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.

∧ E4 protein caused an almost total inhibition of keratin dynamics, despite the phosphorylation of keratin 18 at serine 33, which normally leads to 14-3-3-mediated keratin solubilization. Mutant 16E1 ∧ E4 proteins which lack the LLKLL motif, or which have lost amino acids from their C termini, and which were compromised in the ability to associate with keratins did not disturb normal keratin dynamics. 16E1 ∧ E4 was found to exist as dimers and hexamers, whereas a C-terminal deletion mutant (16E1 ∧ E4⌬87-92) existed as monomers and formed multimeric structures only poorly. Considered together, our results suggest that by associating with keratins through its N terminus, and by associating with itself through its C terminus, 16E1 ∧ E4 may act as a keratin cross-linker and prevent the movement of keratins between the soluble and insoluble compartments. The increase in avidity associated with multimeric binding may contribute to the ability of 16E1 ∧ E4 to sequester its cellular targets in the cytoplasm.Human papillomaviruses (HPVs) are small double-stranded DNA viruses of ϳ8,000 bp. They infect stratified epithelium and produce lesions that range in severity from benign warts to invasive carcinomas (24). HPV DNA has been detected in Ͼ99.7% of cervical cancers, with HPV16 occurring most frequently (28,29,44). HPV16 is a high-risk HPV type which causes cervical lesions that can progress to high-grade neoplasia and cancer (43).The life cycle of HPVs is closely linked to the differentiation status of the host epithelium. After infecting basal cells through a wound, the viral genome maintains itself episomally at a low copy number (5). As the infected cell migrates toward the epithelial surface and undergoes terminal differentiation, the productive stages of the viral life cycle are triggered. Vegetative viral DNA replication is followed by the expression of capsid proteins and the assembly of infectious virions in the superficial cell layers (24).The HPV16 E1 ∧ E4 protein is expressed in abundance during the late stages of the virus life cycle in the upper layer of the epithelium and coincides with the onset of viral genome amplification (12,26,35). Although the precise role of 16E1 ∧ E4 is unclear, previous work has revealed that 16E1 ∧ E4 can induce cell cycle arrest in G 2 (7), can bind to a DEAD box RNA helicase (E4-DBP) (9), and, when expressed in cultured epithelial cells, can interact with keratins and cause the reorganization of the keratin intermediate-filament network (11). Although the mechanism by which 16E1 ∧ E4 mediates keratin filament reorganization is not understood, immunofluorescence staining has shown the LLKLL motif located close to the N terminus to be necessary for filament colocalization and has shown the C terminus to be necessary for filament collapse.Keratins are major structural proteins in epithelial cells and form the cytoplasmic network of intermediate filaments (17). They contain at least 20 members, called keratin 1 (K1) to K20, which are divided into two types according to the sequence...

Human papillomavirus type 16 (HPV16) is the most common cause of cervical carcinoma. Cervical cancer develops from low-grade lesions that support the productive stages of the virus life cycle. The 16E1 ∧ E4 protein is abundantly expressed in such lesions and can be detected in cells supporting vegetative viral genome amplification. Using an inducible mammalian expression system, we have shown that 16E1 ∧ E4 arrests HeLa cervical epithelial cells in G 2 . 16E1 ∧ E4 also caused a G 2 arrest in SiHa, Saos-2 and Saccharomyces pombe cells and, as with HeLa cells, was found in the cytoplasm. However, whereas 16E1 ∧ E4 is found on the keratin networks in HeLa and SiHa cells, in Saos-2 and S. pombe cells that lack keratins, 16E1 ∧ E4 had a punctate distribution. Mutagenesis studies revealed a proline-rich region between amino acids 17 and 45 of 16E1 ∧ E4 to be important for arrest. This region, which we have termed the "arrest domain," contains a putative nuclear localization signal, a cyclin-binding motif, and a single cyclin-dependent kinase (Cdk) phosphorylation site. A single point mutation in the putative Cdk phosphorylation site (T23A) abolished 16E1 ∧ E4-mediated G 2 arrest. Arrest did not involve proteins regulating the phosphorylation state of Cdc2 and does not appear to involve the activation of the DNA damage or incomplete replication checkpoint. G 2 arrest was also mediated by the E1 ∧ E4 protein of HPV11, a low-risk mucosal HPV type that also causes cervical lesions. The E1 ∧ E4 protein of HPV1, which is more distantly related to that of HPV16, did not cause G 2 arrest. We conclude that, like other papillomavirus proteins, 16E1 ∧ E4 affects cell cycle progression and that it targets a conserved component of the cell cycle machinery.Papillomaviruses are small DNA viruses that infect the epithelial tissue of a wide range of vertebrates, including humans (47). Infection begins in cells of the basal layer, with the productive stages of the virus life cycle being initiated as these infected cells migrate toward the epithelial surface. In this way, the virus life cycle is tightly linked to the differentiation of the epithelium, which makes human papillomaviruses (HPVs) difficult to study in the laboratory (37). More than 200 papillomavirus types have been identified (12). These share a common organization of their ϳ8-kb genomes but differ in the types of epithelium they infect and the pathologies that each virus causes (47). Infection occurs at cutaneous epithelial sites, i.e., the skin, for viruses such as HPV type 1 (HPV1) or at mucosal sites (e.g., the anogenital tract or the cervix) for viruses such as HPV11 and HPV16 (47). HPVs are additionally classified from low to high risk, with low-risk viruses such as HPV11 (and HPV1) being associated solely with benign lesions (often known as warts), whereas lesions caused by highrisk viruses such as HPV16 can progress to malignancy (47). HPV16 is the most prevalent high-risk virus and is the most common causative agent of cervical cancers (68).Although the 16E1 ∧ E4 ...

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 ∧

The human papillomavirus (HPV) E1∧ E4 protein is the most abundantly expressed viral protein in HPVinfected epithelia. It possesses diverse activities, including the ability to bind to the cytokeratin network and to DEAD-box proteins, and in some cases induces the collapse of the former. E1∧ E4 is also able to prevent the progression of cells into mitosis by arresting them in the G 2 phase of the cell cycle. In spite of these intriguing properties, the role of this protein in the life cycle of the virus is not clear. Here we report that after binding to and collapsing the cytokeratin network, the HPV type 16 E1 ∧ E4 protein binds to mitochondria. When cytokeratin is not present in the cell, E1∧ E4 appears associated with mitochondria soon after its synthesis. The leucine cluster within the N-terminal portion of the E1 ∧ E4 protein is pivotal in mediating this association. After the initial binding to mitochondria, the E1 ∧ E4 protein induces the detachment of mitochondria from microtubules, causing the organelles to form a single large cluster adjacent to the nucleus. This is followed by a severe reduction in the mitochondrial membrane potential and an induction of apoptosis. HPV DNA replication and virion production occur in terminally differentiating cells which are keratin-rich, rigid squamae that exfoliate after completion of the differentiation process. Perturbation of the cytokeratin network and the eventual induction of apoptotic properties are processes that could render these unyielding cells more fragile and ease the exit of newly synthesized HPVs for subsequent rounds of infection.Papillomaviruses infect the skin and mucosa of many different hosts, including humans (1). In order to multiply in these tissues, these viruses have evolved and adapted their life cycle to the biology of the epithelium. Human papillomaviruses (HPVs) gain entry into the epithelium via microlesions, which allow them to infect the epithelial basal cells. While the viral episomes remain at a low copy number in basal cells, cellular proliferation leads to the generation of a pool of cells that contain viral DNA. Amplification of the HPV genome and the production of infectious virions do not occur until the infected cells undergo terminal differentiation (11,19). The amplification of papillomavirus DNA usually begins when infected keratinocytes differentiate into spinous cells (17). This is followed by the production of viral capsid proteins and the assembly of infectious particles. In order to replicate in differentiating cells, the E6 and E7 proteins of HPV stimulate the production of proteins required for DNA synthesis. This causes the differentiating cells to enter S phase (24, 26). The E6 and E7 proteins are able to do this largely because they can inactivate the p53 (32) and pRb (15, 27) proteins, respectively. It is noteworthy that in spite of inactivating two major tumor suppressor functions in the cell, normal productive HPV infections do not cause uncontrolled cellular proliferation leading to tumorigenesis. At most, hyp...