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.

Animal papillomaviruses are widely used as models to study papillomavirus infection in humans despite differences in genome organization and tissue tropism. Here, we have investigated the extent to which animal models of papillomavirus infection resemble human disease by comparing the life cycles of 10 different papillomavirus types. Three phases in the life cycles of all viruses were apparent using antibodies that distinguish between early events, the onset of viral genome amplification, and the expression of capsid proteins. The initiation of these phases follows a highly ordered pattern that appears important for the production of virus particles. The viruses examined included canine oral papillomavirus, rabbit oral papillomavirus (ROPV), cottontail rabbit papillomavirus (CRPV), bovine papillomavirus type 1, and human papillomavirus types 1, 2, 11, and 16. Each papillomavirus type showed a distinctive gene expression pattern that could be explained in part by differences in tissue tropism, transmission route, and persistence. As the timing of life cycle events affects the accessibility of viral antigens to the immune system, the ideal model system should resemble human mucosal infection if vaccine design is to be effective. Of the model systems examined here, only ROPV had a tissue tropism and a life cycle organization that resembled those of the human mucosal types. ROPV appears most appropriate for studies of the life cycles of mucosal papillomavirus types and for the development of prophylactic vaccines. The persistence of abortive infections caused by CRPV offers advantages for the development of therapeutic vaccines.

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...

Human papillomavirus type 16 (HPV16) can cause cervical cancer. Expression of the viral E1∧ E4 protein is lost during malignant progression, but in premalignant lesions, E1∧ E4 is abundant in cells supporting viral DNA amplification. Expression of 16E1 ∧ E4 in cell culture causes G 2 cell cycle arrest. Here we show that unlike many other G 2 arrest mechanisms, 16E1∧ E4 does not inhibit the kinase activity of the Cdk1/cyclin B1 complex. Instead, 16E1∧ E4 uses a novel mechanism in which it sequesters Cdk1/cyclin B1 onto the cytokeratin network. This prevents the accumulation of active Cdk1/cyclin B1 complexes in the nucleus and hence prevents mitosis. A mutant 16E1∧ E4 (T22A, T23A) which does not bind cyclin B1 or alter its intracellular location fails to induce G 2 arrest. The significance of these results is highlighted by the observation that in lesions induced by HPV16, there is evidence for Cdk1/cyclin B1 activity on the keratins of 16E1 ∧

Expression of the papillomavirus E4 protein correlates with the onset of viral DNA amplification. Using a mutant cottontail rabbit papillomavirus (CRPV) genome incapable of expressing the viral E4 protein, we have shown that E4 is required for the productive stage of the CRPV life cycle in New Zealand White and cottontail rabbits. In these lesions, E4 was not required for papilloma development, but the onset of viral DNA amplification and L1 expression were abolished. Viral genome amplification was partially restored when mutant genomes able to express longer forms of E4 were used. These findings suggest that efficient amplification of the CRPV genome is dependent on the expression of a full-length CRPV E4 protein.Papillomaviruses (PVs) are epitheliotropic viruses that can infect both cutaneous and mucosal surfaces and induce benign and malignant skin tumors (34). There are more than 100 different types of human PVs (HPVs) and many animal-specific PVs (1, 16). The life cycle of PVs can be divided into early and late phases and is dependent on the differentiation program of the infected epithelial cell (28, 49). PVs are highly host and tissue specific. Infections at different histological sites trigger a series of viral life cycle events that are conserved among different virus types (35,39). The early stages begin in the actively proliferating cells in the basal and parabasal layers, where expression of E6 and E7 (the viral transforming proteins), as well as E1 and E2 (the viral replication proteins), induces papilloma formation and maintains the episomal viral genome at a low copy number (19,48,49,53). The late stage of the virus life cycle is triggered when the infected cells further differentiate during their migration toward the epithelial surface (28,49). Although the exact mechanism(s) by which the late events are initiated remains unclear, a change in promoter activity occurs as the virus enters its productive cycle in the upper epithelial layers (2,22). In these differentiated cells, E4 protein expression and the onset of viral DNA amplification begin, and these processes are followed by the expression of the viral capsid proteins (L1 and L2) and the assembly of infectious virions (35,39).In PV-infected regions, the normal differentiation program of the epithelium is disturbed, and alterations in tissue morphology are apparent. Some of the changes are characteristic of PV infections, including acanthosis (thickening of the epithelium), the presence of koilocytes, and parakeratosis (retention of the nuclei in the cornified layers) (34). These changes are partly the consequence of the expression of the viral E6 and E7 proteins in the lower layers of the epithelium (48, 53). E6 and E7 act via different mechanisms to stimulate cell cycle progression, to increase the number of nucleated amplifying cells in the differentiating layers, and to delay keratinocyte terminal differentiation (51, 53). Since PVs rely to a large extent on the host cell machinery to replicate their genomes, the expression of E6 and E7 i...