The ability of human papillomavirus E6 proteins to target p53 for degradation in vivo correlates with their ability to abrogate actinomycin D-induced growth arrest

Functional p53 protein is associated with the ability of cells to arrest in G1 after DNA damage. The E6 protein of cancer-associated human papillomavirus type 16 (HPV-16) binds to p53 and targets its degradation through the ubiquitin pathway. To determine whether the ability of E6 to interact with p53 leads to a disruption of cell cycle control, mutated E6 proteins were tested for p53 binding and p53 degradation targeting in vitro, the ability to reduce intracellular p53 levels in vivo, and the ability to abrogate actinomycin D-induced growth arrest in human keratinocytes. Mutations scattered throughout the amino terminus, either zinc finger or the central region but not the carboxy terminus, severely reduced the ability of E6 to interact with p53. Expression of HPV-16 E6 or mutated E6 proteins that bound and targeted p53 for degradation in vitro sharply reduced the level of intracellular p53 induced by actinomycin D in human keratinocytes. A perfect correlation between the ability of E6 proteins to reduce the level of intracellular p53 and their ability to block actinomycin D-induced cellular growth arrest was observed. These results suggest that interaction with p53 is important for the ability of HPV E6 proteins to circumvent growth arrest.     

Immunoexpression of mutant p53 and human papillomavirus related E6 oncoprotein in anal malignancies

Immunohistochemical expression of mutant p53 protein and human papillomavirus (HPV) 16 and 18 related E6 oncoprotein was studied in 36 biopsy proved anal cancers. Mutant p53 was detected in 61.1% cases. HPV 16 and 18 E6 protein was expressed in 22.2% cases, all of which were squamous cell carcinomas. Coexpression of both mutant p53 and E6 protein was found in only 5 cases (13.8%). In HPV 16/18 positive anal tumors, the degradation of p53 is accelerated by viral E6 oncoprotein. In HPV negative tumors, however, other mutagenic factors probably play a role in carcinogenesis.     

In vitro generation and type-specific neutralization of a human papillomavirus type 16 virion pseudotype

We report a system for generating infectious papillomaviruses in vitro that facilitates the analysis of papillomavirus assembly, infectivity, and serologic relatedness. Cultured hamster BPHE-1 cells harboring autonomously replicating bovine papillomavirus type 1 (BPV1) genomes were infected with recombinant Semliki Forest viruses that express the structural proteins of BPV1. When plated on C127 cells, extracts from cells expressing L1 and L2 together induced numerous transformed foci that could be specifically prevented by BPV neutralizing antibodies, demonstrating that BPV infection was responsible for the focal transformation. Extracts from BPHE-1 cells expressing L1 or L2 separately were not infectious. Although Semliki Forest virus-expressed L1 self-assembled into virus-like particles (VLPs), viral DNA was detected in particles only when L2 was coexpressed with L1, indicating that genome encapsidation requires L2. Expression of human papillomavirus type 16 (HPV16) L1 and L2 together in BPHE-1 cells also yielded infectious virus. These pseudotyped virions were neutralized by antiserum to HPV16 VLPs derived from European (114/K) or African (Z-1194) HPV16 variants but not by antisera to BPV VLPs, to a poorly assembling mutant HPV16 L1 protein, or to VLPs of closely related genital HPV types. Extracts from BPHE-1 cells coexpressing BPV L1 and HPV16 L2 or HPV16 L1 and BPV L2 were not infectious. We conclude that (i) mouse C127 cells express the cell surface receptor for HPV16 and are able to uncoat HPV16 capsids; (ii) if a papillomavirus DNA packaging signal exists, then it is conserved between the BPV and HPV16 genomes; (iii) functional L1-L2 interaction exhibits type specificity; and (iv) protection by HPV virus-like particle vaccines is likely to be type specific.     

Genetic definition of a new bovine papillomavirus type 1 open reading frame, E5B, that encodes a hydrophobic protein involved in altering host-cell protein processing

We have previously observed that bovine papillomavirus type 1 (BPV-1) induces the appearance of five cellular proteins in C127 mouse fibroblasts, four of which appear to arise by altered processing of resident endoplasmic reticulum proteins. Studies of various cell lines revealed that expression of the 3' end of the BPV early region was sufficient for induction of these changes. To identify the BPV gene responsible, we have utilized the simian virus 40 (SV40)/BPV-1 recombinant virus Pava-1, which expresses the 3' end of the BPV early region behind an SV40 early promoter. C127 cells infected with Pava-1 for 48 h show the expected BPV-associated alterations, as do cells infected with Pava constructs mutated in the E5 or E2 genes. However, a mutation in the start codon of a previously ignored open reading frame extending from nucleotides 4013 to 4170 (E5B) eliminated the BPV-associated changes. Similar results were obtained with COS cells infected with the Pava mutants and C127 cells transformed by full-length mutated BPV. Despite its influence on the processing of cellular endoplasmic reticulum proteins, this mutation in E5B did not alter BPV-transforming efficiency or the ability of transformants to form colonies in soft agar. The E5B open reading frame encodes a hydrophobic 52-amino-acid polypeptide that shares structural similarities with HPV6 E5A and HPV16 E5. Speculations on a role for E5B in the viral life cycle are discussed.     

Defining the minimal requirements for papilloma viral E6-mediated inhibition of human p53 activity in fission yeast

The majority of human anogenital carcinomas show evidence of papillomavirus infection. To facilitate viral replication, viruses disable key cellular responses which would otherwise precipitate cell suicide. An obligate factor in one such response is the p53 tumour suppressor protein. p53 gene mutation is an infrequent event in anogenital cancer, apparently due to the action of HPV E6 protein, which inhibits wild-type p53 function by stimulating the degradation of p53 protein. p53 is required for the apoptotic response that is triggered in untransformed cells following inappropriate cell-cycling. E6 directed inhibition of p53 function thus facilitates the survival of transformed cells. We have developed a genetically tractable model that reports E6 protein-mediated human p53 inactivation in the fission yeast Schizosaccharomyces pombe. Functional dissection of the requirements for E6 directed inhibition in this system reveal an absolute requirement for the presence of both E6 protein and the human E3 ubiquitin ligase, E6-AP. Using a defined set of E6 mutants we show that degradation of p53 protein rather than E6/p53 association is likely required for E6-mediated inhibition. This S. pombe based system represents a candidate screen for novel antiviral agents that act by disrupting the E6/E6-AP/p53 interaction.     

Transactivation of the human cdc2 promoter by adenovirus E1A in cycling cells is mediated by induction of a 110-kDa CCAAT-box-binding factor

Cyclin-dependent protein kinases (Cdks) are key regulatory proteins of the eukaryotic cell cycle. Cdc2 is expressed in late G1/S phase and functions in the G2 to M phase transition. Adenovirus E1A proteins are known to induce the expression of p34cdc2 and DNA synthesis in normal quiescent cells. In this study, mutational analysis of the human cdc2 promoter revealed that transactivation of the promoter by the E1A proteins in cycling cells is mediated through the two CCAAT box binding motifs. A 110-kDa protein (CBF/cdc2) was identified in nuclear extracts from monkey kidney (CV-1) cells stably expressing E1A as well as from adenovirus-transformed human 293 cells. Further, we show that this EIA-inducible CBF/cdc2 is related to the CBF which was shown to activate the heat shock protein 70 promoter. Analyses of the functional domain(s) of E1A required for the induction of the CBF and transactivation of the cdc2 promoter in these conditions revealed that E1A mutants which were defective in binding the pRB family of proteins or the cellular p300 protein were still active in assays measuring the induction of the CBF and transactivation of the cdc2 promoter, albeit with reduced efficiencies. But the E1A mutant which lost both functional domains was inactive in these assays. These results suggest that E1A has redundant functional domains for the induction of the 110-kDa CBF and activation of human cdc2 gene expression.     

The polyproline region of p53 is required to activate apoptosis but not growth arrest

p53 is a pivotal regulator of apoptosis but its mechanism of action is obscure. We report that the polyproline (PP) region located between p53's transactivation and DNA binding domains is necessary to induce apoptosis but not cell growth arrest. The PP region was dispensable for DNA binding, inhibition of SAOS-2 tumor cell growth, suppression of E1A + RAS cell transformation, and cell cycle inhibition. A temperature-sensitive dominant inhibitory p53 mutant lacking PP (p53ts deltaPP) retained its ability to cooperate with adenovirus E1A in transformation of primary BRK cells. However, while activation of wt p53 induced apoptosis in E1A + p53ts-transformed cells, activation of p53 deltaPP induced cell cycle arrest but not apoptosis in E1A + p53ts deltaPP-transformed cells. Similarly, PP deletion abolished apoptosis in LoVo colon carcinoma cells, which are killed by wt p53 overexpression. Transactivation was largely unaffected by PP deletion. Significantly, BAX induction was intact, indicating that additional events are required for p53 to induce apoptosis. As a recently described site for familial mutation in at least one breast cancer family, the PP region represents a domain that may be altered in human tumors. We concluded that p53's ability to induce apoptosis is dispensable for inhibiting cell growth and transformation and that the PP region plays a crucial role in apoptotic signaling.     

Elevated wild-type p53 protein levels in human epithelial cell lines immortalized by the human papillomavirus type 16 E7 gene

The role tumor suppressors p53 and retinoblastoma (RB) play in the transformation process has become central to understanding the pathogenesis of DNA tumor viruses. The two oncoproteins of human papillomavirus (HPV)-16, E6 and E7, bind to p53 and RB, respectively, thus inactivating the function of these tumor suppressor genes. Immortalization of primary human foreskin epithelial cells by HPV requires expression of the E7 protein, and the E6 protein greatly enhances the immortalization frequency. Two of three cell lines immortalized by the HPV-16 E7 oncoprotein expressed wild-type p53 and only one of the three cell lines had acquired a p53 mutation and loss of heterozygosity at 17p during the immortalization process. All three E7-immortalized lines contained higher steady-state levels of p53 protein. Mutation of the p53 gene is not required for immortalization in the absence of the HPV-16 E6 inactivation of the p53 protein, and 16E7 expression leads to the stabilization of wild-type p53.