∧ 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) 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 abundant human papillomavirus (HPV) type 16 E4 protein exists as two distinct structural forms in differentiating epithelial cells. Monomeric full-length 16E1∧ E4 contains a limited tertiary fold constrained by the N and C termini. N-terminal deletions facilitate the assembly of E1 ∧ E4 into amyloid-like fibrils, which bind to thioflavin T. The C-terminal region is highly amyloidogenic, and its deletion abolishes amyloid staining and prevents E1 ∧ E4 accumulation. Amyloid-imaging probes can detect 16E1 ∧ E4 in biopsy material, as well as 18E1 ∧ E4 and 33E1 ∧ E4 in monolayer cells, indicating structural conservation. Our results suggest a role for fibril formation in facilitating the accumulation of E1 ∧ E4 during HPV infection.Cervical cancer and its precursors usually arise from areas of low-grade disease caused by high-risk human papillomavirus (HPV) types such as HPV-16 (5, 32). The HPV E4 protein (also known as E1 ∧ E4) is expressed from an E1 ∧ E4 spliced mRNA prior to the assembly of infectious virions and accumulates to very high levels in cells supporting productive infection (10, 11). Abundant E1 ∧ E4 is seen in lesions caused by many different HPV types (10,21,26), suggesting that E1 ∧ E4 accumulation plays an important role in the virus life cycle.Although E1 ∧ E4 function is not yet fully understood, its association with keratin filaments is thought to compromise the mechanical integrity of the cell during the late stages of infection (8,33). Keratin binding is not, however, essential for E1 ∧ E4 accumulation, as keratin-binding mutants still accumulate to wild-type levels (24). Rather, it appears that E1 ∧ E4 accumulation depends on sequences at the C terminus of the protein. To understand 16E1∧ E4 function at the molecular level, and in particular to explain its ability to accumulate within the infected cell, we have carried out a structural analysis of the protein. We have found that monomeric 16E1 ∧ E4 does not have a stable tertiary fold but does contain elements of secondary structure that are key to its assembly into fibrils following N-terminal truncation. This work not only enhances our understanding of the HPV-16 E1 ∧ E4 protein but also contributes a viral protein to the growing family of proteins capable of using the amyloid fold (4, 13).Two distinct forms of 16E1 ∧ E4 are expressed during productive infection. Although the E1 ∧ E4 protein of HPV-1 is known to be N terminally cleaved during natural infection (2, 9), it is not yet clear whether HPV-16 E1∧ E4 is similarly modified. To identify the E1 ∧ E4 species present in productively HPV-16-infected squamous epithelium, NIKS cells harboring the full HPV-16 genome were grown in raft culture (24), harvested at day 11 postlifting, and lysed in phosphate-buffered saline containing 6% sodium dodecyl sulfate (SDS) and 1.5 M urea. The E1 ∧ E4 species were characterized using the 16E1 ∧ E4 antibodies TVG402, which binds an epitope between amino acids 29 and 40 (33), and anti-N-term (10), which binds only to species which retain an intact N-t...

SummaryThe keratin IF network of epidermal keratinocytes provides a protective barrier against mechanical insult, it is also a major player in absorbing stress in these cells. The human papilloma virus (HPV) type 16 E1^E4 protein accumulates in the upper layers of HPV16-infected epithelium and is known to associate with and reorganise the keratin IF network in cells in culture. Here, we show that this function is conserved amongst a number of HPV alpha-group E1^E4 proteins and that the differentiation-dependent keratins are also targeted. Using time-lapse microscopy, HPV16 E1^E4 was found to effect a dramatic cessation of keratin IF network dynamics by associating with both soluble and insoluble keratin. Network disruption was accompanied by keratin hyperphosphorylation at several sites, including K8 S73, which is typically phosphorylated in response to stress stimuli. Keratin immunoprecipitated from E1^E4-expressing cells was also found to be ubiquitylated, indicating that it is targeted for proteasomal degradation. Interestingly, the accumulation of hyperphosphorylated, ubiquitylated E1^E4-keratin structures was found to result in an impairment of proteasomal function. These observations shed new light on the mechanism of keratin IF network reorganisation mediated by HPV16 E1^E4 and provide an insight into the depletion of keratin co-incident with E1^E4 accumulation observed in HPV-infected epithelium.