The E6 protein from high-risk human papillomaviruses appears necessary for persistence of viral episomes in cells but the underlying mechanism is unclear. E6 has many activities, including its ability to bind and degrade PDZ domain-containing proteins, such as hScrib. However little is known about the role of these interactions for E6 function and the viral life cycle. We now show that the levels of expression of wild-type E6 are increased in the presence of hScrib whilst a mutant E6 protein lacking the PDZ-binding motif is found at lower levels as it is turned over more rapidly by the proteasome. This correlates with an inability of genomes containing this mutation to be maintained as episomes. These results show that E6 association with certain PDZ domain-containing proteins can stabilize the levels of E6 expression and provides one explanation as to how the PDZ-binding capacity of E6 might contribute to genome episomal maintenance.

ABSTRACT The human papillomavirus (HPV) type 16 E1^E4 (16E1^E4) protein is expressed in the middle to upper layers of infected epithelium and has several roles within the virus life cycle. It is apparent that within the epithelium there are multiple species of 16E1^E4 that differ in length and/or degree of phosphorylation and that some or all of these can associate with the cellular keratin networks, leading to network disruption. We show here that the cellular cysteine protease calpain cleaves the 16E1^E4 protein after amino acid 17 to generate species that lack the N terminus. These C-terminal fragments are able to multimerize and form amyloid-like fibers. This can lead to accumulation of 16E1^E4 and disruption of the normal dynamics of the keratin networks. The cleavage of E1^E4 proteins by calpain may be a common strategy used by α-group viruses, since we show that cleavage of type 18 E1^E4 in raft culture is also dependent on calpain. Interestingly, the cleavage of 16E1^E4 by calpain appears to be highly regulated as differentiation of HPV genome-containing cells by methylcellulose is insufficient to induce cleavage. We hypothesize that this is important since it ensures that the formation of the amyloid fibers is not prematurely triggered in the lower layers and is restricted to the upper layers, where calpain is active and where disruption of the keratin networks may aid virus release.

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.

ABSTRACT The E1^E4 protein of human papillomavirus type 16 (HPV16) causes cytokeratin reorganization in the middle and upper epithelial layers and is thought to contribute to multiple facets of the virus life cycle. Although little is known as to how HPV16 E1^E4 (16E1^E4) functions are controlled following the first expression of this protein, the finding that low-risk E1^E4 proteins can be phosphorylated in vivo suggests an important role for kinases. Here, we show that 16E1^E4 is phosphorylated by cyclin-dependent kinase 1 (CDK1) and CDK2, extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and PKC α, with CDK1/2 serine 32 and ERK threonine 57 phosphorylations representing the two primary events seen in cells in cycle. Interestingly, T57 phosphorylation was found to trigger a structural change in the 16E1^E4 protein that compacts the central fold region, leading to an increase in 16E1^E4 stability and overall abundance in the cell. When compared to wild-type 16E1^E4, a T57D phosphomimic was found to have greatly enhanced keratin-binding ability and an ability to modulate the binding of the unphosphorylated form, with keratin binding protecting the T57-phosphorylated form of 16E1^E4 from proteasomal degradation. In HPV16 genome-containing organotypic rafts, the T57-phosphorylated form was specifically detected in the intermediate cell layers, where productive infection occurs, suggesting that T57 phosphorylation may have a functional role at this stage of the viral life cycle. Interestingly, coexpression with 16E5 and ERK activation enhanced T57 phosphorylation, suggesting that E1^E4 and E5 may work together in vivo. Our data suggest a model in which the expression of 16E5 from the major E1^E4-E5 mRNA promotes T57 phosphorylation of E1^E4 and keratin binding, with dephosphorylation occurring following the switch to late poly(A) usage. Other forms of E1^E4, with alternative functional roles, may then increase in prevalence in the upper layers of the epithelium.

The E4 (also called E1^E4) and E2 proteins of human papillomavirus type 16 are thought to be expressed within the same cells of a lesion, and their open reading frames overlap, suggesting that they may have a functional relationship. We have examined the effect of co-expression of these two proteins and found that each enhances the level of the other. We also identified the N-terminus of E2 as the first example of a viral protein that directly binds the HPV16 E1^E4 protein. This appears to result in the E2 becoming less soluble and promotes its relocation from the nucleus to the cytoplasm. In addition, the turnover of the E2 protein is decreased in the presence of E1^E4. All this raises the possibility that E1^E4 acts to influence E2 activity by varying the amount of available E2 in the cell.

正 The HPV16 E4 protein has a number of roles during viral infection. E4 contributes to efficient genome amplification in the upper epithelial layers, and can compromise the structure of the infected cell by associating with keratin filaments and the cornified envelope. Our recent work has shown that the structure of the 16E4 protein is highly regulated by post-translational modification, and that this controls the assembly of 16E4 monomers into amyloid-like fibres, which are thought to act as keratin cross-linkers within the cell.

There is increasing evidence that viral infection, expression of viral protein or the presence of viral DNA causes the host cell cycle to arrest during G2/M. The mechanisms used by viruses to cause arrest vary widely; some involve the activation of the cellular pathways that induce arrest in response to DNA damage, while others use completely novel means. The analysis of virus-mediated arrest has not been proven easy, and in most cases the consequences of arrest for the virus life cycle are not well defined. However, a number of effects of arrest are being investigated and it will be interesting to see to what extent perturbation of the G2/M transition is involved in viral infections.

The human papillomavirus type 16 E1^E4 protein is expressed abundantly in cells supporting viral DNA amplification, but its expression is lost during malignant progression. In cell culture, 16E1^E4 causes G2 cell cycle arrest by associating with and preventing the nuclear entry of Cdk1/cyclin B1 complexes. Here, we show that 16E1^E4 is also able to associate with cyclin A and Cdk2 during the G2 phase of the cell cycle. Only a weak association was apparent during S-phase, and progression through S-phase appeared unaffected. As with cyclin B1, the interaction of 16E1^E4 with cyclin A is dependent on residues T22/T23 and results in the accumulation of cyclin A in the cytoplasm where it colocalizes with 16E1^E4. 16E1^E4 serine 32 was found to be phosphorylated by Cdk2/cyclin A. We hypothesize that the interaction of 16E1^E4 with cyclin A may serve to increase the efficiency with which 16E1^E4 is able to prevent mitotic entry.

This text is a comprehensive review of papillomaviruses both human and animal. The book has 25 chapters and 420 pages. It is mostly concerned with human papillomaviruses with chapters ranging from phylogenicity, epidemiology, gene expression and DNA replication. There are individual chapters on essentially each of the early proteins, raft cultures, the life cycle divided into the early and late phases, and transgenic mouse models. There are chapters on the types of cancer that human papillomavirus (HPV) causes in addition to cervical cancer and HPV vaccines. Finally, there are chapters on the bovine papillomaviruses and cottontail rabbit papillomaviruses.

Identification of New Papillomavirus Types Ethel-Michele de Villiers, Corinna Whitley, and Karin Gunst Identification of HPV Variants John Cason, Jon Bible, and Christine Mant Histochemical Analysis of Cutaneous HPV-Associated Lesions Kiyofumi Egawa Histological Analysis of Cervical Intraepithelial Neoplasia Michael Babawale, Rashmi Seth, Adam Christian, Wessam Al-Utayem, Ragini Narula, and David Jenkins Detection of Papillomavirus Proteins and DNA in Paraffin-Embedded Tissue Sections Woei Ling Peh and John Doorbar Detection and Quantitation of HPV Gene Expression Using Real-Time PCR Rashmi Seth, John Rippin, Li Guo, and David Jenkins Analysis of p16INK4a and Integrated HPV Genomes as Progression Markers Svetlana Vinokurova, Nicolas Wentzensen, and Magnus von Knebel Doeberitz Use of Biomarkers in the Evaluation of CIN Grade and Progression of Early CIN Jan P. A. Baak and Arnold-Jan Kruse HPV DNA Detection and Typing in Cervical Scrapes Peter J. F. Snijders, Adriaan J. C. van den Brule, Marcel V. Jacobs, Rene P. Pol, and Chris J. L. M. Meijer HPV DNA Detection and Typing in Inapparent Cutaneous Infections and Premalignant Lesions Maurits de Koning, Linda Struijk, Mariet Feltkamp, and Jan ter Schegget Establishing HPV-Containing Keratinocyte Cell Lines From Tissue Biopsies Margaret Anne Stanley Using an Immortalized Cell Line to Study the HPV Life Cycle in Organotypic 'Raft' Cultures Paul F. Lambert, Michelle A. Ozbun, Asha Collins, Sigrid Holmgren, Denis Lee, and Tomomi Nakahara Differentiation of HPV-Containing Cells Using Organotypic 'Raft' Culture or Methylcellulose Regina Wilson and Laimonis A. Laimins Propagation of Infectious, High-Risk HPV in Organotypic 'Raft' Culture Margaret E. McLaughlin-Drubin and Craig Meyers Retrovirus-Mediated GeneTransfer to Analyze HPV Gene Regulation and Protein Functions in Organotypic 'Raft' Cultures N. Sanjib Banerjee, Louise T. Chow, and Thomas R. Broker The HPV Xenograft Severe Combined Immunodeficiency Mouse Model William Bonnez The Cottontail Rabbit Papillomavirus Model of High-Risk HPV-Induced Disease Janet L. Brandsma Studying the HPV Life Cycle in 3A Trophoblasts and Resulting Pathophysiology Yong Liu, Hong You, and Paul L. Hermonat Replication and Encapsidation of Papillomaviruses in Saccharomyces cerevisiae Peter C. Angeletti Analysis of the Regulation of Viral Transcription Bernd Gloss, Mina Kalantari, and Hans-Ulrich Bernard Analysis of HPV Transcription by RPA Jason M. Bodily and Craig Meyers Analysis of Regulatory Motifs Within HPV Transcripts Sarah A. Cumming and Sheila V. Graham Detection of HPV Transcripts by Nested RT-PCR Christine Mant, Barbara Kell, and John Cason Analysis of HPV DNA Replication Using Transient Transfection and Cell-Free Assays Biing Yuan Lin, Thomas R. Broker, and Louise T. Chow Detection and Quantitation of HPV DNA Replication by Southern Blotting and Real-Time PCR Iain M. Morgan and Ewan R. Taylor Analysis of E7/Rb Associations Sandra Caldeira, Wen Dong, and Massimo Tommasino Transformation Assays for HPV Oncoproteins Paola Massimi and Lawrence Banks Analysis of Adeno-Associated Virus and HPV Interaction Paul L. Hermonat, Hong You, C. Maurizio Chiriva-Internati, and Yong Liu In Vitro Assays of Substrate Degradation Induced by High-Risk HPV E6 Oncoproteins Miranda Thomas and Lawrence Banks Measuring the Induction or Inhibition of Apoptosis by HPV Proteins Anna M. Kowalczyk, Geraldine E. Roeder, Katie Green, David J. Stephens, and Kevin Gaston Codon Optimization of Papillomavirus G

ABSTRACT 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 ∧ E4Δ87-92) 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.

Papillomaviruses are small, DNA viruses that infect epithelial tissue and cause hyperproliferative lesions. The majority of human papillomaviruses (HPV) are associated only with benign lesions (warts). However, a small subset of HPVs, e.g. type 16, are associated with lesions that may become malignant, and these viruses are responsible for greater than 99 % of cervical cancers. The expression of the HPV16 E1^E4 protein occurs late during infection and correlates with the onset of vegetative viral DNA replication. Despite being a highly abundant protein that can associate with the cytokeratins, the role of E1^E4 in the virus life cycle is unknown. The work described in this thesis shows that the 16E1^E4 protein can also cause G2 cell cycle arrest. This is apparent both in human cervical epithelial cancer cells and in the fission yeast Schizosaccharomyces pombe. In S.pombe, arrest appears not to be mediated via the checkpoint pathways that prevent entry into mitosis by maintenance of the inhibitory phosphorylation on the mitosis promoting factor (MPF) complex. The region of 16E1^E4 responsible for S.pombe arrest lies between amino acids 17 to 45, and arrest is critically dependent on the presence of a threonine residue at position 23. Experiments using recombinant adenoviruses to express 16E1^E4 in human cells show that arrest occurs prior to mitotic chromosome condensation and that a potentially active MPF complex is relocalized to the cytokeratins in 16E1^E4-expressing cells. In human cells, arrest appears to be mediated by inhibition of the nuclear import of cyclin B, a component of the MPF complex. Analysis of mutant 16E1^E4 in human cells suggests that a different mechanism may operate in S.pombe cells. It is suggested that the role of the E1^E4 protein in the virus life cycle is to prevent multiple rounds of cellular replication thereby favouring replication of the viral genome. It is also proposed that the loss of the E4 open reading frame is a predisposing factor for the development of cervical cancer.