High-risk (HR) oncogenic mucosal human papillomavirus (HPV) types are the major cause of most carcinomas of the uterine cervix, as well as many other anogenital tumors, and are found in 20 to 30% of cancers of the head and neck (HNC) (36). While many HR HPVs share genomic organization and conserved sequence homologies, they vary significantly in their prevalences in vivo. HPV-16 is the most prevalent HR HPV, as it is present in nearly 50% of cervical and anogenital carcinomas and in more than 90% of HPV-associated HNC (15,46). In contrast, the sequence-related HPV-31 is significantly less prevalent than HPV-16 in cervical carcinomas and is rarely detected in HNC. Mechanisms determining HPV type-specific variations in viral persistence and malignant progression, however, are poorly understood.Papillomaviruses (PVs) replicate as extrachromosomal double-stranded DNA plasmids after infection of the basal keratinocyte host, stably persisting in low copy numbers (10,19,25). Early PV gene expression and plasmid amplification in the initial stages of HPV infection appear to be tightly regulated (18,21,30) through both transcriptional and posttranscriptional mechanisms (reviewed in references 43 and 60). Both the E1 and E2 proteins are required for PV replication (6,14,18), and as shown for HPV-31, transcripts encoding these factors from the plasmid genome are detected early after infection, followed by the production of mRNA encoding other early viral gene products (34). This temporal, regulated expression of limiting levels of these transcription and replication modulators early in HPV infection suggests that these viral gene products are critical to the establishment phase of the viral life cycle. The precise mechanisms that limit HPV early gene expression or initial plasmid amplification and modulate the establishment of a stable viral copy number, however, have not been completely defined.The full-length E2 and spliced E8 ∧ E2 isoforms are conserved in PVs, with E8 ∧ E2 transcripts identified in bovine papillomavirus type 1 (BPV-1) (26, 27), , cottontail rabbit papillomavirus (CRPV) (23), HPV-31 (52), and 45). As in BPV (1,13,16,58), E2 gene products expressed from a variety of HPVs modulate early gene transcription (5,47,48,53,56) and initial plasmid amplification (4,21,29,35,37) by distinct E2 structural domains, as shown in HPV-16 (41) and HPV-31 (51). The HPV E2 protein isoforms exert their transcriptional and replication effects by interacting with defined cis binding sites which are also conserved in mucosal and cutaneous HPVs (42) as well as in animal PVs. E8 ∧ E2 interferes with E2-dependent transcriptional activation by the full-length E2 proteins via competitive binding to conserved E2 binding sites in BPV-1 (T. Haugen, unpublished data) and . Similarly, HPV E8 ∧ E2 products can also inhibit plasmid replication (2, 61). This study uses a newly developed complementation assay for HPV-16 replication to define the structure of the HPV-16

Many human papillomavirus (HPV)-positive high-grade lesions and cancers of the uterine cervix harbor integrated HPV genomes expressing the E6 and E7 oncogenes from chimeric virus-cell mRNAs, but less is known about HPV integration in head and neck cancer (HNC).Mucosal high-risk (HR) human papillomavirus (HPV) types are found in nearly 100% of carcinomas of the uterine cervix, many anogenital cancers, and ϳ25% of head and neck cancers (HNC) (9, 13, 42). The most common HR HPV is type 16, found in over 50% of cervical cancers and 90 to 95% of HPVpositive HNCs. In persistent infection, HPV genomes are maintained as circular, unintegrated plasmids that persist in the nuclei of infected cells (see Fig. 1A). In contrast, HR HPV DNA found in cervical cancer specimens is frequently disrupted and integrated in the cellular genome in a way that potentially alters the expression program of the viral early gene region and/or the host genome (2,5,8,21,23,24,31,43). The integrated HR HPV fragments in high-grade precancerous cervical lesions, cervical carcinomas, and derived cell lines express the viral transforming genes E6 and E7 from chimeric virus-cell mRNAs, while the downstream early genes that are required for viral replication and regulated viral gene expression are disrupted or silenced (33,37,47,55). Further, chimeric virus-cell mRNAs may be more stable than the viral early mRNAs, which harbor a 3Ј destabilization motif (20,53). In addition, some integration sites in cervical cancer and precursor lesions have been found near potential growth control genes (11,40,55).Advances in keratinocyte culture techniques have permitted the study of the life cycle of HR HPV types that replicate efficiently and persist in primary human keratinocytes, such as 34,35), and to a more limited extent 36). However, relatively little is known about the progression of the infected cell toward a malignant phenotype in vivo or in culture. While some cervical cancers have been shown to harbor extrachromosomal HPV DNA, it is apparent that integration of viral DNA accompanied by disruption of the integrity of the HR HPV genomes represents a common, albeit not absolutely obligatory, step associated with carcinogenic progression associated with the genital tract (42, 57). The majority of cervical carcinomas caused by the two most common HR HPV types, HPV-16 and -18, contain integrated viral sequences (52) that express E6-E7 from chimeric, spliced virus-cell transcripts. However, the genetic structure and expres-

Human papillomavirus (HPV) DNAs isolated from cervical and head and neck carcinomas frequently contain nucleotide sequence alterations in the viral upstream regulatory region (URR). Our study has addressed the role such sequence changes may play in the efficiency of establishing HPV persistence and altered keratinocyte growth. Genomic mapping of integrated HPV type 16 (HPV-16) genomes from 32 cervical cancers revealed that the viral E6 and E7 oncogenes, as well as the L1 region/URR, were intact in all of them. The URR sequences from integrated and unintegrated viral DNA were found to harbor distinct sets of nucleotide substitutions. A subset of the altered URRs increased the potential of HPV-16 to establish persistent, cell growth-altering viral-genome replication in the cell. This aggressive phenotype in culture was not solely due to increased viral early gene transcription, but also to augmented initial amplification of the viral genome. As revealed in a novel ori-dependent HPV-16 plasmid amplification assay, the altered motifs that led to increased viral transcription from the intact genome also greatly augmented HPV-16 ori function. The nucleotide sequence changes correlate with those previously described in the distinct geographical North American type 1 and Asian-American variants that are associated with more aggressive disease in epidemiologic studies and encompass, but are not limited to, alterations in previously characterized sites for the negative regulatory protein YY1. Our results thus provide evidence that nucleotide alterations in HPV regulatory sequences could serve as potential prognostic markers of HPV-associated carcinogenesis.

Mucosal high-risk (HR) human papillomaviruses (HPVs) that cause cervical and other anogenital cancers also are found in ϳ25% of head and neck carcinomas (HNCs), especially those arising in the oropharynx and the tonsils. While many HR HPV types are common in anogenital cancer, over 90% of HPV-positive HNCs harbor HPV type 16 (HPV-16). Using a quantitative colony-forming assay, we compared the ability of fulllength mucosal HPV genomes, i.e., the low-risk HPV-11 and HR HPV-16, -18, and -31, to persist in and alter the growth of primary human keratinocytes from the foreskin, cervix, and tonsils. The HR HPV types led to the formation of growing keratinocyte colonies in culture independent of the site of epithelial origin. However, HPV-18 induced colony growth in all keratinocytes >4-fold more effectively than HPV-16 or HPV-31 and >20-fold more efficiently than HPV-11 or controls. HPV-11-transfected or control colonies failed to expand beyond 32 to 36 population doublings postexplantation. In contrast, individual HR HPV-transfected clones exhibited no apparent slowdown of growth or "crisis," and many maintained HPV plasmid persistence beyond 60 population doublings. Keratinocyte clones harboring extrachromosomal HR HPV genomes had shorter population doubling times and formed dysplastic stratified epithelia in organotypic raft cultures, mirroring the pathological features of higher-grade intraepithelial lesions, yet did not exhibit chromosomal instability. We conclude that, in culture, the HR HPV type, rather than the site of epithelial origin of the cells, determines the efficacy of inducing continued growth of individual keratinocytes, with HPV-18 being the most aggressive mucosal HR HPV type tested.Human papillomaviruses (HPVs) are small DNA tumor viruses that infect, persist in, and cause proliferative lesions in the epithelial cells of the skin, ectoderm-derived mucosae, and their adnexa. Mucosal HPV types are associated with most if not all carcinomas of the uterine cervix, many anogenital cancers, and ϳ25% of head and neck cancers (HNCs) (reviewed in reference 55). HNCs arising in the oropharynx or tonsils are HPV positive in over 50% of the cases. Although the same oncogenic or "high-risk" (HR) HPV types are found in the cervix, in the anogenital area, and in HNC, their prevalence is strikingly different: in cervical and anogenital cancers, HPV type 16 (HPV-16) is found in ϳ50% of the tumors, followed by HPV-18 (ϳ20%), while the remaining cases harbor over 15 other HR HPV types, including HPV-31 and other well-characterized as well as additional, novel HPV types. In contrast, over 90 to 95% of HPV-positive HNCs contain HPV-16, whereas HPV-18, HPV-31, or other HR HPVs are infrequent (10,

Replication of the double-stranded, circular human papillomavirus (HPV) genomes requires the viral DNA replicase E1. Here, we report an initial characterization of the E1 cistron of HPV type 16 (HPV-16), the most common oncogenic mucosal HPV type found in cervical and some head and neck cancers. The first step in HPV DNA replication is an initial burst of plasmid viral DNA amplification. Complementation assays between HPV-16 genomes carrying mutations in the early genes confirmed that the expression of E1 was necessary for initial HPV-16 plasmid synthesis. The major early HPV-16 promoter, P97, was dispensable for E1 production in the initial amplification because cis mutations inactivating P97 did not affect the trans complementation of E1؊ mutants. In contrast, E1 expression was abolished by cis mutations in the splice donor site at nucleotide (nt) 226, the splice acceptor site at nt 409, or a TATAA box at nt 7890. The mapping of 5 mRNA ends using rapid amplification of cDNA ends defined a promoter with a transcription start site at HPV-16 nt 14, P14. P14-initiated mRNA levels were low and required intact TATAA (7890). E1 expression required the HPV-16 keratinocyte-dependent enhancer, since cis mutations in its AP-2 and TEF-1 motifs abolished the ability of the mutant genomes to complement E1؊ genomes, and it was further modulated by origin-proximal and -distal binding sites for the viral E2 gene products. We conclude that P14-initiated E1 expression is critical for and limiting in the initial amplification of the HPV-16 genome.High-risk (HR) oncogenic mucosal human papillomavirus (HPV) types are the major cause of most, if not all, carcinomas of the uterine cervix and many other anogenital tumors, and they are found in 20 to 30% of cancers of the head and neck (HNC). HPV type 16 (HPV-16) is the most prevalent HR HPV: it is present in nearly one-half of cervical carcinomas and more than 90% of HPV-associated HNC (reviewed in reference 32). Recently, preventive vaccines against HPV-16 and HPV-18, the second most common HR HPV, have become available. While the vaccines are highly efficacious, they cannot eliminate existing infection. The development of therapies targeted at HPV infection requires a profound understanding of the viral genes involved in replication and their regulation.Upon introduction into the basal layer cell of skin or stratified squamous epithelia, the double-stranded, closed circular HPV plasmid genome undergoes an initial amplification (11,18,45). HPV plasmid amplification in the initial stages of infection appears to be tightly restricted, potentially in part to limit triggering intracellular defense mechanisms by unchecked viral replication and/or to minimize host immune responses to high levels of viral polypeptides. A low steady-state viral copy number in established HPV infections suggests limited levels of cellular factors or the regulated synthesis of viral factors responsible for supporting HPV replication.Papillomaviral DNA replication requires an origin of replication (ori) and two...

Papillomaviral E2 genes encode proteins that regulate viral transcription. While the full-length bovine papillomavirus type 1 (BPV-1) E2 peptide is a strong trans activator, the homologous full-length E2 product of human papillomavirus type 16 (HPV-16) appeared to vary in function in previous studies. Here we show that when expressed from comparable constructs, the full-length E2 products of HPV-16 and BPV-1 trans activate a simple E2-and Spl-dependent promoter up to-100-fold in human keratinocytes and other epithelial cells as well as human and animal fibroblasts. Vaccinia virus-expressed, purified full-length HPV-16 and BPV-1 E2 proteins bound a consensus E2 site with high specific affinities (Kd =-10-9 M) and stimulated in vitro transcription up to sixto eightfold. In vivo and in vitro trans activation by either E2 protein required cooperation with another activator, such as Spl, or other factors that interact with papillomavirus promoters, such as AP-1, Oct-i, nuclear factor 1/CTF, transcriptional enhancer factor 1, or USF. The glutamine-rich domain B of Spl or the mutually unrelated activation domains of other transcription factors were necessary and sufficient for cooperation with either E2 factor. We conclude that like BPV-1 E2, the HPV-16 E2 protein has the potential to function as a strong activator of viral gene expression in cooperation with cellular transcription factors.

Interferon regulatory factors (IRFs) are critical mediators of gene expression, cell growth and immune responses. We previously demonstrated that interferon (IFN) induction of early viral transcription and replication in several mucosal HPVs requires IRF-1 binding to a conserved interferon response element (IRE). Here we show that the IRF-2 protein serves as a baseline transactivator of the HPV-16 major early promoter, P97. Cotransfections in IRF knockout cells confirmed that basal HPV-16 promoter activity was supported by both IRF-1 and IRF-2 complexes interacting with the promoter-proximal IRE in a dose-dependent manner. Furthermore, HPV-16 E7 expression downregulates the IRF-2 promoter, thus linking IRF-2 levels to viral transforming gene expression through a negative feedback mechanism. Taken together, these observations reveal a complex viral strategy utilizing multiple signal transduction pathways during the establishment and maintenance of HPV persistence.

Interferons (IFNs) have been used to treat epithelial lesions caused by human papillomavirus (HPV) persistence. Here, we exposed primary human keratinocytes (HFKs) immortalized by persistently replicating HPV-16 plasmid genomes to increasing levels of IFN-γ. While untreated HFKs retained replicating HPV-16 plasmids for up to 60-120 population doublings, IFN led to rapid HPV-16 plasmid loss. However, treated cultures eventually gave rise to outgrowth of clones harboring integrated HPV-16 genomes expressing viral E6 and E7 oncogenes from chimeric virus-cell mRNAs similar to those in cervical and head and neck cancers. Surprisingly, every HPV-16 integrant that arose after IFN exposure stemmed from an independent integration event into a different cellular gene locus, even within parallel cultures started from small cell inocula and cultured separately for ≥ 25 doublings to permit the rise and expansion of spontaneous integrants. While IFN treatment conferred a growth advantage upon preexisting integrants added to mixed control cultures, our results indicate that IFN exposure directly or indirectly induces HPV-16 integration, rather than only selects preexisting, spontaneous integrants that appear to be much less frequent. We estimate that IFN exposure increased integration rates by ≥ 100-fold. IFN-induced HPV-16 integration involved a wide range of chromosomal loci with less apparent selection for recurrent insertions near genes involved in cancer-related pathways. We conclude that IFNs and other potential treatments targeting high-risk HPV persistence that disrupt viral genome replication may promote increased high-risk HPV integration as a step in cancer progression. Therapies against high-risk HPV persistence thus need to be evaluated for their integration-inducing potential.