The productive cycle of human papillomaviruses (HPVs) can be divided into discrete phases. Cell proliferation and episomal maintenance in the lower epithelial layers are followed by genome amplification and the expression of capsid proteins. These events, which occur in all productive infections, can be distinguished by using antibodies to viral gene products or to surrogate markers of their expression. Here we have compared precancerous lesions caused by HPV type 16 (HPV16) with lesions caused by HPV types that are not generally associated with human cancer. These include HPV2 and HPV11, which are related to HPV16 (supergroup A), as well as HPV1 and HPV65, which are evolutionarily divergent (supergroups E and B). HPV16-induced low-grade squamous intraepithelial lesions (CIN1) are productive infections which resemble those caused by other HPV types. During progression to cancer, however, the activation of late events is delayed, and the thickness of the proliferative compartment is progressively increased. In many HPV16-induced high-grade squamous intraepithelial lesions (CIN3), late events are restricted to small areas close to the epithelial surface. Such heterogeneity in the organization of the productive cycle was seen only in lesions caused by HPV16 and was not apparent when lesions caused by other HPV types were compared. By contrast, the order in which events in the productive cycle were initiated was invariant and did not depend on the infecting HPV type or the severity of disease. The distribution of viral gene products in the infected cervix depends on the extent to which the virus can complete its productive cycle, which in turn reflects the severity of cervical neoplasia. It appears from our work that the presence of such proteins in cells at the epithelial surface allows the severity of the underlying disease to be predicted and that markers of viral gene expression may improve cervical screening.

Fanconi anemia (FA) patients have an increased risk for squamous cell carcinomas (SCCs) at sites of predilection for infection with high-risk human papillomavirus (HPV) types, including the oral cavity and the anogenital tract. We show here that activation of the FA pathway is a frequent event in cervical SCCs. We found that FA pathway activation is triggered mainly by the HPV type 16 (HPV-16) E7 oncoprotein and is associated with an enhanced formation of large FANCD2 foci and recruitment of FANCD2 as well as FANCD1/BRCA2 to chromatin. Episomal expression of HPV-16 oncoproteins was sufficient to activate the FA pathway. Importantly, the expression of HPV-16 E7 in FA-deficient cells led to accelerated chromosomal instability. Taken together, our findings establish the FA pathway as an early host cell response to high-risk HPV infection and may help to explain the greatly enhanced susceptibility of FA patients to squamous cell carcinogenesis at anatomic sites that are frequently infected by high-risk HPVs.Fanconi anemia (FA) is a rare autosomal recessive or Xlinked cancer susceptibility syndrome characterized by congenital abnormalities, progressive bone marrow failure, and cellular sensitivity to DNA cross-linking agents (10). FA patients have a greatly increased susceptibility to squamous cell carcinomas (SCCs) of the anogenital tract and the oral cavity (24), sites with a predilection for infection with high-risk human papillomaviruses (HPVs) (33). An analysis of head and neck SCCs from FA patients showed that over 80% of the tumors contained high-risk HPV DNA, in particular that of HPV type 16 (HPV-16) (13). However, these results have been challenged by a study in which HPVs were undetectable in four cell lines derived from head and neck SCCs that developed in FA patients (31). Nonetheless, there is evidence that FA genes can become epigenetically inactivated during cervical carcinogenesis in the general population (22). The reason for the increased susceptibility of FA patients to SCCs that frequently arise at sites of HPV infection and why they develop these tumors at a significantly younger age than the general population is currently unknown (16).High-risk HPV types, such as HPV-16, encode two oncoproteins, E6 and E7, which have important functions during the viral life cycle by reinitiating DNA replication in terminally growth-arrested host keratinocytes (1,15,20). The efficiency of HPV-16 oncoproteins to subvert cell cycle checkpoints in order to create an S-phase-like milieu supportive of viral DNA replication is likely to be critical for their oncogenic potential. Unscheduled entry into S phase and the deregulation of cyclin expression, characteristics of HPV-16 E7-expressing cells (17,18,25), have previously been suggested to result in perturbations of DNA replication, increased stalling of replication forks, and chromosomal instability (4,26).Previous studies have shown that the FA pathway can become activated by stalled replication forks (9). Upon activation, the core complex of FA proteins mediate...

The life cycle of human papillomaviruses (HPVs) is tightly linked to the differentiation program of the host's stratified epithelia that it infects. E1∧ E4 is a viral protein that has been ascribed multiple biochemical properties of potential biological relevance to the viral life cycle. To identify the role(s) of the viral E1 ∧ E4 protein in the HPV life cycle, we characterized the properties of HPV type 16 (HPV16) genomes harboring mutations in the E4 gene in NIKS cells, a spontaneously immortalized keratinocyte cell line that when grown in organotypic raft cultures supports the HPV life cycle. We learned that E1 ∧ E4 contributes to the replication of the viral plasmid genome as a nuclear plasmid in basal cells, in which we also found E1 ∧ E4 protein to be expressed at low levels. In the suprabasal compartment of organotypic raft cultures harboring E1 ∧ E4 mutant HPV16 genomes there were alterations in the frequency of suprabasal cells supporting DNA synthesis, the levels of viral DNA amplification, and the degree to which the virus perturbs differentiation. Interestingly, the comparison of the phenotypes of various mutations in E4 indicated that the E1 ∧ E4 protein-encoding requirements for these various processes differed. These data support the hypothesis that E1 ∧ E4 is a multifunctional protein and that the different properties of E1 ∧ E4 contribute to different processes in both the early and late stages of the virus life cycle.Human papillomaviruses (HPVs) are small, doublestranded DNA viruses that infect the stratified epithelium lining the skin, anogenital tract, and oral cavity. Viral infection generally causes hyperproliferative lesions such as warts and condyloma. High-risk, mucosotropic (previously termed anogenital) HPVs, most commonly HPV type 16 (HPV16), are also associated with malignant tumors of the anogenital tract and oral cavity and are now accepted as the major causative agent of cervical cancer (64, 69). The life cycle of HPVs is tightly linked to the differentiation program of the host epithelium. HPVs infect basal keratinocytes, presumably at a site of wounding, and they establish their double-stranded, circular DNA genome as an extrachromosomal nuclear plasmid (replicon) at a low copy number. In these proliferating basal cells, early viral genes are selectively expressed, and viral DNA replication occurs along with cellular chromosomal DNA replication to maintain viral DNA copy numbers in both parent and daughter cells. This stage of the viral life cycle within basal cells is called the nonproductive or early stage because no new virus is made. As the infected cells migrate upward and undergo terminal differentiation, the productive or late stage of the viral life cycle begins. In the suprabasal compartment of the epithelium, the viral DNA is amplified, and this is followed by the expression of the late viral genes, including those encoding the structural proteins that form the capsid. Viral DNA is packaged into these capsids to form progeny virions that accumulate in the most superf...

The papillomavirus life cycle is tied to the differentiation of the stratified squamous epithelium that this virus infects. The ability to study the papillomavirus life cycle is facilitated by organotypic culturing techniques that allow one to closely recapitulate this terminal differentiation process in the laboratory. Current techniques allow for the establishment of recombinant wild-type or mutant human papillomavirus (HPV) genomes in transfected early-passage human foreskin keratinocytes (HFKs). These cells can then be used in organotypic culture to investigate the role of individual viral genes in different aspects of the viral life cycle. When using early-passage HFKs, there is a need for the transfected HPV genome to extend the life span of the cells in order to have sufficient cell generations in which to carry out organotypic culturing. The recent isolation of a spontaneously immortalized HFK cell line that supports the complete HPV life cycle has further allowed investigators to study wild-type or mutant papillomaviral genomes that do not confer immortalization. In this chapter, we describe the methodologies that permit the study of the HPV life cycle in this HFK cell line.

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