Papilloma virus assembly

The papillomavirus life cycle. Identification of proteins encoded by the L1 and L2 open reading frames of human papillomavirus 1a. Identification of the human papilloma virus-1a E4 gene products. Characterization of events during the late stages of HPV16 infection in vivo using high-affinity synthetic Fabs to E4. The human papilloma virus E7 oncoprotein is able to bind to the retinoblastoma gene product. Do human papillomaviruses target epidermal stem cells?

Differential antibody responses to a distinct region of human papillomavirus minor capsid proteins. Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial.

Structural polypeptides of rabbit, bovine, and human papillomaviruses. Human papillomavirus DNA: physical map. The positively charged termini of L2 minor capsid protein required for bovine papillomavirus infection function separately in nuclear import and DNA binding. Human papillomavirus type 31 E5 protein supports cell cycle progression and activates late viral functions upon epithelial differentiation. Interactions between papillomavirus L1 and L2 capsid proteins.

DNA-induced structural changes in the papillomavirus capsid. Evidence for a switch in the mode of human papillomavirus type 16 DNA replication during the viral life cycle.

A protective and broadly cross-neutralizing epitope of human papillomavirus L2. Human papillomavirus-associated head and neck cancer is a distinct epidemiologic, clinical, and molecular entity.

Human papillomavirus infection requires cell surface heparan sulfate. Identification of a differentiation-inducible promoter in the E7 open reading frame of human papillomavirus type 16 HPV in raft cultures of a new cell line containing high copy numbers of episomal HPV DNA. Self-assembly of human papillomavirus type 1 capsids by expression of the L1 protein alone or by coexpression of the L1 and L2 capsid proteins.

Three-dimensional structure of vaccinia virus-produced human papillomavirus type 1 capsids. Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial.

Human papillomavirus and oral cancer: the International Agency for Research on Cancer multicenter study. Virol J 4 Enhancers and trans-acting E2 transcriptional factors of papillomaviruses. The minor capsid protein L2 contributes to two steps in the human papillomavirus type 31 life cycle.

Detection of human papillomavirus DNA in laryngeal squamous cell carcinomas by polymerase chain reaction. Differentiation-induced and constitutive transcription of human papillomavirus type 31b in cell lines containing viral episomes. Human papillomaviruses. Thiol-reactive reagents inhibits [ sic ] intracellular trafficking of human papillomavirus type 16 pseudovirions by binding to cysteine residues of major capsid protein L1. Cys9, Cys and Cys of simian virus 40 Vp1 are essential for inter-pentamer disulfide-linkage and stabilization in cell-free lysates.

Adhesive properties of human basal epidermal cells: an analysis of keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells.

Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic. Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles. Role for Wee1 in inhibition of G2-to-M transition through the cooperation of distinct human papillomavirus type 1 E4 proteins.

Neutralization of HPV16, 18, 31, and 58 pseudovirions with antisera induced by immunizing rabbits with synthetic peptides representing segments of the HPV16 minor capsid protein L2 surface region.

The rolling circle. Transcriptional activity of human papillomavirus type 31b enhancer is regulated through synergistic interaction of AP1 with two novel cellular factors. Bovine papillomavirus type 1: from clathrin to caveolin. Proteins present in bovine papillomavirus particles. Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation. Pathogenesis of human papillomaviruses in differentiating epithelia.

Risk factors for invasive squamous cell carcinoma of the vulva and vagina—population-based case-control study in Denmark. Evidence for the coexistence of two genital HPV types within the same host cell in vitro.

Human papillomavirus type 45 propagation, infection, and neutralization. Propagation, infection, and neutralization of authentic HPV16 virus. Brd4 is required for e2-mediated transcriptional activation but not genome partitioning of all papillomaviruses. Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation. Atomic model of the papillomavirus capsid. Targeting the E1 replication protein to the papillomavirus origin of replication by complex formation with the E2 transactivator.

Human papillomavirus infection as a risk factor for squamous-cell carcinoma of the head and neck. Human papillomavirus type 16 E7 regulates E2F and contributes to mitogenic signalling. Human papillomavirus infection and other risk factors for cervical neoplasia: a case-control study.

Open reading frames E6 and E7 of bovine papillomavirus type 1 are both required for full transformation of mouse C cells. Detection of human papillomavirus type 16 DNA in carcinomas of the palatine tonsil. Human papillomavirus DNA in oral squamous cell carcinomas and normal mucosa. Characterization of late gene transcripts expressed during vegetative replication of human papillomavirus type 31b. Temporal usage of multiple promoters during the life cycle of human papillomavirus type 31b.

Two novel promoters in the upstream regulatory region of human papillomavirus type 31b are negatively regulated by epithelial differentiation. Suprabasal change and subsequent formation of disulfide-stabilized homo- and hetero-dimers of keratins during esophageal epithelial differentiation.

Mutational analyses of differentiation-dependent human papillomavirus type 18 enhancer elements in epithelial raft cultures of neonatal foreskin keratinocytes. Human papillomavirus type 31b infection of human keratinocytes does not require heparan sulfate. The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similar to those of adenovirus E1A. Production of infectious human papillomavirus independently of viral replication and epithelial cell differentiation.

Invasion of host cells by JC virus identifies a novel role for caveolae in endosomal sorting of noncaveolar ligands. Cleavage of the papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for infection.

Reconstitution of pubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein E6AP.

Mutational analysis of cis elements involved in E2 modulation of human papillomavirus type 16 P97 and type 18 P promoters. Expression of human papillomavirus type 11 L1 protein in insect cells: in vivo and in vitro assembly of viruslike particles. Human papillomavirus HPV type 11 recombinant virus-like particles induce the formation of neutralizing antibodies and detect HPV-specific antibodies in human sera. Papillomavirus assembly requires trimerization of the major capsid protein by disulfides between two highly conserved cysteines.

Simian Virus 40 depends on ER protein folding and quality control factors for entry into host cells. Pang, S. Frye, P. Jensen, D. Papillomavirus-like particles induce acute activation of dendritic cells.

Liu, W. Gissmann, X. Sun, A. Kanjanahaluethai, M. Muller, J. Doorbar, and J. Sequence close to the N-terminus of L2 protein is displayed on the surface of bovine papillomavirus type 1 virions. Loeken, M. Bikel, D. Livingston, and J. Cell 55 : McBride, A. Bolen, and P. Phosphorylation sites of the E2 transcriptional regulatory proteins of bovine papillomavirus type 1. Meyers, C. In vitro systems for the study and propagation of human papillomaviruses.

Muller, M. Gissmann, R. Cristiano, X. Sun, I. Frazer, A. Jenson, A. Alonso, H. Zentgraf, and J. Papillomavirus capsid binding and uptake by cells from different tissues and species. Penrose, K. Proteasome-mediated degradation of the papillomavirus E2-TA protein is regulated by phosphorylation and can modulate viral genome copy number. Roden, R. Kirnbauer, F. Booy, J. Jessie, D. In vitro generation and type-specific neutralization of a human papillomavirus type 16 virion pseudotype.

Weissinger, D. Henderson, F. Booy, R. Kirnbauer, J. Mushinski, D. Neutralization of bovine papillomavirus by antibodies to L1 and L2 capsid proteins.

Rudolf, M. Fausch, D. Da Silva, and W. Human dendritic cells are activated by chimeric human papillomavirus type virus-like particles and induce epitope-specific human T cell responses in vitro. Schwartz, S. Regulation of human papillomavirus late gene expression. Upsala J. Shi, W. Liu, Y. Huang, and L. Papillomavirus pseudovirus: a novel vaccine to induce mucosal and systemic cytotoxic T-lymphocyte responses.

Smith, D. Problems of translating heterologous genes in expression systems: the role of tRNA. Sokolowski, M. Tan, M. Jellne, and S. Stauffer, Y. Raj, K. Masternak, and P. Infectious human papillomavirus type 18 pseudovirions. Stubenrauch, F.

Human papillomavirus life cycle: active and latent phases. Cancer Biol. Tai, H. Smith, P. Sharp, and J. Sequence heterogeneity in closed simian virus 40 deoxyribonucleic acid. Tan, W. Felber, A. Zolotukhin, G. Pavlakis, and S. Efficient expression of the human papillomavirus type 16 L1 protein in epithelial cells by using Rev and the Rev-responsive element of human immunodeficiency virus or the cis -acting transactivation element of simian retrovirus type 1. Touze, A. In vitro gene transfer using human papillomavirus-like particles.

Nucleic Acids Res. Unckell, F. Streeck, and M. Generation and neutralization of pseudovirions of human papillomavirus type Volkin, D. Shi, and G. Stabilized human papillomavirus formulations. Yeager, M. Aste-Amezaga, D. Brown, M. Martin, M. Shah, J. Cook, N. Christensen, C. Ackerson, R. Lowe, J. Keller, and K. Neutralization of human papillomavirus HPV pseudovirions: a novel and efficient approach to detect and characterize HPV neutralizing antibodies.

Yoshiike, K. Heterogeneous defective virions produced by successive undiluted passages. Virology 34 : Zhao, K. Frazer, W. Jun Liu, M. V5 and H H5, H J4 and H O7 were significantly reduced or abolished in the case of H A pair wise mapping study delineated the approximate footprint size and relative overlaps of the four mAbs Figure 4. An image of the sample Figure 5A shows that in addition to particles with the expected 55 nm diameter a substantial fraction of the particles have a smaller diameter of nm and a few particles with a nm diameter are also visible.

V5 Fab show that all particles are densely decorated by the antibody regardless of their size Figure 5D-F. However, we cannot rule out that other antibodies may be sensitive to structural differences between the and nm particles.

B , C Larger scale images of individual VLP particles show that the individual capsomeres are arranged with a high degree of order. V5 Fab shows a similar field of particles densely decorated with antibody fragment. E , F Larger scale images of individual particles show that the antibody attachment is independent of the diameter of the individual particles.

Scale bar is nm. A significant fraction of the particles in the cryoEM images have a diameter and capsomere arrangement that is consistent with the atomic force microscopy AFM data Figure 6A, B. The atomic homology model of the HPV16 VLP was constructed by superimposing the crystal structure of the HPV16 L1 pentamer [ 38 ] onto the core pentamer in the asymmetric unit of the high-resolution cryoEM structure of bovine papillomavirus type [ 39 ]. The scale bar is 50 nm long.

The model was generated as described in the Methods. A A single subunit of L1 in the standard orientation. Residues are in red and form part of the H J4 epitopes. Residues are in pink and form part of the H A2, H E70 epitopes. Residues are in dark green and form the H H5 epitope. Residues are in blue and form the H I23 epitope.

B A pentameric L1 capsomere with the same coloring scheme as in A and with the subunit in the foreground in approximately the same orientation as in A. The atomic model was generated as described in the Methods.

The epitopes of the antibodies studied here have been coarsely mapped by measuring antibody reactivity against overlapping synthetic linear peptides see Table 1 for references. The H H5 and H O7 epitopes were mapped in this manner to residues , in the EF loop [ 23 ]. While H H5 showed significant binding on the purified VLPs from yeast, H O7 binding Figures 3 , 4. Residues and are not solvent-accessible in the pentameric L1 capsomere and are therefore unlikely to be part of any antibody epitope Figure 7B.

H5 binding or for optimal H O7 binding. Moreover, we note that in our atomic model Ser forms a hydrogen bond with His from the C-terminal arm of an adjacent capsomere Figure 8B. This interaction results in the occlusion of Ser upon VLP assembly.

Fine mapping of antibody epitopes on the HPV16 capsid. A Same view as in Figure 7B, with the H H5 epitope residues shown in red. Side chains in the C-terminal arms of neighboring capsomeres that partially occluding the H H5 epitope are shown as colored spheres not red. The conserved disulfide bond between Cys and Cys is highlighted with green spheres. The HPVspecific Val is shown in black.

B Close-up of the H H5 epitope from the subunit in the foreground of A with the same coloring scheme as in A. C Same view as in A , with the H J4 epitopes highlighted: residues H J4 epitope only are in dark green, residues in all four antibody epitopes are in red, residues not part of the H J4 epitope are in magenta. Residues are in pink not part of the H J4 epitope.

Glu is shown in black. D Same view as in A , with the H I23 epitope residues in red. J4 is a weakly neutralizing antibody originally identified by the immunoreactivity of the sera from HPV infected patients with cervical cancer [ 17 , 30 ]. Like H O7, H J4 epitope has been mapped to residues [ 17 , 25 ], in the first part of the FG loop, which partially overlaps with the epitopes of H The epitope is located in a loop on the crown of the L1 capsomere Figure 7B and is conserved across several types.

Most of the residues are exposed on the viral surface in the HPV16 VLP atomic model, with the exception of residues and These residues form packing interactions that anchor the loop onto the core of the capsomere.

Glu is partially occluded by Arg, with which it forms a salt bridge. Additionally, accessibility of Glu is restricted by a number of other neighboring side chains including that of Gln from the C-terminal arm of an adjacent capsomere Figure 8C.

We conclude that the partial occlusion of Glu upon completion of VLP assembly may be responsible for the decrease in binding to H J4, and that the epitope may also include residues and see Discussion. In contrast to H O7 and H J4, antibodies H We have selectively deleted each of the three helices from papillomavirus L1.

The biochemical and structural characterization of the deletion mutant proteins demonstrated the important roles of these C-terminal helices in the folding and assembly of L1 capsid. The mutant proteins with partial or entire h4 deletion, D1-L1 and D2-L2, were similar to the full-length wild-type L1 in protein expression and behavior by size-exclusion chromatography.

Like wild type, D1-L1 and D2-L2 were expressed at quite high levels, and a good fraction of each expressed proteins was soluble and could be purified as pentamers Fig.

This result demonstrated that h4 is not required for L1 folding and pentamerization. Because only a GST-fusion can bind the glutathione resin of the column used for purification, the smaller protein bands must contain intact GST plus L1 fragments of various lengths due to C-terminal degradation. Thrombin treatment of these GST-fusions did not produce discreet L1 fragments, indicating that both D3-L1 and D4-L1 were unfolded and sensitive to protease degradation.

Consistent with this severe degradation of these deletion mutants of L1 is that there are six potential thrombin cleavage sites within the HPV16 L1 sequence and many Arg and Lys residues can serve as cleavage sites for other proteases. Thus, we conclude that h2 and h3 are important for the folding and integrity of HPV L1 structure. Interestingly, a small helix at the C-terminus, h5 Fig.

A common feature of h2, h3, and h5 in the x-ray structure [ 2 ] is that all helices have a highly hydrophobic side that packs with the core structure of L1. Disrupting these hydrophobic packing interactions will obviously affect the folding of L1.

This result confirmed that the L1 structure is conserved among different HPV types, which is not surprising considering the amino acid sequences of L1 proteins are highly conserved. We predict that the same D2 deletion of any other papillomavirus L1 should have the same properties. If the deletion mutants assemble into particles, these particles might be unstable to the acidic conditions used during EM sample preparation, which requires uranyl acetate treatment.

To rule out this possibility, size-exclusion column chromatography was used to analyze the deletion constructs after assembly treatment Fig. The size-exclusion analysis showed that, in contrast to the full length L1, the D1-L1 and D2-L2 mutants lacking h4 failed to form assemblies larger than the pentamers, confirming the results from EM study.

In conclusion, we have demonstrated that the structural elements h2, h3, and h4 near the C-terminal end of L1 are important for the assembly of papillomaviruses into particles. The h2 and h3 regions are essential for L1 folding and pentamer formation, whereas the h4 region is indispensable for the assembly of not only T1, but also of the T7 virus-like particle. The standard molecular cloning methods, including PCR amplification, enzyme digestion, DNA end ligation, and transformation, was used to obtain the deletion mutants of L1 as shown in Fig.

All deletion clones were confirmed by sequencing the whole DNA insert to ensure the correct deletion and the wilt type sequences for this study. The protein expression and purification of all L1 deletion mutants was carried out essentially as described preciously [ 1 , 2 ]. Briefly, 0. The mixture was incubated at RT for one hour with gentle shaking, and then dialyzed against three changes of buffer over an 18 hour period. Usually, 10 ml glutathione resin was used for supernatant from 12 liter cell culture.

Wild-type L1 can assemble into particles under known in vitro assembly conditions [ 1 , 2 ]. The assembly reaction was performed by incubating purified L1 protein in assembly buffer 1 M NaCl, 40 mM sodium acetate, pH 5.

The protein peaks were detected by an UV monitor at a wavelength of nM. In Fields Virology. Volume 1. Forth edition. Google Scholar. Volume 2.