An association between human papillomavirus (HPV) infection and the development of cervical cancer was initially reported over 30 years ago, and today there is overwhelming evidence that certain subtypes of HPV are the causative agents of these malignancies. The p53 and retinoblastoma proteins are well-characterized targets of the HPV E6 and E7 oncoproteins, but recent studies have shown that the alteration of additional pathways are equally important for transformation. These additional factors are crucial regulators of cell cycle progression, telomere maintenance, apoptosis and chromosomal stability. Understanding how HPV oncoproteins modify these activities provides novel insights into the basic mechanisms of oncogenesis.

HUMAN PAPILLOMAVIRUSES AND CERVICAL CANCERHuman papillomaviruses (HPVs) are small, double-stranded DNA viruses that infect cutaneous and mucosal epithelial tissues of the anogenital tract, the hands, or the feet. A subset of HPV types are the causative agents of cervical cancer, since 99% of tumors are positive for HPV DNA (150). To date, over 100 different viral types have been identified, and about onethird of these infect epithelial cells in the genital tract. The viral types that infect the genital tract fall into two categories: high risk and low risk. The high-risk types are associated with the development of anogenital cancers including those of the cervix, while infections by the low-risk HPVs induce only benign genital warts. The high-risk types include HPV-16, HPV-18, HPV-31, HPV-33, and HPV-45, while the low-risk types are HPV-6 and HPV-11. HPVs that infect the genital tract are sexually transmitted, and it is estimated that about two-thirds of individuals who have sexual relations with an infected partner will themselves become infected. However, the majority of infections are subclinical (137). Infection by high-risk HPVs is not limited to the genital tract, since approximately 20% of cancers of the oropharynx contain DNA from these HPV types (61).Infection of the genital tract by HPVs can initially result in low-grade lesions termed dysplasias or cervical intraepithelial neoplasia grade I. These lesions exhibit only mildly altered patterns of differentiation, and many of them are cleared by the immune system in less than a year (62, 71). The mechanisms by which the cellular immune response clears HPV infections are still not clearly understood. Some of these lesions, however, are not cleared by the immune system and can persist for periods as long as several decades. Persistence of infection by high-risk HPV types is the greatest risk factor for development of genital malignancies such as squamous cell carcinoma or, less commonly, adenocarcinoma of the cervix (161). Cervical cancer is the second most prevalent cancer worldwide and is the fifth leading cause of cancer deaths in women (120,124). Approximately 470,000 new cases of cervical cancer are diagnosed yearly, with the mean age for the development of malignancy being 52 years (8, 124). Risk factors for tumor development include persistent infection with high-risk viral types, a large number of lifetime sexual partners, coinfection with human immunodeficiency virus, immunosuppression, and cigarette smoking (81). Most cases of cervical cancer are found outside of the United States and Western Europe. In the United States, the number of cases of cervical cancer has declined by over 80% in the last 50 years due to the implementation of the Pap smear as a diagnostic (137). While the number of cases has significantly decreased, approximately 10,000 women are diagnosed with cervical cancer and 5,000 die of this disease annually (120). HUMAN PAPILLOMAVIRUS LIFE CYCLEHPVs are nonenveloped viruses with icosahedral capsids that replicate their genomes wi...

Human papillomaviruses (HPV) are the causative agents of cervical cancers. The infectious HPV life cycle is closely linked to the differentiation state of the host epithelia, with viral genome amplification, late gene expression and virion production restricted to suprabasal cells. The E6 and E7 proteins provide an environment conducive to DNA synthesis upon differentiation, but little is known concerning the mechanisms that regulate productive viral genome amplification. Using keratinocytes that stably maintain HPV-31 episomes, and chemical inhibitors, we demonstrate that viral proteins activate the ATM DNA damage response in differentiating cells, as indicated by phosphorylation of CHK2, BRCA1 and NBS1. This activation is necessary for viral genome amplification, as well as for formation of viral replication foci. In contrast, inhibition of ATM kinase activity in undifferentiated keratinocytes had no effect on the stable maintenance of viral genomes. Previous studies have shown that HPVs induce low levels of caspase 3/7 activation upon differentiation and that this is important for cleavage of the E1 replication protein and genome amplification. Our studies demonstrate that caspase cleavage is induced upon differentiation of HPV positive cells through the action of the DNA damage protein kinase CHK2, which may be activated as a result of E7 binding to the ATM kinase. These findings identify a major regulatory mechanism responsible for productive HPV replication in differentiating cells. Our results have potential implications for the development of anti-viral therapies to treat HPV infections.

The study of the human pathogen papillomaviruses (HPVs) has been hampered by the inability to propagate the virus in tissue culture. The addition of 12-O-tetradecanoyl phorbol-13-acetate to the media of organotypic (raft) cultures increased expression of physiological markers of keratinocyte differentiation and concomitantly induced production of virions. Capsid production was detected in differentiated suprabasal cells. Virions approximately 54 nanometers in size were observed by electron microscopy in raft tissue cross sections in the suprabasal layers. Virions purified through isopycnic gradients were found to contain type 31b DNA and exhibited an icosahedral shape similar to that of papillomaviruses found in clinical samples.

The dominant transcriptional regulator of the papillomaviruses, E2, binds to its specific DNA target through a previously unobserved dimeric antiparallel beta-barrel. The DNA is severely but smoothly bent over the barrel by the interaction of successive major grooves with a pair of symmetrically disposed alpha-helices. The specific interface is an 'interwoven' network of interactions where the identifying base pairs of the target contact more than one amino-acid side chain and the discriminating amino acids interact with more than one base pair.

The simian virus (SV40) 72-base pair (bp) tandem repeated sequences have recently been shown to function as activators or enhancers of early viral transcription. A recombinant viral genome was recently constructed by inserting 72-bp tandem repeats from the Moloney murine sarcoma virus (MSV) in place of the 72-bp repeats of SV40. Although this genome replicates in monkey kidney cells, its rateof large tumor antigen expression and replication is considerably slower than that of wild-type SV40. In mouse cells, however, equivalent levels of large tumor antigen appear to be expressed from both wild-type and recombinant genomes, suggesting a relationship between the level of enhancer activity and the host cell. To confirm this observation, we have applied a sensitive quantitative assay for gene expression based on the conversion of chloramphenicol to its acetylated forms. The gene encoding the enzymatic function chloramphenicol acetyltransferase was inserted into two vectors in which the enhancer sequences from SV40 or MSV were placed adjacent to the early SV40 promoter. The SV40.tandem repeats appear to activate gene expression to significantly higher levels in monkey kidney cells, but-the MSV repeats are more active in two lines of mouse cells. These findings suggest that the tandem repeat elements may interact with host-specific molecules and, furthermore, may constitute one of the elements determining the host range of these eukaryotic viruses.Characterization of the nucleotide signals that constitute eukaryotic promoters is essential to an-understanding ofgene regulation. In addition to the Goldberg-Hogness sequence (or T-A-T-A box) which participates 'in the precise positioning of the 5' ends of RNA-molecules (1-3), upstream elements have been implicated in the activation or enhancement oftranscription for certain viral or eukaryotic genes by a number of studies (4)(5)(6)(7)(8)(9)(10)(11)(12). It was initially demonstrated that the simian virus 40 (SV40) 72-base-pair (bp) repeats activate early viral gene expression (5, 6). Further studies indicated that these sequences could also function when they were removed from their original location and placed at positions distant from the other promoter elements (8, 9). The tandem repeats also retained activity when inserted in an inverse orientation (9, 13). Other studies have indicated that the 72-bp repeats ofSV40 could increase the transformation efficiency of the herpes simplex virus thymidine kinase gene (10). An analogous fragment from the polyoma virus genome was shown to enhance the transcriptional activity of heterologous genes such as rabbit /3-globin gene (11).We have recently demonstrated that a retrovirus, the Moloney murine sarcoma virus (MSV), contains sequences in the long terminal repeats (LTRs) which can functionally replace the 72-bp repeats of SV40 (12). Preliminary data on large tumor antigen (T antigen) expression has suggested the possibility that the tandem repeats of SV40 and MSV activate gene expression in a host-specific manner.In th...

Human papillomaviruses (HPVs) are the etiologic agents of cervical and other epithelial cancers. Persistence of infections by high-risk HPV types is the single greatest risk factor for malignant progression. Although prophylactic vaccines have been developed targeting high risk HPV types, there is a continuing need to better understand the cell-host interactions that underlie persistent benign infection and progression to cancer. In this review, we summarize the molecular events that facilitate the differentiation-dependent HPV life cycle, how the life cycle is organized to facilitate viral persistence, and how the activities of HPV regulatory proteins result in malignancy. Human papillomavirus infection leading to cancerViruses cause approximately 15% of human cancers, and of these nearly half are attributable to human papillomaviruses (HPVs) 1 . Infections by HPVs in the genital tract are the most common sexually transmitted viral infection 2 . Although progression to cancer is a rare event, the high prevalence of HPV infection makes HPV-related cancers, including cervical and other anogenital cancers, among the most common malignancies. It is estimated that over 450,000 new cases of cervical cancer are diagnosed each year making this the second most prevalent cancer in women worldwide 1 . The effective utilization of the Papanicolaou (Pap) smear to recognize cellular abnormalities associated with HPV infection has resulted in a reduction in the number of cases of cervical cancer in the US by 80% in the last 50 years. Despite this decrease, approximately 11,000 women are diagnosed with cervical cancer annually in the US and approximately 3700 die of this disease 2 . In addition, highrisk HPV infection is responsible for approximately 3,000 cases of anal cancer and over 3500 cases of oropharyngeal cancer annually in the United States 2 .Papillomaviruses are small, non-enveloped DNA viruses that infect stratified squamous and cutaneous epithelia 3 . Over 120 different types of HPVs have been identified and approximately one-third of these types specifically target epithelial cells in the genital tract. The remaining types infect epithelial cells in other tissues including cutaneous epithelia. The HPV types that infect the genital mucosa can be divided into two groups. The high-risk (HR) types, including HPV16, 18, 31 and others, are frequently found in cervical cancers. By contrast, the low-risk (LR) types, including HPV6 and 11, also infect the genital epithelia, but are rarely detected in malignancies 4 . Two prophylactic vaccines (Gardasil (Merck) and Ceravrix (GSK)) against the HR types HPV16 and 18 were recently approved

Human papillomaviruses (HPVs) are small double-stranded DNA viruses that infect the cutaneous and mucosal epithelium. Infection by specific HPV types has been linked to the development of cervical carcinoma. HPV infects epithelial cells that undergo terminal differentiation and so encode multiple mechanisms to override the normal regulation of differentiation to produce progeny virions. Two viral proteins, E6 and E7, alter cell cycle control and are the main arbitrators of HPV-induced oncogenesis. Recent data suggest that E6 and E7 also play a major role in the inhibition of the host cell innate immune response to HPV. The E1 and E2 proteins, in combination with various cellular factors, mediate viral replication. In addition, E2 has been implicated in both viral and cellular transcriptional control. Despite decades of research, the function of other viral proteins still remains unclear. While prophylactic vaccines to block genital HPV infection will soon be available, the widespread nature of HPV infection requires greater understanding of both the HPV life cycle as well as the mechanisms underlying HPV-induced carcinogenesis.