In vitro generation and type-specific neutralization of a human papillomavirus type 16 virion pseudotype.

We report a system for generating infectious papillomaviruses in vitro that facilitates the analysis of papillomavirus assembly, infectivity, and serologic relatedness. Cultured hamster BPHE-1 cells harboring autonomously replicating bovine papillomavirus type 1 (BPV1) genomes were infected with recombinant Semliki Forest viruses that express the structural proteins of BPV1. When plated on C127 cells, extracts from cells expressing L1 and L2 together induced numerous transformed foci that could be specifically prevented by BPV neutralizing antibodies, demonstrating that BPV infection was responsible for the focal transformation. Extracts from BPHE-1 cells expressing L1 or L2 separately were not infectious. Although Semliki Forest virus-expressed L1 self-assembled into virus-like particles (VLPs), viral DNA was detected in particles only when L2 was coexpressed with L1, indicating that genome encapsidation requires L2. Expression of human papillomavirus type 16 (HPV16) L1 and L2 together in BPHE-1 cells also yielded infectious virus. These pseudotyped virions were neutralized by antiserum to HPV16 VLPs derived from European (114/K) or African (Z-1194) HPV16 variants but not by antisera to BPV VLPs, to a poorly assembling mutant HPV16 L1 protein, or to VLPs of closely related genital HPV types. Extracts from BPHE-1 cells coexpressing BPV L1 and HPV16 L2 or HPV16 L1 and BPV L2 were not infectious. We conclude that (i) mouse C127 cells express the cell surface receptor for HPV16 and are able to uncoat HPV16 capsids; (ii) if a papillomavirus DNA packaging signal exists, then it is conserved between the BPV and HPV16 genomes; (iii) functional L1-L2 interaction exhibits type specificity; and (iv) protection by HPV virus-like particle vaccines is likely to be type specific.     

Arrangement of L2 within the papillomavirus capsid

Papillomaviruses are a family of nonenveloped DNA tumor viruses. Some sexually transmitted human papillomavirus (HPV) types, including HPV type 16 (HPV16), cause cancer of the uterine cervix. Papillomaviruses encode two capsid proteins, L1 and L2. The major capsid protein, L1, can assemble spontaneously into a 72-pentamer icosahedral structure that closely resembles native virions. Although the minor capsid protein, L2, is not required for capsid formation, it is thought to participate in encapsidation of the viral genome and plays a number of essential roles in the viral infectious entry pathway. The abundance of L2 and its arrangement within the virion remain unclear. To address these questions, we developed methods for serial propagation of infectious HPV16 capsids (pseudoviruses) in cultured human cell lines. Biochemical analysis of capsid preparations produced using various methods showed that up to 72 molecules of L2 can be incorporated per capsid. Cryoelectron microscopy and image reconstruction analysis of purified capsids revealed an icosahedrally ordered L2-specific density beneath the axial lumen of each L1 capsomer. The relatively close proximity of these L2 density buttons to one another raised the possibility of homotypic L2 interactions within assembled virions. The concept that the N and C termini of neighboring L2 molecules can be closely apposed within the capsid was supported using bimolecular fluorescence complementation or "split GFP" technology. This structural information should facilitate investigation of L2 function during the assembly and entry phases of the papillomavirus life cycle.     

Bovine papillomavirus type 1 infection is mediated by SNARE syntaxin 18

Events that lead to viral infections include the binding of the virus to the target cells, internalization of the virus into the cells, and the ability of the viral genome to be expressed. These steps are mediated by cellular and viral proteins and are temporally regulated. The papillomavirus capsid consists of two virally encoded capsid proteins, L1 and L2. Much is known about the role of the major capsid protein L1 compared to what is known of the role of the L2 protein. We identified the interaction of the L2 protein with SNARE protein syntaxin 18, which mediates the trafficking of vesicles and their cargo between the endoplasmic reticulum, the cis-Golgi compartment, and possibly the plasma membrane. Mutations of L2 residues 41 to 44 prevented the interaction of L2 protein with syntaxin 18 in cotransfection experiments and resulted in noninfectious pseudovirions. In this paper, we describe that syntaxin 18 colocalizes with infectious bovine papillomavirus type 1 (BPV1) pseudovirions during infection but does not colocalize with the noninfectious BPV1 pseudovirions made with an L2 mutant at residues 41 to 44. We show that an antibody against BPV1 L2 residues 36 to 49 (alpha L2 36-49) binds to in vitro-generated BPV1 pseudoviral capsids and does not coimmunoprecipitate syntaxin 18- and BPV1 L2-transfected proteins. alpha L2 36-49 was able to partially or completely neutralize infection of BPV1 pseudovirions and genuine virions. These results support the dependence of syntaxin 18 during BPV1 infection and the ability to interfere with infection by targeting the L2-syntaxin 18 interaction and further define the infectious route of BPV1 mediated by the L2 protein.     

Cell surface-binding motifs of L2 that facilitate papillomavirus infection

Human papillomavirus type 16 (HPV16) is the primary etiologic agent of cervical carcinoma, whereas bovine papillomavirus type 1 (BPV1) causes benign fibropapillomas. However, the capsid proteins, L1 and L2, of these divergent papillomaviruses exhibit functional conservation. A peptide comprising residues 1 to 88 of BPV1 L2 binds to a variety of cell lines, but not to the monocyte-derived cell line D32, and blocks BPV1 infection of mouse C127 cells. Residues 13 to 31 of HPV16 L2 and BPV1 L2 residues 1 to 88 compete for binding to the cell surface, and their binding, unlike that of HPV16 L1/L2 virus-like particles, is unaffected by heparinase or trypsin pretreatment of HeLa cells. A fusion of HPV16 L2 peptide 13-31 and GFP binds (K(d), approximately 1 nM) to approximately 45,000 receptors per HeLa cell. Furthermore, mutation of L2 residues 18 and 19 or 21 and 22 significantly reduces both the ability of the HPV16 L2 13-31-GFP fusion protein to bind to SiHa cells and the infectivity of HPV16 pseudovirions. Antibody to BPV1 L2 peptides comprising residues 115 to 135 binds to intact BPV1 virions, but fails to neutralize at a 1:10 dilution. However, deletion of residues 91 to 129 from L2 abolishes the infectivity of BPV1, but not their binding to the cell surface. In summary, L2 residues 91 to 129 contain epitopes displayed on the virion surface and are required for infection, but not virion binding to the cell surface. Upon the binding of papillomavirus to the cell surface, residues 13 to 31 of L2 interact with a widely expressed, trypsin- and heparinase-resistant cell surface molecule and facilitate infection.     

Positively charged termini of the L2 minor capsid protein are necessary for papillomavirus infection

Coexpression of bovine papillomavirus L1 with L2 mutants lacking either eight N-terminal or nine C-terminal amino acids that encode positively charged domains resulted in wild-type levels of viral genome encapsidation. Despite wild-type binding to the cell surface, the resulting virions were noninfectious. An L2 mutant encoding a scrambled version of the nine C-terminal residues restored infectivity, in contrast to an L2 mutant encoding a scrambled version of the N-terminal residues.     

The positively charged termini of L2 minor capsid protein required for bovine papillomavirus infection function separately in nuclear import and DNA binding

During the papillomavirus (PV) life cycle, the L2 minor capsid protein enters the nucleus twice: in the initial phase after entry of virions into cells and in the productive phase to mediate encapsidation of the newly replicated viral genome. Therefore, we investigated the interactions of the L2 protein of bovine PV type 1 (BPV1) with the nuclear import machinery and the viral DNA. We found that BPV1 L2 bound to the karyopherin alpha2 (Kap alpha2) adapter and formed a complex with Kap alpha2beta1 heterodimers. Previous data have shown that the positively charged termini of BPV1 L2 are required for BPV1 infection after the binding of the virions to the cell surface. We determined that these BPV1 L2 termini function as nuclear localization signals (NLSs). Both the N-terminal NLS (nNLS) and the C-terminal NLS (cNLS) interacted with Kap alpha2, formed a complex with Kap alpha2beta1 heterodimers, and mediated nuclear import via a Kap alpha2beta1 pathway. Interestingly, the cNLS was also the major DNA binding site of BPV1 L2. Consistent with the promiscuous DNA encapsidation by BPV1 pseudovirions, this DNA binding occurred without nucleotide sequence specificity. Moreover, an L2 mutant encoding a scrambled version of the cNLS, which supports production of virions, rescued the DNA binding but not the Kap alpha2 interaction. These data support a model in which BPV1 L2 functions as an adapter between the viral DNA via the cNLS and the Kaps via the nNLS and facilitates nuclear import of the DNA during infection.     

Expression of human papillomavirus types 6b and 16 L1 open reading frames in Escherichia coli: detection of a 56,000-dalton polypeptide containing genus-specific (common) antigens.

The human papillomavirus (HPV) genome contains two large open reading frames (ORFs), designated L1 and L2. To characterize the antigenic properties of the L1 ORF-encoded proteins, we cloned the L1 ORFs of HPV6b and HPV16 in plasmids, and these were expressed in Escherichia coli. First, the HPV6b DNA, representing 85.2% of the L1 ORF, was cloned in pUC19 and expressed in E. coli JM83 and RB791 as a 160,000-molecular-weight (160K) fusion protein with E. coli beta-galactosidase (6bL1/beta-gal). Second, the HPV16 DNA, representing 89.8% of the L1 ORF, was cloned in pKK233-2 and expressed as a 56K protein (16L1) in strain RB791. Both the 6bL1/beta-gal and 16L1 proteins cross-reacted with anti-bovine papillomavirus type 1 (BPV1) antibody raised against disrupted BPV1 particles. An antibody raised against the 6bL1/beta-gal fusion protein reacted with the 16L1 protein and also with native papillomavirus antigens in human genital condyloma and bovine fibropapilloma tissues, as determined by biotin-streptavidin staining. Furthermore, the anti-6bL1/beta-gal antibody recognized a 54K protein which seemed to be a major capsid protein of BPV1 and also a 56K protein of biopsies harboring HPV6 or HPV11. From these results we concluded that the papillomavirus L1 gene product contains genus-specific (common) antigens and that the HPV6 and HPV11 L1 genes specify the 56K capsid protein.     

Interaction of human papillomavirus (HPV) type 16 capsid proteins with HPV DNA requires an intact L2 N-terminal sequence.

Encapsidation of papillomavirus DNA involves DNA-protein and protein-protein interactions. We sought to define the role of each human papillomavirus (HPV) capsid protein in HPV DNA encapsidation. HPV16 major (L1) and minor (L2) capsid proteins purified from recombinant vaccinia virus-infected cells were compared for their ability to bind nucleic acids. L2 protein, but not L1 protein, could bind HPV DNA. To map the DNA-binding region of L2, a series of truncated or point-mutated L2 protein open reading frames were used to show that only the N terminal of L2 was required for L2-DNA binding. This interaction depends critically on charged amino acids (Lys or Arg) in the first 12 amino acids of the N terminal of the protein. Several techniques were used to show that L2 interaction with DNA did not require specific DNA sequences. We propose that HPV L2 protein may play a major role in papillomavirus capsid assembly by introducing HPV DNA to the virus particles formed by the self assembly of the L1 major structural protein.     

The minor capsid protein L2 contributes to two steps in the human papillomavirus type 31 life cycle

Prior studies, which have relied upon the use of pseudovirions generated in heterologous cell types, have led to sometimes conflicting conclusions regarding the role of the minor capsid protein of papillomaviruses, L2, in the viral life cycle. In this study we carry out analyses with true virus particles assembled in the natural host cell to assess L2's role in the viral infectious life cycle. For these studies we used the organotypic (raft) culture system to recapitulate the full viral life cycle of the high-risk human papillomavirus HPV31, which was either wild type or mutant for L2. After transfection, the L2 mutant HPV31 genome was able to establish itself as a nuclear plasmid in proliferating populations of poorly differentiated (basal-like) human keratinocytes and to amplify its genome to high copy number, support late viral gene expression, and cause formation of virus particles in human keratinocytes that had been induced to undergo terminal differentiation. These results indicate that aspects of both the nonproductive and productive phases of the viral life cycle occur normally in the absence of functional L2. However, upon the analysis of the virus particles generated, we found an approximate 10-fold reduction in the amount of viral DNA encapsidated into L2-deficient virions. Furthermore, there was an over-100-fold reduction in the infectivity of L2-deficient virus. Because the latter deficiency cannot be accounted for solely by the 10-fold decrease in encapsidation, we conclude that L2 contributes to at least two steps in the production of infectious virus.     

Characterization of Mus musculus Papillomavirus 1 Infection In Situ Reveals an Unusual Pattern of Late Gene Expression and Capsid Protein Localization

Full-length genomic DNA of the recently identified laboratory mouse papillomavirus 1 (MusPV1) was synthesized in vitro and was used to establish and characterize a mouse model of papillomavirus pathobiology. MusPV1 DNA, whether naked or encapsidated by MusPV1 or human papillomavirus 16 (HPV 16) capsids, efficiently induced the outgrowth of papillomas as early as 3 weeks after application to abraded skin on the muzzles and tails of athymic NCr nude mice. High concentrations of virions were extracted from homogenized papillomatous tissues and were serially passaged for >10 generations. Neutralization by L1 antisera confirmed that infectious transmission was capsid mediated. Unexpectedly, the skin of the murine back was much less susceptible to virion-induced papillomas than the muzzle or tail. Although reporter pseudovirions readily transduced the skin of the back, infection with native MusPV1 resulted in less viral genome amplification and gene expression on the back, including reduced expression of the L1 protein and very low expression of the L2 protein, results that imply skin region-specific control of postentry aspects of the viral life cycle. Unexpectedly, L1 protein on the back was predominantly cytoplasmic, while on the tail the abundant L1 was cytoplasmic in the lower epithelial layers and nuclear in the upper layers. Nuclear localization of L1 occurred only in cells that coexpressed the minor capsid protein, L2. The pattern of L1 protein staining in the infected epithelium suggests that L1 expression occurs earlier in the MusPV1 life cycle than in the life cycle of high-risk HPV and that virion assembly is regulated by a previously undescribed mechanism.     

Identification of TRAPPC8 as a Host Factor Required for Human Papillomavirus Cell Entry

Human papillomavirus (HPV) is a non-enveloped virus composed of a circular DNA genome and two capsid proteins, L1 and L2. Multiple interactions between its capsid proteins and host cellular proteins are required for infectious HPV entry, including cell attachment and internalization, intracellular trafficking and viral genome transfer into the nucleus. Using two variants of HPV type 51, the Ma and Nu strains, we have previously reported that MaL2 is required for efficient pseudovirus (PsV) transduction. However, the cellular factors that confer this L2 dependency have not yet been identified. Here we report that the transport protein particle complex subunit 8 (TRAPPC8) specifically interacts with MaL2. TRAPPC8 knockdown in HeLa cells yielded reduced levels of reporter gene expression when inoculated with HPV51Ma, HPV16, and HPV31 PsVs. TRAPPC8 knockdown in HaCaT cells also showed reduced susceptibility to infection with authentic HPV31 virions, indicating that TRAPPC8 plays a crucial role in native HPV infection. Immunofluorescence microscopy revealed that the central region of TRAPPC8 was exposed on the cell surface and colocalized with inoculated PsVs. The entry of Ma, Nu, and L2-lacking PsVs into cells was equally impaired in TRAPPC8 knockdown HeLa cells, suggesting that TRAPPC8-dependent endocytosis plays an important role in HPV entry that is independent of L2 interaction. Finally, expression of GFP-fused L2 that can also interact with TRAPPC8 induced dispersal of the Golgi stack structure in HeLa cells, a phenotype also observed by TRAPPC8 knockdown. These results suggest that during viral intracellular trafficking, binding of L2 to TRAPPC8 inhibits its function resulting in Golgi destabilization, a process that may assist HPV genome escape from the trans-Golgi network.     

Interaction of L2 with beta-actin directs intracellular transport of papillomavirus and infection

Viruses that replicate in the nucleus, including the primary causative agent of cervical cancer, human papillomavirus type 16 (HPV16), must first cross the cytoplasm. We compared the uptake of HPV16 virus-like particles (VLPs) either with or without the minor capsid protein L2. Whereas VLPs containing only the major capsid protein L1 were diffusely distributed within the cytoplasm even 6 h post-infection, VLPs comprising both L1 and L2 exhibited a radial distribution in the cytoplasm and accumulated in the perinuclear region of BPHE-1 cells within 2 h. L2 of HPV16 or bovine papillomavirus was shown to bind to a 43-kDa cellular protein that was subsequently identified as beta-actin by matrix-assisted laser desorption ionization time-of-flight analysis. A conserved domain comprising residues 25-45 of HPV16 L2 was sufficient for interaction with beta-actin. HPV16 L2 residues 25-45 fused to green fluorescent protein, but not green fluorescent protein alone, colocalized with actin and caused cell retraction and disruption of the microfilament network. Finally, wild-type L2, but not L2 with residues 25-45 deleted, facilitated HPV16 pseudovirion infection. Thus, binding of beta-actin by L2 residues 25-45 facilitates transport of HPV16 across the cytoplasm during infection, and blockade of this novel interaction may be useful for prophylaxis.     

Interactions between papillomavirus L1 and L2 capsid proteins

The human papillomavirus (HPV) capsid consists of 360 copies of the major capsid protein, L1, arranged as 72 pentamers on a T=7 icosahedral lattice, with substoichiometric amounts of the minor capsid protein, L2. In order to understand the arrangement of L2 within the HPV virion, we have defined and biochemically characterized a domain of L2 that interacts with L1 pentamers. We utilized an in vivo binding assay involving the coexpression of recombinant HPV type 11 (HPV11) L1 and HPV11 glutathione S-transferase (GST) L2 fusion proteins in Escherichia coli. In this system, L1 forms pentamers, GST=L2 associates with these pentamers, and L1+L2 complexes are subsequently isolated by using the GST tag on L2. The stoichiometry of L1:L2 in purified L1+L2 complexes was 5:1, indicating that a single molecule of L2 interacts with an L1 pentamer. Coexpression of HPV11 L1 with deletion mutants of HPV11 L2 defined an L1-binding domain contained within amino acids 396 to 439 near the carboxy terminus of L2. L2 proteins from eight different human and animal papillomavirus serotypes were tested for their ability to interact with HPV11 L1. This analysis targeted a hydrophobic region within the L1-binding domain of L2 as critical for L1 binding. Introduction of negative charges into this hydrophobic region by site-directed mutagenesis disrupted L1 binding. L1-L2 interactions were not significantly disrupted by treatment with high salt concentrations (2 M NaCl), weak detergents, and urea concentrations of up to 2 M, further indicating that L1 binding by this domain is mediated by strong hydrophobic interactions. L1+L2 protein complexes were able to form virus-like particles in vitro at pH 5.2 and also at pH 6.8, a pH that is nonpermissive for assembly of L1 protein alone. Thus, L1/L2 interactions are primarily hydrophobic, encompass a relatively short stretch of amino acids, and have significant effects upon in vitro assembly.     

Cyclophilins Facilitate Dissociation of the Human Papillomavirus Type 16 Capsid Protein L1 from the L2/DNA Complex following Virus Entry

Human papillomaviruses (HPV) are composed of the major and minor capsid proteins, L1 and L2, that encapsidate a chromatinized, circular double-stranded DNA genome. At the outset of infection, the interaction of HPV type 16 (HPV16) (pseudo)virions with heparan sulfate proteoglycans triggers a conformational change in L2 that is facilitated by the host cell chaperone cyclophilin B (CyPB). This conformational change results in exposure of the L2 N terminus, which is required for infectious internalization. Following internalization, L2 facilitates egress of the viral genome from acidified endosomes, and the L2/DNA complex accumulates at PML nuclear bodies. We recently described a mutant virus that bypasses the requirement for cell surface CyPB but remains sensitive to cyclosporine for infection, indicating an additional role for CyP following endocytic uptake of virions. We now report that the L1 protein dissociates from the L2/DNA complex following infectious internalization. Inhibition and small interfering RNA (siRNA)-mediated knockdown of CyPs blocked dissociation of L1 from the L2/DNA complex. In vitro, purified CyPs facilitated the dissociation of L1 pentamers from recombinant HPV11 L1/L2 complexes in a pH-dependent manner. Furthermore, CyPs released L1 capsomeres from partially disassembled HPV16 pseudovirions at slightly acidic pH. Taken together, these data suggest that CyPs mediate the dissociation of HPV L1 and L2 capsid proteins following acidification of endocytic vesicles.     

Polyglutamine aggregation behavior in vitro supports a recruitment mechanism of cytotoxicity

The L1 major capsid proteins of human papillomavirus (HPV) types 11 and 16 were purified and analyzed for structural integrity and in vitro self-assembly. Proteins were expressed in Escherichia coli as glutathione-S-transferase-L1 (GST-L1) fusions and purified to near homogeneity as pentamers (equivalent to viral capsomeres), after thrombin cleavage from the GST moiety and removal of tightly associated GroEL protein. Sequences at the amino and carboxy termini contributing to formation of L1 pentamers and to in vitro capsid assembly were identified by deletion analysis. For both HPV11 and HPV16 L1, up to at least ten residues could be deleted from the amino terminus (Delta N10) and 30 residues from the carboxy terminus (Delta C30) without affecting pentamer formation. The HPV16 pentamers assembled into relatively regular, 72-pentamer shells ("virus-like particles" or VLPs) at low pH, with the exception of HPV16 L1 Delta N10, which assembled into a 12-pentamer, T=1 capsid (small VLP) under all conditions tested. The production of large quantities of assembly-competent L1, using the expression and purification protocol described here, has been useful for crystallographic analysis, and will be valuable for studies of virus-receptor interactions and potentially for vaccine design.     

Demonstration of evolutionary differences between conserved antigenic epitopes in the minor nucleocapsid protein of human papillomavirus types 6b, 16 and 18.

We studied human papillomavirus (HPV) minor nucleocapsid protein (L2) by epitope scanning. Conserved antigenic epitopes identified by rabbit antiserum to bovine papillomavirus (BPV) were revealed in HPV-6b (amino acids, aa, 196-205); HPV-16 (aa:s 376-85) and HPV-18 (aa:s 221-230). L2 proteins. The first two epitopes were situated in hydrophilic regions of the proteins. Aligning the aa-sequences that corresponded to the epitopes with the total L2 sequences of BPV and HPV1a revealed consensus motifs between BPV, HPV1a and the reactive HPV type. In the non-reactive types amino acid alterations were noted. Mismatch between HPV1a sequences and the corresponding HPV-6b and HPV-16, HPV-6b and HPV-18, and HPV-16 and HPV-18 sequences suggests that the alterations may have evolved to facilitate immune surveillance of the genital HPV types.     

Nuclear import strategies of high risk HPV16 L1 major capsid protein

During the late phase of human papillomavirus (HPV) infection, the L1 major capsid proteins enter the nuclei of host epithelial cells and, together with the L2 minor capsid proteins, assemble the replicated viral DNA into virions. We investigated the nuclear import of the L1 major capsid protein of high risk HPV16. When digitonin-permeabilized HeLa cells were incubated with HPV16 L1 capsomeres, the L1 protein was imported into the nucleus in a receptor-mediated manner. HPV16 L1 capsomeres formed complexes with Kap alpha2beta1 heterodimers via interaction with Kap alpha2. Accordingly, nuclear import of HPV16 L1 capsomeres was mediated by Kap alpha2beta1 heterodimers, required RanGDP and free GTP, and was independent of GTP hydrolysis. Remarkably, HPV16 L1 capsomeres also interacted with Kap beta2 and binding of RanGTP to Kap beta2 did not dissociate the HPV16 L1.Kap beta2 complex. Significantly, HPV16 L1 capsomeres inhibited the nuclear import of Kap beta2 and of a Kap beta2-specific M9-containing cargo. These data suggest that, during the productive stage of infection, while the HPV16 L1 major capsid protein enters the nucleus via the Kap alpha2beta1-mediated pathway to assemble the virions, it also inhibits the Kap beta2-mediated nuclear import of host hnRNP A1 protein and, in this way, favors virion formation.