‐16‐transformed human keratinocyte cell line.

The E6 protein of human papillomavirus types 16 and 18 (HPV‐16 and HPV‐18) can stably associate with the p53 protein in vitro. In the presence of rabbit reticulocyte lysate, this association leads to the specific degradation of p53 through the ubiquitin‐dependent proteolysis system. We have examined the E6‐p53 complex in more detail and have found that association of E6 with p53 is mediated by an additional cellular factor. This factor is present in rabbit reticulocyte lysate, primary human keratinocytes and in each of five human cell lines examined. The factor is designated E6‐AP, for E6‐associated protein, based on the observation that the E6 proteins of HPV‐16 and 18 can form a stable complex with the factor in the absence of p53, whereas p53 association with the factor can be detected only in the presence of E6. Gel filtration and coprecipitation experiments indicate that E6‐AP is a monomeric protein of approximately 100 kDa.

The early human papillomavirus type 16 genes that directly participate in the in vitro transformation of primary human keratinocytes have been defined. In the context of the full viral genome, mutations in either the E6 or E7 open reading frame completely abrogated transformation of these cells. Mutations in the El, E2, and E2-E4 open reading frames, on the other hand, had no effect. Thus, both the full-length E6 and E7 genes were required for the induction of keratinocyte immortalization and resistance to terminal differentiation. The E6 and E7 genes expressed together from the human ,-actin promoter were sufficient for this transformation; mutation of either gene in the context of this recombinant plasmid eliminated the ability to induce stable differentiation-resistant transformants.

No abstract

We cloned and analyzed the integrated human papillomavirus type 16 (HPV-16) genomes that are present in the human cervical carcinoma cell lines SiHa and CaSki. The single HPV-16 genome in the SiHa line was cloned as a 10-kilobase (kb) Hindlll fragment. Integration of the HPV-16 genome occurred at bases 3132 and 3384 with disruption of the E2 and E4 open reading frames (ORFs). An additional 52-base-pair deletion of HPV-16 sequences fused the E2 and E4 ORFs. The 5' portion of the disrupted E2 ORF terminated immediately in the contiguous human right-flanking sequences. Heteroduplex analysis of this cloned integrated viral genome with the prototype HPV-16 DNA revealed no other deletions, insertions, or rearrangements. DNA sequence analysis of the El ORF, however, revealed the presence of an additional guanine at nucleotide 1138, resulting in the fusion of the Ela and Elb ORFs into a single El ORF. Sequence analysis of the human flanking sequences revealed one-half of an Alu sequence at the left junction and a sequence highly homologous to the human 0 repeat in the right-flanking region. Analysis of the three most abundant BamHI clones from the CaSki line showed that these consisted of (i) full-length, 7.9-kb HPV-16 DNA; (ii) a 6.5-kb genome resulting from a 1.4-kb deletion of the long control region; and (iii) a 10.5-kb clone generated by a 2.6-kb tandem repeat of the 3' early region. These HPV-16 genomes were arranged in the host chromosomes as head-to-tail, tandemly repeated arrays. Transcription analysis revealed expression of the HPV-16 genome in each of these two cervical carcinoma cell lines, albeit at significantly different levels. Preliminary mapping of the viral RNA with subgenomic strand-specific probes indicated that viral transcription appeared to be derived primarily from the E6 and E7 ORFs.

No abstract

Expression of the ‘late’ genes of bovine papillomavirus type 1 (BPV‐1) occurs only in the differentiated keratinocytes of the productively infected fibropapilloma. A detailed analysis of viral transcription in the fibropapilloma was performed and compared to BPV‐1 specific transcription in transformed C127 cells. A cDNA library was constructed from bovine fibropapilloma mRNA using the method of Okayama and Berg. Analysis of full length cDNAs showed that the majority of viral transcripts in the fibropapilloma have 5′ termini near nt 7250 and utilize a common splice donor site at nt 7385. This mRNA start site was confirmed by the combination of primer extension and nuclease S1 analyses; it is not utilized in the BPV‐1‐transformed C127 cell, thus identifying it as a wart specific, ‘late’ promoter. Upstream of this mRNA start site is a tandemly repeated sequence element homologous to the SV40 late promoter sequence GGTACCTAACC, which has been shown to be important for the efficient utilization of the SV40 major late start site. Two additional mRNA start sites at nt 7185 and nt 7940 in the long control region (LCR) were identified and were found to be used in bovine warts as well as in BPV‐1‐transformed mouse cells. The promoter region upstream of the nt 7940 mRNA start site contains the E2 responsive enhancer mapping between nt 7611 and nt 7805 [Spalholz, B.A., Lambert, P.F., Yee, C. and Howley, P.M. (1987) J. Virol., in press].(ABSTRACT TRUNCATED AT 250 WORDS)