E1--E4-mediated keratin phosphorylation and ubiquitylation: a mechanism for keratin depletion in HPV16-infected epithelium.

The 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.

Evidence for enhanced characterization of cortical bone using novel pQCT shape software.

Bone shape, mass, structural geometry, and material properties determine bone strength. This study describes novel software that uses peripheral quantitative computed tomography (pQCT) images to quantify cortical bone shape and investigates whether the combination of shape-sensitive and manufacturer's software enhances the characterization of tibiae from contrasting populations. Existing tibial pQCT scans (4% and 50% sites) from Gambian (n=38) and British (n=38) women were used. Bone mass, cross-sectional area (CSA), and geometry were determined using manufacturer's software; cross-sectional shape was quantified using shape-sensitive software. At 4% site, Gambian women had lower total bone mineral content (BMC: -15.4%), CSA (-13.4%), and trabecular bone mineral density (BMD: -19%), but higher cortical subcortical BMD (6.1%). At 50% site, Gambian women had lower cortical BMC (-7.6%), cortical CSA (-12.6%), and mean cortical thickness (-15.0%), but higher cortical BMD (4.9%) and endosteal circumference (8.0%). Shape-sensitive software supported the finding that Gambian women had larger tibial endosteal circumference (9.8%), thinner mean cortical thickness (-26.5%) but smaller periosteal circumference (-5.6%). Shape-sensitive software revealed that Gambian women had tibiae with shorter maximum width (-7.6%) and thinner cortices (-22% to -41.2%) and more closely resembled a circle or ellipse. Significant differences remained after adjusting for age, height, and weight. In conclusion, shape-sensitive software enhanced the characterization of tibiae in 2 contrasting groups of women.

Structural analysis reveals an amyloid form of the human papillomavirus type 16 E1--E4 protein and provides a molecular basis for its accumulation.

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.

Human papillomavirus type 16 E1 E4-induced G2 arrest is associated with cytoplasmic retention of active Cdk1/cyclin B1 complexes.

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 G2 cell cycle arrest. Here we show that unlike many other G2 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 G2 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 E4-expressing cells. We hypothesize that E1 E4-induced G2 arrest may play a role in creating an environment optimal for viral DNA replication and that loss of E1 E4 expression may contribute to malignant progression.

Functional analysis of the human papillomavirus type 16 E1=E4 protein provides a mechanism for in vivo and in vitro keratin filament reorganization.

High-risk human papillomaviruses, such as human papillomavirus type 16 (HPV16), are the primary cause of cervical cancer. The HPV16 E1=E4 protein associates with keratin intermediate filaments and causes network collapse when expressed in epithelial cells in vitro. Here, we show that keratin association and network reorganization also occur in vivo in low-grade cervical neoplasia caused by HPV16. The 16E1=E4 protein binds to keratins directly and interacts strongly with keratin 18, a member of the type I intermediate-filament family. By contrast, 16E1=E4 bound only weakly to keratin 8, a type II intermediate-filament protein, and showed no detectable affinity for the type III protein, vimentin. The N-terminal 16 amino acids of the 16E1=E4 protein, which contains the YPLLXLL motif that is conserved among supergroup A viruses, were sufficient to target green fluorescent protein to the keratin network. When expressed in the SiHa cervical epithelial cell line, the full-length 16E1=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=E4Delta87-92) 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.