Efficient infection of cells by human papillomaviruses (HPVs) and pseudovirions requires primary interaction with cell surface proteoglycans with apparent preference for species carrying heparan sulfate (HS) side chains. To identify residues contributing to virus/cell interaction, we performed point mutational analysis of the HPV16 major capsid protein, L1, targeting surface-exposed amino acid residues. Replacement of lysine residues 278, 356, or 361 for alanine reduced cell binding and infectivity of pseudovirions. Various combinations of these amino acid exchanges further decreased cell attachment and infectivity with residual infectivity of less than 5% for the triple mutant, suggesting that these lysine residues cooperate in HS binding. Single, double, or triple exchanges for arginine did not impair infectivity, demonstrating that interaction is dependent on charge distribution rather than sequence-specific. The lysine residues are located within a pocket on the capsomere surface, which was previously proposed as the putative receptor binding site. Fab fragments of binding-neutralizing antibody H16.56E that recognize an epitope directly adjacent to lysine residues strongly reduced HS-mediated cell binding, further corroborating our findings. In contrast, mutation of basic surface residues located in the cleft between capsomeres outside this pocket did not significantly reduce interaction with HS or resulted in assembly-deficient proteins. Computer-simulated heparin docking suggested that all three lysine residues can form hydrogen bonds with 2-O-, 6-O-, and N-sulfate groups of a single HS molecule with a minimal saccharide domain length of eight monomer units. This prediction was experimentally confirmed in binding experiments using capsid protein, heparin molecules of defined length, and sulfate group modifications.

Studying viral infection in the laboratory requires viruses, permissive cells, and an assay of infection. None of these elements has been easily available for papillomaviruses. Only a few types of papillomavirus can be obtained in sufficiently large quantity, e.g., bovine papillomavirus types 1 and 4 (BPV1, 4), cottontail rabbit papillomavirus (CRPV), and human papillomavirus types 1 or 11 (HPV1, 11), prepared from cutaneous warts and squamous lesions, respectively. Therefore these viruses have been the first to be used in studies of papillomavirus infection. A few additional viruses, including HPV16, 18, and 31, have been prepared in small quantity using the xenograft and the raft culture system. A more recent system for in vivo studies has been canine oral papillomavirus (CoPV). Because of the strict epitheliotropism of papillomaviruses, matching epithelia were used in early studies of viral infection and neutralization: fetal bovine skin for BPV1, rabbit ear for CRPV, and neonatal human foreskin for HPV11 (Christensen and Kreider, 1990). The infected epithelia were transplanted beneath the renal capsule of athymic mice, where the tissues develop into lesions similar to naturally occurring lesions in the course of three to five months. These lesions have been a valuable source of a small number of authentic virions, most notably HPV11, and this system has been used to identify neutralizing antibodies. However, xenografts are not easily amenable to genetic or biochemical approaches and therefore are of limited value for the analysis of molecular mechanisms of viral entry into cells. The organotypic (raft) epithelial culture system (Meyers et al., 1992) has been most valuable for the analysis of the papillomavirus life cycle (Laimins, this volume) and the synthesis of infectious papillomavirus (Meyers et al., 1997, 2002; Ozbun, 2002), but has not been used for the study of viral entry. Substituting cell culture for skin grafts has been a major advancement for the analysis of papillomavirus infection. Primary keratinocyte, but also keratinocytes

ABSTRACT Papillomaviruses enter cells via endocytosis (H. C. Selinka et al., Virology 299:279-287, 2002). After egress from endosomes, the minor capsid protein L2 accompanies the viral DNA to the nucleus and subsequently to the subnuclear promyelocytic leukemia protein bodies (P. M. Day et al., Proc. Natl. Acad. Sci. USA 101:14252-14257, 2004), suggesting that this protein may be involved in the intracytoplasmic transport of the viral genome. We now demonstrate that the L2 protein is able to interact with the microtubule network via the motor protein dynein. L2 protein was found attached to microtubules after uncoating of incoming human papillomavirus pseudovirions. Based on immunofluorescence and coimmunoprecipitation analyses, the L2 region interacting with dynein is mapped to the C-terminal 40 amino acids. Mutations within this region abrogating the L2/dynein interaction strongly reduce the infectivity of pseudoviruses, indicating that this interaction mediates the minus-end-directed transport of the viral genome along microtubules towards the nucleus.

Abstract Background Infections with papillomaviruses induce type-specific immune responses, mainly directed against the major capsid protein, L1. Based on the propensity of the L1 protein to self-assemble into virus-like particles (VLPs), type-specific vaccines have already been developed. In order to generate vaccines that target a broader spectrum of HPV types, extended knowledge of neutralizing epitopes is required. Despite the association of human papillomavirus type 33 (HPV33) with cervical carcinomas, fine mapping of neutralizing conformational epitopes on HPV33 has not been reported yet. By loop swapping between HPV33 and HPV16 capsid proteins, we have identified amino acid sequences critical for the binding of conformation-dependent type-specific neutralizing antibodies to surface-exposed hyper variable loops of HPV33 capsid protein L1. Results Reactivities of monoclonal antibodies (mAbs) H33.B6, H33.E12, H33.J3 and H16.56E with HPV16:33 and HPV33:16 hybrid L1 VLPs revealed the complex structures of their conformational epitopes as well as the major residues contributing to their binding sites. Whereas the epitope of mAb H33.J3 was determined by amino acids (aa) 51–58 in the BC loop of HPV33 L1, sequences of at least two hyper variable loops, DE (aa 132–140) and FGb (aa 282–291), were found to be essential for binding of H33.B6. The epitope of H33.E12 was even more complex, requiring sequences of the FGa loop (aa 260–270), in addition to loops DE and FGb. Conclusion These data demonstrate that neutralizing epitopes in HPV33 L1 are mainly located on the tip of the capsomere and that several hyper variable loops contribute to form these conformational epitopes. Knowledge of the antigenic structure of HPV is crucial for designing hybrid particles as a basis for intertypic HPV vaccines.

adenovirus type 3 inverted terminal repeat deletion 188 -spongiform encephalopathy 56 Bunyaviridae 318 Caprine arthritis encephalitis virus 177 Capsid protein(s) 287 --VP1 308 Carmovirus 160 Cell-to-cell infection 164 Cervical carcinoma 287 -screening 111 Chemical coupling of peptides to virus capsids 362 Chimera 371 Chimeric virus 233 Chronic hepatitis C 11, 33, 105 Cirrhosis and hepatocellular carcinoma 11 Conformational change 142 Core protein 105 Cowpea mosaic virus 362 Cr release 290 Cyclophosphamide 119 Cytotoxic T cell 300

Nuclear domains (ND) 10 are associated with proteins implicated in transcriptional regulation, growth suppression, and apoptosis. We now show that the minor capsid protein L2 of human papillomavirus (HPV) type 33 induces a reorganization of ND10-associated proteins. Whereas the promyelocytic leukemia protein, the major structural component of ND10, was unaffected by L2, Sp100 was released from ND10 upon L2 expression. The total cellular amount of Sp100, but not of Sp100 mRNA, decreased significantly, suggesting degradation of Sp100. Proteasome inhibitors induced the dispersal of Sp100 and inhibited the nuclear translocation of L2. In contrast to Sp100, Daxx was recruited to ND10 by L2 expression. Coimmunoprecipitation demonstrated interaction of the two proteins. L2-induced reorganization of ND10 was observed both in cell culture and in natural HPV lesions. The differential change in protein composition observed provides further evidence to suggest that the ND10-associated proteins are an important interface of viral life cycle and host cell.

Using pseudoinfection of cell lines, we demonstrate that cell surface heparan sulfate is required for infection by human papillomavirus type 16 (HPV-16) and HPV-33 pseudovirions. Pseudoinfection was inhibited by heparin but not dermatan or chondroitin sulfate, reduced by reducing the level of surface sulfation, and abolished by heparinase treatment. Carboxy-terminally deleted HPV-33 virus-like particles still bound efficiently to heparin. The kinetics of postattachment neutralization by antiserum or heparin indicated that pseudovirions were shifted on the cell surface from a heparin-sensitive into a heparin-resistant mode of binding, possibly involving a secondary receptor. Alpha-6 integrin is not a receptor for HPV-33 pseudoinfection.

The envelope proteins of hepadnaviruses are highly cross-linked by disulfide bonds in complete virions and 20 nm subviral envelope particles. We have previously shown which of the cysteines in the envelope proteins of the human hepatitis B virus (HBV) are essential for assembly and secretion of 20 nm particles and for the structure of the major antigenic determinants (HBsAg). Now we have analyzed the intermolecular disulfide bonds between S proteins. We have constructed mutants lacking cysteines and have analyzed their capacity for oligomerization in COS-7 cells. We demonstrate that C121 and C147 located in the second hydrophilic region carrying the major antigenic determinants of the HBV S protein participate in intermolecular disulfide bonding. A disulfide bond involving C124 blocks the accessibility of arginine/lysine at position 122, as shown by trypsin digestion of cysteine mutants. Alkylation studies using N-ethyl-maleimide indicate that C76, C90, and/or C221 carry the only free sulfhydryl group(s) present in 20 nm particles secreted from cell lines.

AIDS 72 Antigen delivery system 93 Assembly 32 Baculovirus 40, 126 - vectors 62 Bluetongue virus 62 Carcinogenesis 54 CD8+cytotoxicTlymphocytes 111 Chimera 126 Chimeric proteins 9, 16 - viruses 72 Circumsporozoite antigen 104 Cloning 9, 16 Coat proteins 9 Combinatorial libraries 72 Core-like particles 62 Core particles 104 Cowpea mosaic virus 79 - plants 79 Cytotoxic T lymphocytes 120 Disulfide bonds 49 DNA-based immunization 120 DNA vaccine 120 Endocytic vesicles 49 Epitope presentation 104 Epitopes 126 Gag proteins 32 Genetic engineering 9, 16 HBeAg 104 Hepatitis B core antigen 16,104 - surface antigen 23 - virus 23, 104, 120, 126 - – small surface antigen 111 - – surface protein, large 23 Human immunodeficiency virus 32, 40 - – typel 79,93 Immunodominant epitopes 16 Immunoelectron microscopy 40 Immunogenicity 93 Internet, DNA vaccines 120 Luteinizmg hormone releasing hormone 85 Malaria 85 …