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T R Broker

Publications and source records attributed to T R Broker.

At least 55 records · Page 3Linked to original sources
PubMed1992

Human papillomavirus type 16 and 18 gene expression in cervical neoplasias.

Human papillomavirus (HPV) types 16 and 18 are strongly implicated in the generation of progressive cervical neoplasms. The viruses produce complex families of overlapping messenger RNAs that are linked to differentiation, making it necessary to analyze gene expression in the context of morphology. We have developed HPV type 16 and type 18 subgenomic clones from which 3H-labeled riboprobes specific to individual mRNA families can be generated in vitro. Using these probes for in situ hybridization, we examined serial sections of archival biopsy specimens of the spectrum of genital lesions. In low-grade squamous lesions, all viral open reading frames were expressed, and the most abundant transcription spanned the E4 and E5 open reading frames at the 3' end of the E region. L region transcription coding for the capsid proteins was restricted to terminally differentiated keratinocytes. As the grade of neoplasia increased, cellular differentiation and overall viral transcription decreased and, with few exceptions, the L2 and L1 transcripts ceased to exist. The E6-E7 transforming region was invariably derepressed. Interestingly, the patterns of HPV-16 gene expression suggested the coexistence of episomal and integrated viral DNAs. In contrast, in HPV-18 lesions, all the viral template DNA appeared to have integrated. Integration was deduced to have occurred near the boundary of the E1 and E2 open reading frames. Viral transcription patterns were similar in carcinomas in situ and in invasive carcinomas, regardless of the histologic cell types or the associated virus types, consistent with the notion that additional host gene alterations were necessary for progression. On the basis of viral gene expression in vivo and the E6 promoter regulation previously characterized in vitro, we discuss a molecular mechanism for HPV-associated carcinogenesis.

Adenocarcinoma↗
PubMed1992

Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. OFF.

Human papillomaviruses trophic for anogenital epithelia cause benign warts, and certain genotypes are closely associated with cervical neoplasia. By using our modifications of the epithelial raft culture system, we were able to recapitulate and modulate the infectious program of a papillomavirus in vitro for the first time. Small pieces of a condyloma containing human papillomavirus type 11 were explanted onto a dermal equivalent consisting of a collagen matrix with fibroblasts and were cultured at the medium-air interface. The infected stem cells proliferated rapidly across the matrix, stratified, and differentiated, as judged by histology. The results correlated with the state of epithelial differentiation, which, in turn, was dependent on the type of fibroblast in the matrix. Under conditions where the epithelial outgrowth underwent terminal differentiation, the entire productive program took place, leading to virion assembly. In contrast, using an alternative condition where the outgrowth failed to achieve terminal differentiation, only the E-region RNAs from the E1 promoter accumulated to any appreciable extent. The proliferating cell nuclear antigen was induced in the differentiated suprabasal cells in the productive cyst growth, which also exhibited high copy viral DNA and abundant E6-E7 RNAs. Comparable cells in the nonproductive cyst outgrowth were negative for all three. These results suggest that the E6 and E7 proteins may play a role in establishing a cellular environment conducive to vegetative viral replication. The culture conditions described should be useful for genetic analysis of this family of important human pathogens and for testing potential pharmacological agents.

3T3 Cells↗
PubMed1992

Control of human papillomavirus type 11 origin of replication by the E2 family of transcription regulatory proteins.

Replication of human papillomavirus type 11 (HPV-11) DNA requires the full-length viral E1 and E2 proteins (C.-M. Chiang, M. Ustav, A. Stenlund, T. F. Ho, T. R. Broker, and L. T. Chow, Proc. Natl. Acad. Sci. USA 89:5799-5803, 1992). Using transient transfection of subgenomic HPV DNA into hamster CHO and human 293 cells, we have localized an origin of replication (ori) to an 80-bp segment in the upstream regulatory region spanning nucleotide 1. It overlaps the E6 promoter region and contains a short A + T-rich segment and a sequence which is homologous to the binding site of the bovine papillomavirus type 1 (BPV-1) E1 protein in the BPV-1 ori. However, unlike the BPV-1 ori, for which half an E2-responsive sequence (E2-RS) or binding site suffices, an intact binding site is essential for the HPV-11 ori. Replication was more efficient when additional E2-RSs were present. The intact HPV-11 genome also replicated in both cell lines when supplied with E1 and E2 proteins. Expression vectors of transcription repressor proteins that lack the N-terminal domain essential for E2 transcriptional trans activation did not support replication in collaboration with the E1 expression vector. Rather, cotransfection with the repressor expression vectors inhibited ori replication by the E1 and E2 proteins. These results demonstrate the importance of the N-terminal domain of the E2 protein in DNA replication and indicate that the family of E2 proteins positively and negatively regulates both viral DNA replication and E6 promoter transcription.

Animals↗
PubMed1992

Recombinant retroviruses encoding human papillomavirus type 18 E6 and E7 genes stimulate proliferation and delay differentiation of human keratinocytes early after infection.

Human papillomavirus (HPV) DNAs are detected in most genital dysplasias and cancers, suggesting that these viruses perturb epithelial growth and differentiation. The E6 and E7 genes of HPV type 18 induce immortality in keratinocytes cultured from genital tract epithelia, and the immortal cell lines display aberrant squamous differentiation. To examine whether the E6 and E7 proteins directly alter keratinocyte growth and differentiation, high-titer recombinant retroviruses were constructed for efficient transfer and expression of HPV-18 genes E6, E6* and E7 in cultures of normal human keratinocytes. Infection with retroviruses encoding E6 and E7 stimulated cell proliferation, reduced the requirement for bovine pituitary extract and induced immortality. E6 and E7 also delayed but did not prevent the onset of terminal squamous differentiation. The magnitude of effects on growth and differentiation of cultured cells was directly related to levels of E7 protein expression. Thus, expression of the HPV-18 E6 and E7 genes stimulates cell proliferation and delays differentiation of keratinocytes in vitro.

Blotting, Southern↗
PubMed1992

Epithelial-specific gene expression during differentiation of stratified primary human keratinocyte cultures.

Cultured epithelial cells are used to generate extensive patches of autologous skin equivalent for patients with burns or wounds and to investigate the growth and differentiation of epithelia in vitro. We have undertaken a comprehensive study of the morphological and molecular events that occur during culturing of human foreskin keratinocytes at the liquid-air interface on a dermal equivalent consisting of a collagen matrix containing fibroblasts. Using radioactively labeled RNA probes for mRNAs and monoclonal antibodies for proteins, we found that the expression of a comprehensive set of differentiation stage-specific genes was affected by the type of fibroblasts included in the matrix as well as by the age of the culture. The expression of these genes was not always coordinated and could not be predicted from the histological appearance of the stratified epithelium. Surprisingly, the mouse fibroblasts promoted epithelial differentiation much more closely resembling foreskin than did the homologous primary foreskin fibroblasts.

3T3 Cells↗
PubMed1991

An E1M--E2C fusion protein encoded by human papillomavirus type 11 is asequence-specific transcription repressor.

We have isolated a putative, spliced E5 cDNA of human papillomavirus type 11 (HPV-11) by polymerase chain reaction amplification of cDNAs from an experimental condyloma. Using retrovirus-mediated gene transfer, we isolated two novel HPV-11 cDNAs, one of which had a splice linking nucleotides 1272 and 3377. This transcript also existed in experimental condylomata and in cervical carcinoma cells transfected with cloned genomic HPV-11 DNAs. The 5' end of the transcript in transfected cells originated upstream of the initiation codon of the E1 open reading frame (ORF). It could conceptually encode a fusion protein consisting of the amino-terminal 23% of the E1 ORF and the carboxy-terminal 40% of the E2 ORF. This E1M--E2C fusion protein contained both the DNA replication modulator domain E1M, as defined in the bovine papillomavirus system, and the DNA binding domain of the E2 protein, which regulates viral transcriptional activities. Indirect immunofluorescence with polyclonal antibodies raised against the bacterially expressed TrpE-HPV-11 E2 protein demonstrated nuclear localization of the E1M--E2C protein in cells transiently transfected with an expression plasmid. Immunoprecipitation revealed a specific protein with an apparent molecular weight of 42,000 in transfected cells. The chloramphenicol acetyltransferase assay established that the putative E1M--E2C protein was a potent transcriptional repressor of both E2-dependent and E2-independent HPV-11 enhancer/promoter activities. Northern (RNA) blot hybridization indicated the repression was on the transcriptional level. Mutational analysis suggested that the E1M--E2C protein is an E2-binding site-specific repressor. The fusion protein also repressed bovine papillomavirus type 1 (BPV-1) E2 protein-dependent BPV-1 enhancer activity. When constitutively expressed in mouse C127 cells, the E1M--E2C protein inhibited BPV-1 transformation and episomal DNA replication, consistent with a role in the modulation of replication.

Condylomata Acuminata↗
PubMed1990

Human papillomavirus type 1 produces redundant as well as polycistronic mRNAs in plantar warts.

Human papillomavirus type 1 (HPV-1) causes plantar warts. On the basis of previously mapped mRNAs and sequence homologies of HPV-1 to other papillomaviruses, we designed oligonucleotide primers and employed the polymerase chain reaction to recover HPV-1 cDNAs from plantar warts. Seven spliced RNA species were characterized, including three not previously detected, and the coding potentials of each were deduced. The most abundant viral mRNA encodes an E1i--E4 protein. One new species is predicted to encode the full-length E2 protein, and another can, theoretically, encode the E2-C or E1-M proteins, three products that regulate mRNA transcription and DNA replication. One RNA species originating from a novel HPV promoter in the upstream regulatory region has the potential to encode the minor capsid protein L2. A newly recognized E5a open reading frame (ORF) is contained in all mRNAs that are polyadenylated at the E-region poly(A) site and also in a putative L2 mRNA. Three distinct species, two of which are derived from the upstream regulatory region promoter, have the potential to encode the L1 protein; the third species also contains the entire coding region of the E1i--E4 protein 5' to the L1 ORF. Both the E1i--E4 mRNA and the potentially bicistronic L1 mRNA are derived from a promoter located in the E7 ORF. We uncovered no evidence of alternatively spliced mRNAs that could account for the multiple, abundant E4 proteins in plantar warts, suggesting that posttranslational modification is mainly responsible for the observed protein heterogeneity.

Base Sequence↗
PubMed1990

Infectious cycle of human papillomavirus type 11 in human foreskin xenografts in nude mice.

We have performed the first molecular analysis of a time course of infection by a papillomavirus. The Hershey isolate of the human papillomavirus type 11 was used to infect human foreskin tissues, which were then implanted under the renal capsules of nude mice. The xenografts were recovered every 2 weeks for 14 weeks, fixed in formalin, and embedded in paraffin. Four-micrometer serial sections were examined by light microscopy for morphological changes, by immunocytochemistry for virion antigen production, and by in situ hybridization with 3H-labeled RNA probes for viral DNA replication and expression of the major mRNA species. After a lag period, probes spanning the E4 and E5 open reading frames, which are present in all E region viral mRNAs, generated the first detectable signals at week 4. Signals of other E region probes were minimally detected at week 6. Between weeks 6 and 8, there was an abrupt change in the implant such that cellular proliferation, viral DNA replication, and E and L region mRNA transcription were robust and reached a plateau. By weeks 10 to 12, the experimental condylomata were morphologically and histologically indistinguishable from naturally occurring condylomata acuminata. These findings suggest that cellular hyperproliferation and the morphologic features of condylomata are direct results of viral genetic activities. Unlike other DNA viruses, the E region transcripts increased with cell age and cellular differentiation and persisted throughout the entire experiment. In particular, the mRNA encoding the E1iE4 and perhaps E5 proteins remained overwhelmingly abundant. In contrast, viral DNA replication, L region mRNA synthesis, and virion antigen production were restricted to the most differentiated, superficial cells.

Animals↗
PubMed1989

Papillomavirus-associated inductions of cellular proteins in mouse C127 cells: correlation with the presence of open reading frame E2.

Bovine papillomavirus type 1 (BPV-1) readily transforms mouse C127 cells, conferring the ability to grow in soft agar and to form tumors in athymic (nu/nu) mice. Electrophoresis of total cellular proteins from these BPV-transformed lines on ultra-high resolution, giant two-dimensional gels displays the presence of novel, papillomavirus-related protein phenotypes. Analysis of the established BPV-1-transformed C127 cell lines, ID13 and ID14, reveals a set of six proteins which are either absent or synthesized at extremely low levels in the parental cell line. One of these proteins is also present in v-ras-transformed C127 cells, but none of the others are found in cells transformed by a variety of viral oncogenes, including the polyomavirus middle T, v-mos, or v-fes. The genome of BPV-1 contains two separate open reading frames (ORFs), E5 and E6, which can act independently to transform C127 cells. In addition, trans-activator and repressor proteins encoded respectively by the full-length and carboxy-terminal E2 ORF regulate the level of expression of other BPV-1 genes. We examined 34 cell lines transformed by intact and subgenomic recombinant DNAs of BPV-1. Cells harboring BPV-1 DNAs engineered to eliminate the expression of ORFs E4, E5, E6, or E7 display five of the PV-associated proteins, but these proteins are not seen in lines lacking the full E2 ORF. Moreover, G418-selected nontransformed cells expressing E2 cDNA from an SV40 promoter exhibit these proteins at high levels. Surprisingly, these proteins are also present in cells containing BPV-1 DNAs with amino-terminal E2 deletions, suggesting that these PV-associated proteins represent novel cellular responses to a factor encoded within the E2-C gene region.

Animals↗
PubMed1989

Differentiation-linked human papillomavirus types 6 and 11 transcription in genital condylomata revealed by in situ hybridization with message-specific RNA probes.

Human papillomaviruses (HPVs) infect specific human epithelial tissues. Because viral propagation in cultured cells has not been achieved, studies of HPV genetic activities have been difficult and rely largely on analyses of patient specimens by conventional biochemical methods. HPV type 6 and type 11 infections often result in genital warts (condylomata acuminata). Structural mapping of RNAs from such warts reveals that they use alternative promoters, splice sites, and polyadenylation sites to produce complex families of overlapping mRNAs that span multiple open reading frames. Based on the mRNA structures, we have developed message-specific subgenomic clones of HPV-6 and HPV-11 in pGEM vectors. Tritium-labeled, single-stranded RNA probes were synthesized in vitro and applied to serial thin sections of patient specimens for in situ hybridization. Our results reveal the qualitative and quantitative transcription patterns of different viral messages in relationship to one another, to viral DNA replication, and to cellular differentiation. The viral "E region" is transcribed before the onset of vegetative DNA replication and continues to be expressed in increasing amounts in the maturing epithelium. Even in mature epithelia, E region messengers are far more abundant than "L region" mRNAs. The L region messages encoding capsid proteins are truly late in that they appear concomitant with or after the onset of vegetative viral DNA replication and are only present in the superficial strata of the epithelium, which contain the oldest and most differentiated keratinocytes. Abundant intron material derived from processing E region transcripts accumulates in the nuclei. Strictly nuclear signals from the L region transcripts in the midepithelium suggest that regulation of their expression is at the level of transcription elongation.

Condylomata Acuminata↗
PubMed1989

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.

Base Sequence↗
PubMed1989

Characterization of rare human papillomavirus type 11 mRNAs coding for regulatory and structural proteins, using the polymerase chain reaction.

Certain human papillomavirus (HPV) types cause warts, dysplasias, and carcinomas of the ano-genital and oral mucosa. Because of the inability to propagate HPVs in cultured cells, the paucity of viral mRNAs in human lesions, and the complexity of alternatively spliced transcripts derived from different promoters, it has not been possible to ascertain the exact structures of the majority of the mRNA species and the proteins encoded. We have adapted the recently developed polymerase chain reaction to amplify cDNAs of rare, type 11 HPV mRNAs isolated from a productively infected human foreskin xenograft in an athymic mouse. The oligonucleotide primers were designed to flank each of the mRNA splice sites previously mapped by electron microscopic analysis of heteroduplexes formed between cloned HPV-11 DNA and viral mRNAs isolated from genital warts. The splice junctions were determined by direct sequencing of the PCR-amplified cDNA products or after the cDNA was cloned into a plasmid vector. We provide the first direct evidence for the existence of rare mRNAs with the potential to encode regulatory proteins that have been hypothesized to exist for HPVs. Depending on the lengths of the upstream exons, the translation frame used and the possibility of internal reinitiation during translation, one pair of mRNAs with the same splice junction could encode the viral DNA copy number modulating protein E1-M, the enhancer repression protein E2-C, or both. A second pair of mRNAs, also with identical splice junctions, encode the enhancer-regulating protein E2; the longer of the two could also encode, in its 5' exon, either or both of the E6 and E7 proteins. Finally, we demonstrate that the doubly spliced late message for the major virion capsid protein L1 also contains the entire coding region for the early E1 E4 protein in the first two exons, with the initiation codon for the L1 protein located precisely at the splice acceptor of the third exon. The potential of this late mRNA to encode both the E1 E4 protein and the capsid protein could contribute to the preponderance of the E4 protein in the lesion.

Base Sequence↗
PubMed1989

Characterization of an HPV type 11 isolate propagated in human foreskin implants in nude mice.

Human papillomavirus type 11-Hershey (HPV-11-H) from an extract of genital warts has been serially passaged by infecting human foreskin chips that are then implanted in athymic mice (J. W. Kreider, M. K. Howett, A. E. Leure-Dupree, R. J. Zaino, and J. A. Weber (1987), J. Virol. 61, 590). Subsequent attempts to propagate HPVs present in other human lesions have not been successful. In an effort to identify the basis for the seemingly unique ability of HPV-11-H to propagate in the xenografts, we carried out extensive physical and functional characterizations of the cloned DNA, including restriction digestions, DNA sequencing of transcriptional control regions, and E2 trans-activation of the upstream regulatory region. We uncovered no significant differences compared to the prototype HPV-11 (K. Dartmann, E. Schwarz, L. Gissmann, and H. zur Hausen (1986) Virology 151, 124; H. Hirochika, T. R. Broker, and L. T. Chow (1987) J. Virol. 61, 2599). Moreover, viral enhancer assays indicated that the observed stimulatory effect of pregnancy on condylomata and of estradiol on experimental cysts is likely an indirect one and could not be attributed to up-regulation of the HPV-11 transcriptional enhancer.

Animals↗
PubMed1989

Identification of a novel constitutive enhancer element and an associated binding protein: implications for human papillomavirus type 11 enhancer regulation.

The human papillomavirus type 11 enhancer, when linked to the minimal simian virus 40 early promoter, has been dissected into two domains in monkey kidney CV-1 cells, one being constitutive (designated CEI) and the other inducible by trans-acting E2 proteins encoded by homologous and heterologous papillomaviruses (H. Hirochika, T.R. Broker, and L.T. Chow, J. Virol. 61:2599-2606, 1987; H. Hirochika, R. Hirochika, T.R. Broker, and L.T. Chow, Genes Dev. 2:54-67, 1988). We have demonstrated that the natural promoter regulated by this enhancer is located immediately upstream of the E6 open reading frame (the E6 promoter). We have mapped the cap site to nucleotide 99 by RNase protection. We further demonstrate a second constitutive enhancer element, CEII, which is required for transcription from the E6 promoter in the human cervical carcinoma cell lines C-33A and HeLa but not in CV-1 cells. By deletion mapping, we have localized this cell type-specific domain to 71 base pairs by using chloramphenicol acetyltransferase assays. Deletion of either CEI or CEII dramatically decreased the constitutive activity of the enhancer and the E6 promoter, whereas multimerization of either domain in the absence of the other could independently restore expression. Furthermore, when either of these elements was deleted, the full-length E2 protein of human papillomavirus type 11 abolished the remaining basal E6 promoter activity, demonstrating for the first time that the enhancer-activating E2 protein of human papillomaviruses can also function as a transcriptional repressor for the homologous E6 viral promoter. The presence of multiple copies of each element in tandem overcomes the repression by the E2 protein. The effects of CEII are at the level of transcription, without changing the cap site. By gel shift assay, we have shown that a protein present in nuclear extracts of C-33A and HeLa cervical carcinoma cells binds to the newly identified constitutive element II. This protein did not bind the simian virus 40 enhancer, nor did it bind to the enhancer region of many other papillomaviruses tested. UV cross-linking experiments revealed major 44-kilodalton and minor 34-kilodalton proteins that bound specifically to CEII. These two proteins are either related or bind to CEII with high cooperativity. We conclude that transcriptional activities directed by the enhancer and E6 promoter reflect an intricate balance among viral and cellular factors. We present a model on the regulation of the E6 promoter by host and viral transcription factors.

Animals↗
PubMed1988

Functional mapping of the human papillomavirus type 11 transcriptional enhancer and its interaction with the trans-acting E2 proteins.

The transcriptional enhancer sequences of the papillomaviruses are regulated by trans-acting factors encoded by the viral E2 open reading frame. We have performed detailed functional and physical analyses of the enhancer of the human papillomavirus type 11 (HPV-11). Using the chloramphenicol acetyltransferase (CAT) assay in transiently transfected monkey CV-1 cells, the enhancer region has been localized to a 270-bp tract immediately preceding the E6 open reading frame, and it consists of two functional components. The first is a constitutive enhancer containing sequences homologous to the GT-, Sph-, and P-motifs found in the SV40 and polyomavirus enhancers; others resemble the recognition sequence for CTF (NF-1), a factor which stimulates transcription of certain eukaryotic genes and replication of adenovirus DNA. The second component is an inducible enhancer with a consensus sequence ACCN6GGT responsive to the E2 protein encoded by papillomaviruses. Tandem copies of portions of the constitutive enhancer function as an E2-independent enhancer, whereas multiple copies of HPV-11 DNA restriction fragments or synthetic oligonucleotides containing the E2-responsive sequence (E2-RS) act as an enhancer in the presence of the E2 protein encoded by HPV-1, HPV-11, or bovine papillomavirus type 1 (BPV-1). The inducible activity is lost when mutations are introduced into the E2-RS or when a mutant palindromic sequence is substituted. We have also expressed the E2 proteins of HPV-1, HPV-11, and BPV-1 in Escherichia coli and studied their physical interactions with the E2-responsive sequence in vitro. Filter-binding analyses with crude Escherichia coli lysates show that the E2 proteins bind to the E2-RS, but not to mutated motifs, with an affinity proportional to the copy number. These E2 proteins have been purified to near-homogeneity by sequence-specific DNA affinity chromatography using the synthetic E2-RS as a ligand. The purified proteins protect a DNA segment containing the E2-RS and several flanking nucleotides in pancreatic DNase I footprinting analyses. Based on these results, we conclude that E2 proteins activate the enhancer by binding directly to the E2-RS and interacting with other transcriptional factors and that the sequence ACCN6GGT is both necessary and sufficient for the E2 protein binding in vitro and for activation of RNA transcription in vivo.

Chromosome Deletion↗
PubMed1988

Regulation of human papillomavirus type 11 enhancer and E6 promoter by activating and repressing proteins from the E2 open reading frame: functional and biochemical studies.

E2-C, a protein consisting mainly of the carboxy-terminal 45% of the human papillomavirus type 11 (HPV-11) E2 protein, was expressed from the Rous sarcoma virus long terminal repeat in mammalian cells. It competitively repressed the stimulatory action of the full-length E2 protein on the HPV-11 enhancer located in the upstream regulatory region, as assayed by the expression of a reporter gene from the simian virus 40 (SV40) early promoter in transiently transfected monkey CV-1 cells. A mutation in the initiation codon for E2-C protein eliminated repression. In the human cervical carcinoma cell line C-33A, which apparently lacks endogenous HPV DNA, the HPV-11 enhancer-SV40 promoter and the HPV-11 enhancer in its normal association with the E6 promoter had high constitutive activity. In these cells, E2 proteins had little or no stimulatory effect on the transcriptional activity of the HPV-11 enhancer-SV40 promoter. In contrast, the HPV-11 enhancer-E6 promoter was stimulated by the HPV-11 E2 protein but repressed by the bovine papillomavirus type 1 E2 protein, an effect due either to a quantitative difference in E2 expression levels or to a qualitative difference in the trans-activating abilities of the two E2 proteins. In this cell line, the HPV-11 E2-C protein suppressed both the constitutive activity and the HPV-11 E2 trans activation. The E2-C protein was also produced from an expression vector in Escherichia coli. The E2-C protein present in crude E. coli lysates or purified by DNA affinity chromatography associated in vitro with a specific sequence, ACCN6GGT, in filter-binding assays. Moreover, the protein generated DNase I footprints spanning this motif identical to those of bacterially expressed full-length E2 proteins. This DNA sequence motif is necessary and sufficient for E2 binding in vitro and enhancer trans activation in vivo (H. Hirochika, R. Hirochika, T. R. Broker, and L. T. Chow, Genes Dev. 2:54-67, 1988). Mutations in this sequence that abolished interactions with E2 also precluded binding to the E2-C protein. These data strongly suggest that the full-length E2 protein consists of two functional domains: the amino-terminal half for trans activation and the carboxy-terminal half for DNA binding. The mechanism by which E2-C represses E2-dependent enhancer activity most likely involves competition with E2 for binding to a common transcriptional regulatory site.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning, Molecular↗
PubMed1987

A human papilloma virus type 11 transcript encoding an E1--E4 protein.

The human papilloma virus (HPV) associated with a genital wart (condyloma acuminatum) was determined to be type 11. The majority of the viral DNA molecules were monomeric circles present in the cells at high copy number, as demonstrated by one- and two-dimensional agarose gell electrophoretic separation followed by Southern blot analysis. A cDNA library in phage lambda gt11 was constructed from poly(A)-selected mRNA recovered from the tissue. Recombinant clones corresponding to the most abundant 1.2-kb viral mRNA species detected by Northern blot hybridization and by electron microscopic analysis of R loops were isolated and their nucleotide sequence was determined. Comparison to the prototype HPV-11 DNA sequence revealed that this message consisted of two exons. The promotor-proximal exon spanned nucleotides 716 through 847 and the distal exon included nucleotides 3325 through 4390 or 4392. The mRNAs were alternatively polyadenylated after either of these latter two sites, in both cases following a G and preceding a U residue. Fourteen or sixteen bases upstream from the poly(A) was the hexanucleotide AGUAAA, which apparently serves as the signal for cleavage and polyadenylation of the nascent message. The splice donor and acceptor sites conformed to the usual /GU. . .AG/pattern. The exons joined open reading frame (ORF) E1, which contributed the initiation codon and four additional triplets, to ORF E4, which specified 85 amino acids to encode a protein of 10,022 Da. The cDNA also contained the ORFs E5a and E5b toward the 3' end. The complete sequence of the cDNA revealed three single-base changes from the prototype HPV-11, two resulting in altered amino acids in E4. Neither affects the coding potential of the overlapping E2 ORF. The function of the E1--E4 protein is unknown.

Base Sequence↗