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W C Phelps

Publications and source records attributed to W C Phelps.

34 records · Page 2Linked to original sources

Adenovirus E1A, simian virus 40 tumor antigen, and human papillomavirus E7 protein share the capacity to disrupt the interaction between transcription factor E2F and the retinoblastoma gene product.

The adenovirus E1A gene product, the simian virus 40 large tumor antigen, and the human papillomavirus E7 protein share a short amino acid sequence that constitutes a domain required for the transforming activity of these proteins. These sequences are also required for these proteins to bind to the retinoblastoma gene product (pRb). Recent experiments have shown that E1A can dissociate complexes containing the transcription factor E2F bound to pRb, dependent on this conserved sequence element. We now show that the E7 protein and the simian virus 40 large tumor antigen can dissociate the E2F-pRb complex, dependent on this conserved sequence element. We also find that the E2F-pRb complex is absent in various human cervical carcinoma cell lines that either express the E7 protein or harbor an RB1 mutation, suggesting that the loss of the E2F-pRb interaction may be an important aspect in human cervical carcinogenesis. We suggest that the ability of E1A, the simian virus 40 large tumor antigen, and E7 to dissociate the E2F-pRb complex may be a common activity of these viral proteins that has evolved to stimulate quiescent cells into a proliferating state so that viral replication can proceed efficiently. In circumstances in which a lytic infection does not proceed, the consequence of this action may be to initiate the oncogenic process in a manner analogous to the mutation of the RB1 gene.

Adenovirus Early Proteins↗

Evaluation of sexually abused and nonabused young girls for intravaginal human papillomavirus infection.

OBJECTIVE: The objective of this study was to compare the prevalence of intravaginal human papillomavirus-associated disease in two groups of girls to develop information regarding the means of transmission of anal-genital human papillomavirus disease. DESIGN: A pair of parallel studies of prevalence of human papillomavirus infections in two populations of prospectively enrolled girls. PATIENTS: Index patients consisted of 15 consecutive girls aged 11 years or younger who were confirmed to have been sexually abused, had signs or symptoms of vaginal disease, and required generalized anesthesia for evaluation. Selection of nonabused control patients was based on negative findings from screening evaluations and physical examinations. MAIN OUTCOME MEASURES: Prevalences of cervical-vaginal human papillomavirus infections in the two populations were compared. Vaginal wash samples from index and control patients were assayed for human papillomavirus 1, 2, 4, 6, 11, and 16 by reverse-blot and Southern transfer hybridization methods. Papanicolaou smears were examined from index patients. RESULTS: Vaginal wash samples from five (33%) of 15 index patients were positive for human papillomavirus 6, 11, or 16, compared with none of 17 controls. The presence or absence of external anal-genital warts was not correlated with results from the assay of intravaginal samples. Blinded readings of vaginal exfoliative cytologic findings of the index patients showed koilocytosis, atypia, or inflammatory reactions in four of five human papillomavirus-positive girls, and normal cytologic findings in one human papillomavirus-positive girl. CONCLUSION: These findings support other studies that indicate that sexual contact is a major route in the transmission of anal-genital human papillomavirus-related disease in children. Evaluation of intravaginal specimens was required to identify human papillomavirus-infected girls since the results of the wash samples were not correlated with the presence or absence of external anal-genital warts.

Academic Medical Centers↗

Structure-function analysis of the human papillomavirus type 16 E7 oncoprotein.

The E7 gene of human papillomavirus type 16 encodes a multifunctional nuclear phosphoprotein that is functionally and structurally similar to the adenovirus (Ad) E1A proteins and the T antigens of other papovaviruses. E7 can cooperate with an activated ras oncogene to transform primary rodent cells, trans activate the Ad E2 promoter, and abrogate transforming growth factor beta-mediated repression of c-myc. Recent studies suggest that these functions may in part be a consequence of the ability of E7 to associate with the product of the retinoblastoma tumor suppressor gene (pRB). In this study, a series of site-specific mutations of the human papillomavirus type 16 E7 gene product were constructed and assessed for their effects on intracellular protein stability, ras cooperativity, transcriptional trans activation, pRB association, and phosphorylation. The results of these studies indicate that the transforming and trans-activating domains extensively overlap within a region of the protein analogous to conserved region 2 of Ad E1A, suggesting that pRB binding is necessary for both activities. Deletion of sequences in conserved region 1 abrogates cellular transformation but has only a marginal effect on trans activation. These data suggest that E7 trans activation and cellular transformation are interrelated but separable functions.

Amino Acid Sequence↗

Analysis of trans activation by human papillomavirus type 16 E7 and adenovirus 12S E1A suggests a common mechanism.

The human papillomavirus E7 gene product is an oncoprotein with properties similar to those of the adenovirus E1A proteins. The human papillomavirus E7 proteins possess substantial amino acid sequence similarity to portions of conserved regions 1 and 2 of E1A, and the human papillomavirus type 16 E7 protein trans-activates the adenovirus E2 early promoter. Analysis of point mutations in the E2 promoter indicated that the E2F recognition sites were critical to E7 stimulation. In contrast to the activation of the E2 promoter, E7 could not trans-activate various other E1A-inducible promoters. Although the promoter specificity for E7 differs from that of 13S E1A trans activation, it is very similar to activation by the E1A 12S product. Moreover, analysis of the E7 protein has suggested that amino acid sequences critical for trans activation include those shared with E1A within conserved region 2. Biochemical studies demonstrate that the E7 protein, like the 12S E1A product, can alter the interaction of cellular factors with the E2F transcription factor. We therefore conclude that E7 trans activation is functionally related to that mediated by the 12S E1A product.

Adenovirus Early Proteins↗

Biochemical and biological differences between E7 oncoproteins of the high- and low-risk human papillomavirus types are determined by amino-terminal sequences.

Differences in the biological characteristics of the high-risk human papillomavirus type 16 (HPV-16) and the low-risk HPV-6 E7 proteins were analyzed and shown to correlate with certain biochemical properties. To ascertain which region of E7 conferred these properties, chimeric E7 genes were constructed by the exchange of the amino and carboxyl coding halves of the HPV-6 and HPV-16 E7 genes. The amino-terminal half of E7 determined the affinity for binding to the retinoblastoma protein pRB, the transformation properties, and the ability to abrogate transforming growth factor beta-mediated repression of the c-myc promoter. This region of E7 is therefore responsible for the biological and biochemical differences between the E7 proteins of the low-risk and the high-risk HPVs and consequently is one of the critical determinants distinguishing these two groups of viruses. Transcriptional transactivation of the adenovirus E2 promoter, in contrast, was a property shared by E7 proteins of both low-risk and high-risk HPVs.

Adenovirus Early Proteins↗

Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product.

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.

Amino Acid Sequence↗

The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes.

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 E1, 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 beta-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.

Cell Transformation, Viral↗

Molecular mechanisms of transformation by the human papillomaviruses.

A variety of studies have now indicated that the papillomaviruses associated with cervical carcinoma, HPV-16 and HPV-18, contain two genes (E6 and E7) which have transforming properties. These genes are generally expressed in cervical carcinoma cells. The HPV-16 E7 protein has recently been shown to be a multi-functional protein possessing both transcriptional modulatory and cellular transforming properties similar to those described for adenovirus E1A proteins (1). E7 is able to transactivate the adenovirus E2 promoter and can cooperate with an activated ras oncogene to transform primary baby rat kidney cells. The N-terminal 37 amino acids of all of the E7 proteins of the genital associated HPVs contain regions which are highly conserved and which are quite similar to portions of conserved domains 1 and 2 of adenovirus E1A. These domains in E1A are critical for cellular transformation properties and contain the amino acid sequences involved in binding the product of the retinoblastoma tumor suppressor gene (pRB). Results from a collaborative study with Ed Harlow and Nick Dyson (Cold Spring Harbor Laboratory) have shown that the E7 oncoprotein of HPV-16 can associate with the retinoblastoma gene product in vitro (2). The ability of the E7 proteins encoded by various HPVs to bind pRB has been examined using an in vitro complexing assay. E7 is not sufficient for transformation of human keratinocytes. The co-operation of the HPV-16 E6 and E7 genes has been shown to be important for transformation of these cells (3). Potential intracellular protein targets for E6 are being assessed.

Animals↗

The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similar to those of adenovirus E1A.

Clinical and epidemiological data have implicated the human papillomaviruses (HPVs) as having an etiologic role in some anogenital malignancies, with HPV-16 being most frequently (greater than 60%) detected in cervical carcinoma. HPV-16 is actively transcribed in the cancers; the most abundant transcripts map to the E6 and E7 early open reading frames. Evidence is presented that the HPV-16 E7 open reading frame encodes transcriptional transactivation and cellular transformation functions analogous to those of adenovirus E1A proteins. Specifically, the HPV-16 E7 gene product could transactivate the adenovirus E2 promoter and cooperate with an activated ras oncogene to transform primary baby rat kidney cells. The E7 transforming function differed somewhat from that of adenovirus E1A in that E7 was also able to transform established mouse cells. Examination of the amino acid sequence of HPV-16 E7 revealed striking similarities with conserved domains 1 and 2 of adenovirus E1A proteins.

Adenoviruses, Human↗

Quantitative keratinocyte assay detects two biological activities of human papillomavirus DNA and identifies viral types associated with cervical carcinoma.

Keratinocytes electroporated with human papillomavirus (HPV) DNA (HPV-6, 11, 16 and 18) exhibited an increased cellular proliferation which was quantitated as microcolony and macrocolony formation. However, only macrocolonies induced by HPV-16 or HPV-18 DNA (the two viral types most commonly found in human cervical carcinomas) gave rise to proliferating, poorly-stratified colonies when grown in the presence of serum and calcium. Hydrocortisone increased the frequency of these differentiation-resistant colonies, and studies showed that they were immortalized, contained one copy of viral DNA per cell, expressed three discrete species of viral RNA and synthesized the viral E7 protein. HPV-induced cellular proliferation and altered differentiation are therefore separable events and may represent the activity of different viral genes.

Biological Assay↗

Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines.

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) HindIII 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 E1 ORF, however, revealed the presence of an additional guanine at nucleotide 1138, resulting in the fusion of the E1a and E1b ORFs into a single E1 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 O repeat in the right-flanking region. Analysis of the three most abundant BamHI clones from the CaSki line showed that these consisted of full-length, 7.9-kb HPV-16 DNA; a 6.5-kb genome resulting from a 1.4-kb deletion of the long control region; and 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.

Amino Acid Sequence↗

Transcriptional trans-activation by the human papillomavirus type 16 E2 gene product.

We identified a conditional transcriptional enhancer in the long control region (LCR) of human papillomavirus type 16 (HPV-16). This conditional enhancer requires activation in trans by a product of the viral early-region open reading frames (ORFs). Primer extension analysis of chloramphenicol acetyltransferase RNA isolated from transiently transfected CV-1 cells demonstrated that trans-activation of the HPV-16 LCR enhancer operated at the transcriptional level. Mutational analysis of the early ORFs demonstrated that the conditional enhancer of the LCR was trans-activated by the product of the E2 ORF. The E2 gene product of bovine papillomavirus type 1, which can trans-activate the conditional enhancer in the bovine papillomavirus type 1 LCR, was also capable of trans-activating the E2-responsive enhancer of HPV-16. The activity of the HPV-16 LCR enhancer was also assayed in two human cervical carcinoma cell lines, HeLa and SiHa, which harbor transcriptionally active, integrated HPV-18 and HPV-16 DNA sequences, respectively. No endogenous E2 or E2-like activity was detected in either cell line.

Bovine papillomavirus 1↗

Shope papillomavirus transcription in benign and malignant rabbit tumors.

Shope papillomavirus induced benign and malignant tumors from both wild and domestic rabbits were analyzed by the Northern blotting technique for the presence of viral-specific RNAs. Virus-producing benign tumors of wild cottontail rabbits are shown to contain two major RNA species approximately 5000 and 3000 bases in length that originate in the early region and include the majority of the L1 and L2 late open reading frames. Non-productive tumors including wild-rabbit carcinomas, and benign warts and primary and metastatic carcinomas of domestic rabbits uniformly are shown to contain two major viral-specific RNA species, 2400 and 1400 bases in length. These transcripts map entirely within the early region of the viral genome. In addition, Southern blot analysis of metastatic tumors indicates that the viral DNA remains exclusively extrachromosomal and apparently unrearranged supporting previous findings with epithelial malignancies.

Animals↗

Cellular transformation by human papillomavirus DNA in vitro.

Molecularly cloned DNA's of human papillomaviruses HPV-5 and HPV-l induced morphological transformation of mouse C127 cells in culture. Single-cell clones of cells transformed by papillomavirus contained multiple persistent episomal copies of the transfected DNA species and were analyzed for growth characteristics indicating malignant potential.

Animals↗

Free cottontail rabbit papillomavirus DNA persists in warts and carcinomas of infected rabbits and in cells in culture transformed with virus or viral DNA.

We have compared warts and carcinomas from cottontail and domestic rabbits for the presence of cottontail rabbit papillomavirus (CRPV) and the status of the viral DNA genome. Our studies indicate that benign warts from cottontail rabbits, whether found naturally or induced in the laboratory, contain large amounts of virus and on the average 1000 copies of the virus genome per cell. Both benign warts and carcinomas from domestic rabbits contain significantly reduced levels of virus relative to cottontail rabbit warts and an average of 100 copies of the virus genome per cell. A single sample of a naturally occurring cottontail rabbit carcinoma contained approximately 80 copies of the viral genome per cell. None of the tumors that we have analyzed thus far appear to have integrated viral genomes by Southern blot analysis of undigested and restriction endonuclease-digested DNA samples. Furthermore, the CRPV genome present in domestic rabbit carcinomas and a cottontail rabbit carcinoma appears identical by restriction endonuclease mapping to that present in papillomas of cottontail and domestic rabbits indicating that no major deletions or rearrangements of the CRPV genome had occurred during the progression of benign to malignant tumors nor was a variant of wild-type CRPV responsible for this phenomenon. Finally, we have demonstrated morphological transformation in vitro of NIH 3T3 and C127 cells upon infection with purified CRPV and upon transfection with purified CRPV DNA. Furthermore, single cell clones derived from transformed foci contain free forms of CRPV DNA that persist through continued passage in culture. Cells transformed by CRPV grow in soft agar in vitro and produce tumors in athymic nude mice.

Animals↗