Are transforming properties of the bovine papillomavirus E5 protein shared by E5 from high-risk human papillomavirus type 16?

The E5 proteins of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 16 (HPV-16) are small (44-83 amino acids), hydrophobic polypeptides that localize to membranes of the Golgi apparatus and endoplasmic reticulum, respectively. While the oncogenic properties of BPV-1 E5 have been characterized in detail, less is known about HPV-16 E5 due to its low expression in mammalian cells. Using codon-optimized HPV-16 E5 DNA, we have generated stable fibroblast cell lines that express equivalent levels of epitope-tagged BPV-1 and HPV-16 E5 proteins. In contrast to BPV-1 E5, HPV-16 E5 does not activate growth factor receptors, phosphoinositide 3-kinase or c-Src, and fails to induce focus formation, although it does promote anchorage-independent growth in soft agar. These variant activities are apparently unrelated to differences in intracellular localization of the E5 proteins since retargeting HPV-16 E5 to the Golgi apparatus does not induce focus formation.     

Cloning and molecular characterization of an oral papillomavirus of domestic rabbits

DNA obtained from New Zealand white rabbit oral papillomas was analyzed for the presence of papillomavirus DNA. The viral genome was cloned as three separate subclones, which were each mapped and oriented with respect to one another. Comparisons with other papillomavirus DNAs by Southern blot hybridization under various conditions of stringency revealed a strong area of conservation among the DNAs of the rabbit oral papillomavirus (ROPV) and CRPV, HPV-1a, HPV-16, and BPV-5, but not with 12 other papillomavirus DNAs. This region, which spans the junction of the presumptive E2 and L2 open reading frames of ROPV, was sequenced and compared to other known papillomavirus sequences. These analyses revealed a high degree of DNA homology in the C-terminal E2 and N-terminal L2 regions between ROPV and both HPV-1a and CRPV. The homology with HPV-16 was limited to the L2 open reading frame. The predicted amino acid sequences of each region were also compared and bore out the same conclusions. In addition, no E5 open reading frame was detected in the ROPV sequence.     

Competitive binding to a charged leucine motif represses transformation by a papillomavirus E6 oncoprotein

E6 oncoproteins from HPV-16 and bovine papillomavirus type 1 (BPV-1) bind to similar leucine-rich peptides termed charged leucine motifs found on the cellular focal adhesion protein paxillin and the E3 ubiquitin ligase E6AP. BPV-1 E6 (BE6) mutants that do not bind to paxillin are defective at inducing cellular transformation. It is possible, however, that BE6 mutants that do not bind paxillin are defective for transformation for an unrelated reason than the ability to bind to charged leucine motifs. To address the role of BE6 interaction with charged leucine motifs, we fused a BE6-binding charged leucine motif to the amino terminus of BE6, thereby creating an autoinhibitory binding domain. We found that the fusion protein failed to bind to paxillin or transform murine C127 cells. Mutation of the amino terminal binding motif in the fusion protein restored both interaction with paxillin and transformation. This demonstrates that BE6 transformation requires binding to charged leucine motifs on particular cellular proteins and that transformation by papillomavirus oncoproteins can be repressed by competitive interactions with charged leucine motifs.     

The E5 Protein of HPV-6, but Not HPV-16, Associates Efficiently with Cellular Growth Factor Receptors

The transforming activity of the prototype E5 protein of bovine papillomavirus type 1 (BPV-1) is associated with its binding to, and activation of, both the platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. The E5 proteins of human papillomavirus types 6 and 16 (HPV-6, HPV-16) also transform rodent cells in the presence of the EGF receptor. In this study we examined whether epitope-tagged HPV E5 proteins could associate with three different tyrosine kinase-containing growth factor receptors: the EGF receptor, the erbB2 receptor, and the PDGF receptor. The HPV-6 E5 protein was found to associate efficiently with all three of these growth factor receptors, while the HPV-16 E5 protein did not. These findings suggest either that the in vitro transforming activities of HPV-6 and HPV-16 E5 proteins involve a similar mechanism unrelated to receptor binding (e.g., binding to the 16-kDa membrane pore protein) or that they proceed along distinct pathways, with receptor binding being important for HPV-6. Regardless of the ultimate mechanisms, the differences between the HPV-6 and HPV-16 E5 proteins in binding to growth factor receptors may potentially contribute to the distinctive morphologies of their respective neoplastic lesions.     

Properties of bovine papillomavirus E1 mutants.

Ostensibly comparable mutants of bovine papillomavirus type 1 (BPV-1) affecting the E1 open reading frame that were constructed in several laboratories have been reported to exhibit either reduced or increased transformation efficiencies in established mouse cell lines relative to wild-type BPV-1 DNA. To resolve these discrepancies, we have reexamined many of the mutants in mouse C127 cells by using focus formation assays. Our primary conclusions are that all E1 mutants tested consistently generated reduced numbers of transformants and that the reduced transformation was not due to cell toxicity associated with E1 mutations, as had been proposed. Our results can best be explained by the inability of the E1 mutants to replicate extrachromosomally, therefore leading to a rapid loss of the BPV-1 DNA and consequently, reduced transformation. In support of this hypothesis, we demonstrated that the human papillomavirus type 11 E1 protein was able to suppress BPV-1 transformation, probably because of interference with BPV-1 replication. Therefore, we attribute the phenotypic disparities reported by the various laboratories to still undefined differences in assay conditions.     

Conservation of HPV-16 E6/E7 ORF sequences in a cervical carcinoma.

A cervical carcinoma that contained human papillomavirus (HPV)-16 homologous DNA was analyzed. Each tumor cell genome contained a single, incomplete copy of HPV-16 DNA. The E6 and E7 open reading frames (ORFs) were completely conserved relative to other published HPV-16 sequences. Much of the non-coding region (NCR) was free of base changes, including complete conservation of several regulatory elements. Multiple mutations were identified in the remaining integrated HPV-16 DNA, which was composed of parts of the L1 and E1 ORFs. The extraordinary conservation of the E6/E7 DNA sequence, as compared with other regions of the integrated HPV-16 DNA, supports the role of E6/E7 in tumorigenesis.     

Down-regulation of MHC class I is a property common to papillomavirus E5 proteins

The E5 protein family of papillomaviruses comprises small hydrophobic proteins which are associated with the cell endomembrane compartments. The functions of the E5 proteins, particularly those of HPV, are still far from clear. We have reported that the E5 proteins of BPV-1, BPV-4, HPV-16 and HPV-6 down-regulate MHC class I, potentially helping the virus evade the host immune response. Others have described MHC class I down-regulation by HPV-2 E5. We report here that another E5 protein, HPV-83 E5, likewise down-regulates MHC class I and propose that interference with expression, assembly and/or transport of MHC class I is a common property of all E5 proteins evolved by the virus to circumvent host immunosurveillance and thus establish productive infection.     

In vitro transformation and molecular characterization of Colobus monkey venereal papillomavirus DNA

The DNA of a monkey papillomavirus (CgPV-1), originally isolated from a penile lesion on a Colobus monkey was cloned into the EcoRI site of the pUC18 vector and characterized. Using a variety of restriction enzymes a physical map of the DNA was constructed. Cross-hybridization with a variety of animal and human papillomaviruses under high (Tm-22 degrees C) and low (Tm-40 degrees C) stringency conditions indicated various degrees of homology. CgPV-1 showed higher homology with HPVs than it did with any other animal papillomaviruses tested. DNA similarities with the human papillomaviruses HPV-16 and HPV-18 that are frequently associated with cervical cancer, were manifested by extensive cross-hybridization under stringent conditions. Functional alignment of the genomic map of CgPV-1 with that of HPV-16 was carried out by determination of homology between specific restriction fragments of the two viral genomes in cross-hybridization analyses. This alignment was refined by sequencing two regions of approximately 200 bp of the CgPV-1 DNA, and aligning them by computer with their homologous HPV-16 counterparts. CgPV-1 DNA in its pUC18 vector, transformed NIH 3T3 cells with roughly the same efficiency as BPV-1, as determined by the number of transformed foci generated per ug of DNA. The data presented indicate that the state of the CgPV-1 viral DNA in these transformed cells is integrated and partially deleted, not unlike the genomes of HPV-16 and HPV-18 characterized in cell lines derived from cervical cancers.     

DNA sequence of the HPV-16 E5 ORF and the structural conservation of its encoded protein

Infection of cervical epithelium by human papillomavirus type 16 (HPV-16) appears to be closely associated with the development of cervical dysplasia and carcinoma. By inference from genetic and biochemical studies of the bovine papillomavirus, the E5 ORF of the human papillomaviruses is anticipated to encode a "transforming" protein. In an effort to compare the E5 ORF of HPV-16 with other human papillomaviruses and bovine papillomavirus, we sequenced this region from a new isolate of HPV-16 which was derived from extrachromosomal viral DNA within a premalignant cervical lesion (cervical intraepithelial neoplasia, grade III, or CIN III). In addition, we also sequenced the original isolate of HPV-16 (derived from integrated viral DNA by Durst et al. [Proc. Natl. Acad. Sci. USA 80, 3812-3815 (1983)] and sequenced by Seedorf et al. [Virology 145, 181-185 (1985)]. Both HPV-16 isolates contained an additional nucleotide (T) at bp 3906. This nucleotide addition caused a frameshift in the E5 ORF such that it now contains an initiation codon at bp 3849; the frameshift also alters the predicted E5 NH2 terminus but retains the original COOH half of the protein. E5 proteins encoded by several HPVs which infect the genital region (e.g., types 6, 11, 16, 18, 33) exhibit a conserved trimodal hydrophobic structure, but not a conserved amino acid sequence.     

DNA replication specificity and functional E2 interaction of the E1 proteins of human papillomavirus types 1a and 18 are determined by their carboxyl-terminal halves

Replication of most papillomaviruses (PVs) requires the viral-encoded E1 and E2 proteins that bind to the origin of replication (ori) containing the E1- and E2-binding sites and help recruit host replication factors during the initiation of DNA replication. We studied the ability of heterologous E1 and E2 proteins to interact in vivo and support replication, using the human papillomavirus (HPV) types 1a and 18 as model systems. The E1 protein of HPV-1a in combination with HPV-18 E2 supported high-level replication of various ori plasmids. In contrast, the HPV-18 E1 protein interacted weakly with HPV-1a E2 during the replication of ori plasmids. We have previously shown that the E1 protein of HPV-1a alone is sufficient for replication of HPV-1a ori plasmids, whereas HPV-18 replication requires both the E1 and E2 proteins. However, in the latter case, E2-binding sites alone in the absence of the E1-binding site can function as the minimal ori. Based on the above observations, we generated hybrids between HPV-1a and HPV-18 E1 proteins in an effort to identify their "replication specificity" domains using a transient replication assay. These hybrids were also used to localize the domains in the E1 proteins that are involved in their functional interaction with the E2 protein during replication. Our results suggest that the "replication specificity" and functional E2 interaction domains of the HPV-1a and HPV-18 E1 proteins are located in their carboxyl-terminal halves.     

E6 and E7 oncoproteins from human papillomavirus type 16 induce activation of human transforming growth factor beta1 promoter throughout Sp1 recognition sequence

Human Papillomavirus (HPV) infection is the main etiologic agent of cervical cancer and HPV E6 and E7 oncogenes trans-regulate many cellular genes. An association between TGF-beta1 gene expression and cervical cancer development has been suggested; however, the mechanisms by which HPV influences TGF-beta1 expression remain unclear. In the present study we analyzed the mechanism through which HPV-16 E6 and E7 oncoproteins regulate the TGF-beta1 promoter in cervical tumor cells. Our results showed that E6 and E7 increased TGF-beta1 promoter activity. Furthermore, we identified a specific DNA sequence motif in the TGF-beta1 core promoter that is responsible for trans-activation and that corresponds to the Sp1e-binding site associated with HPV-16 E6 and E7 oncoproteins. Mutational analysis showed that the Sp1e recognition site abolished the trans-activation caused by E6 and E7. These results suggest a physical interaction and functional cooperation between viral oncoproteins and cellular regulatory elements of the TGF-beta1 promoter, and may explain the contribution of HPV-16 to TGF-beta1 gene expression in cervical cancer.     

Genus specific features of bovine papillomavirus E6, E7, E5 and E8 proteins

Six bovine papillomavirus (BPV) types, BPV-1 to -6, have been classified in genera Delta-papillomavirus (BPV-1 and -2), Epsilon-papillomavirus (BPV-5) and Xi-papillomavirus (BPV-3, -4 and -6). In addition, 16 unclassified putative BPV types have been reported. In the present study, we characterized genus specific features of E6, E7, E5 (formerly E8) and E8 proteins of seven putative BPV types, BAPV-1, -2, -3, -4 and -10, BAA-5 and BPV-3c. These putative BPV types were classified in genera Epsilon- or Xi-papillomavirus. The E6 proteins of BPV and putative BPV types in Epsilon-papillomavirus showed high sequence similarities, and contained two typical zinc-binding domains. However, E7 proteins contained atypical zinc-binding domains, and lacked the canonical retinoblastoma tumor suppressor protein (pRB)-binding motif. BPV and putative BPV types in Xi-papillomavirus contained E5 or E8 open reading frame (ORF) in the E6 position. The E5 ORFs encoded proteins consist of 42-amino acid with a hydrophobic transmembrane and a hydrophilic C-terminal domain. But the E8 ORFs encoded protein which have two transmembrane domains. Our results demonstrated that E5, E8, E6, E7 proteins of the putative BPV types, which are presumably classified in genera Epsilon- or Xi-papillomavirus, retained the some genus specific features.     

Inhibition of CDK activity and PCNA-dependent DNA replication by p21 is blocked by interaction with the HPV-16 E7 oncoprotein

p21 inhibits cyclin-dependent kinase (CDK) activity and proliferating cell nuclear antigen (PCNA)-dependent DNA replication by binding to CDK/cyclin complexes and to PCNA through distinct domains. The human papillomavirus (HPV)-16 E7 oncoprotein (16E7) abrogated a DNA damage-induced cell cycle arrest in vivo, despite high levels of p21. Using cell lysates and purified proteins we show that 16E7 prevented p21 both from inhibiting CDK2/cyclin E activity and PCNA-dependent DNA replication, whereas the nononcogenic HPV-6 E7 had reduced effects. Inactivation of both inhibitory functions of p21 was attained through binding between 16E7 and sequences in the carboxy-terminal end of p21 that overlap with the PCNA-binding site and the second p21 cyclin-binding motif. These data imply that the carboxyl terminus of p21 simultaneously modulates both CDK activity and PCNA-dependent DNA replication and that a single protein, 16E7, can override this modulation to disrupt normal cell cycle control.