The l2 minor capsid protein of human papillomavirus type 16 interacts with a network of nuclear import receptors

The L2 minor capsid proteins enter the nucleus twice during viral infection: in the initial phase after virion disassembly and in the productive phase when, together with the L1 major capsid proteins, they assemble the replicated viral DNA into virions. In this study we investigated the interactions between the L2 protein of high-risk human papillomavirus type 16 (HPV16) and nuclear import receptors. We discovered that HPV16 L2 interacts directly with both Kapbeta(2) and Kapbeta(3). Moreover, binding of Ran-GTP to either Kapbeta(2) or Kapbeta(3) inhibits its interaction with L2, suggesting that the Kapbeta/L2 complex is import competent. In addition, we found that L2 forms a complex with the Kapalpha(2)beta(1) heterodimer via interaction with the Kapalpha(2) adapter. In agreement with the binding data, nuclear import of L2 in digitonin-permeabilized cells could be mediated by either Kapalpha(2)beta(1) heterodimers, Kapbeta(2), or Kapbeta(3). Mapping studies revealed that HPV16 L2 contains two nuclear localization signals (NLSs), in the N terminus (nNLS) and C terminus (cNLS), that could mediate its nuclear import. Together the data suggest that HPV16 L2 interacts via its NLSs with a network of karyopherins and can enter the nucleus via several import pathways mediated by Kapalpha(2)beta(1) heterodimers, Kapbeta(2), and Kapbeta(3).     

Chimeric human papillomavirus type 16 (HPV-16) L1 particles presenting the common neutralizing epitope for the L2 minor capsid protein of HPV-6 and HPV-16

Both the Human papillomavirus (HPV) major (L1) and minor (L2) capsid proteins have been well investigated as potential vaccine candidates. The L1 protein first oligomerizes into pentamers, and these capsomers assemble into virus-like particles (VLPs) that are highly immunogenic. Here we examine the potential of using HPV type 16 (HPV-16) L1 subunits to display a well-characterized HPV-16 L2 epitope (LVEETSFIDAGAP), which is a common-neutralizing epitope for HPV types 6 and 16, in various regions of the L1 structure. The L2 sequence was introduced by PCR (by replacing 13 codons) into sequences coding for L1 surface loops D-E (chideltaC-L2), E-F (chideltaA-L2), and an internal loop C-D (chideltaH-L2); into the h4 helix (chideltaF-L2); and between h4 and beta-J structural regions (chideltaE-L2). The chimeric protein product was characterized using a panel of monoclonal antibodies (MAbs) that bind to conformational and linear epitopes, as well as a polyclonal antiserum raised to the L2 epitope. All five chimeras reacted with the L2 serum. ChideltaA-L2, chideltaE-L2, and chideltaF-L2 reacted with all the L1 antibodies, chideltaC-L2 did not bind H16:V5 and H16:E70, and chideltaH-L2 did not bind any conformation-dependent MAb. The chimeric particles elicited high-titer anti-L1 immune responses in BALB/c mice. Of the five chimeras tested only chideltaH-L2 did not elicit an L2 response, while chideltaF-L2 elicited the highest L2 response. This study provides support for the use of PV particles as vectors to deliver various epitopes in a number of locations internal to the L1 protein and for the potential of using chimeric PV particles as multivalent vaccines. Moreover, it contributes to knowledge of the structure of HPV-16 L1 VLPs and their derivatives.     

Identification of a dynein interacting domain in the papillomavirus minor capsid protein l2

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.     

Mechanisms of human papillomavirus type 16 neutralization by l2 cross-neutralizing and l1 type-specific antibodies

Pseudovirions of human papillomavirus type 16 (HPV16), the principal etiologic agent in 50% of cervical cancers, were used as a model system to investigate the cell surface interactions involved in the exposure of the broadly cross-neutralizing papillomavirus L2 epitopes. These neutralizing epitopes were exposed only after cell surface binding and a subsequent change in capsid conformation that permitted cleavage by the cellular protease furin at a specific highly conserved site in L2 that is immediately upstream of the cross-neutralizing epitopes. Unexpectedly, binding of L2 antibodies led to the release of the capsid/antibody complexes from the cell surface and their accumulation on the extracellular matrix. Study of the dynamics of exposure of the L2 epitopes further revealed that representatives of the apparently dominant class of L1-specific neutralizing antibodies induced by virus-like particle vaccination prevent infection, not by preventing cell surface binding but rather by preventing the conformation change involved in exposure of the L2 neutralizing epitope. These findings suggest a dynamic model of virion-cell surface interactions that has implications for both evolution of viral serotypes and the efficacy of current and future HPV vaccines.     

Evaluation of serum antibody response to a newly identified B-cell epitope in the minor nucleocapsid protein L2 of human papillomavirus type 16

The aim of this work was to identify B-cell epitopes in the minor nucleocapsid (L2) protein of human papillomavirus (HPV) type 16 and characterization of allied antibody response. Serum samples of 513 individuals (323 women with various degrees of cervical atypia, 150 men and 40 small children) were available for the study. Synthetic peptides overlapping the L2 protein of HPV 16 twice were applied in ELISA for epitope scanning and antibody determination. An HPV 16 L2 derived dodecamer SGYIPANTTIPF (amino acids 391-402) proved to be the major B-cell epitope. Both IgA antipeptide antibody positivity (range 7-28%) and mean IgA antibody levels (range 13.2 EIU to 42.4 EIU, P < 0.05) increased with the degree of cervical atypia, whereas antipeptide IgG antibodies showed an opposite trend. During a 2-years follow-up significantly (P < 0.0005) decreasing IgA antibody levels to the SGYIPANTTIPF peptide were associated with regression of koilocytotic atypia. Analysis of anti-peptide IgA antibodies of 118 women with known HPV type revealed that a majority of positives had HPV 16/18 DNA. It was concluded that antibody response to the newly discovered peptide was partially type- and disease-specific. Our results also suggest an impairment of the IgG but not IgA class antibody response to HPV 16 in patients with persistent cervical HPV infection.     

Identification of a human papillomavirus type 16-specific epitope on the C-terminal arm of the major capsid protein L1

To characterize epitopes on human papillomavirus (HPV) virus-like particles (VLPs), a panel of mutated HPV-16 VLPs was created. Each mutated VLP had residues substituted from HPV-31 or HPV-52 L1 sequences to the HPV-16 L1 backbone. Mutations were created on the HPV-31 and -52 L1 proteins to determine if HPV-16 type-specific recognition could be transferred. Correct folding of the mutated proteins was verified by resistance to trypsin digestion and by binding to one or more conformation-dependent monoclonal antibodies. Several of the antibodies tested were found to bind to regions already identified as being important for HPV VLP recognition (loops DE, EF, FG, and HI). Sequences at both ends of the long FG loop (amino acids 260 to 290) were required for both H16.V5 and H16.E70 reactivity. A new antibody-binding site was discovered on the C-terminal arm of L1 between positions 427 and 445. Recognition of these residues by the H16.U4 antibody suggests that this region is surface exposed and supports a recently proposed molecular model of HPV VLPs.     

The l2 minor capsid protein of low-risk human papillomavirus type 11 interacts with host nuclear import receptors and viral DNA

Analysis of the interactions of low-risk human papillomavirus type 11 (HPV11) L2 with karyopherin beta (Kap beta) nuclear import receptors revealed that L2 interacted with Kap beta 1, Kap beta 2, and Kap beta 3 and formed a complex with the Kap alpha 2 beta 1 heterodimer. HPV11 L2 contains two nuclear localization signals (NLSs)-in the N terminus and the C terminus-that could mediate its nuclear import via a classical pathway. Each NLS was functional in vivo, and deletion of both of them abolished L2 nuclear localization. Both NLSs interacted with the viral DNA. Thus, HPV11 L2 can interact with several karyopherins and the viral DNA and may enter the nucleus via multiple pathways.     

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.     

Human papillomavirus type 16 E7 oncoprotein binds and inactivates growth-inhibitory insulin-like growth factor binding protein 3

The E7 protein encoded by human papillomavirus type 16 is one of the few viral genes that can immortalize primary human cells and thereby override cellular senescence. While it is generally assumed that this property of E7 depends on its interaction with regulators of the cell cycle, we show here that E7 targets insulin-like growth factor binding protein 3 (IGFBP-3), the product of a p53-inducible gene that is overexpressed in senescent cells. IGFBP-3 can suppress cell proliferation and induce apoptosis; we show here that IGFBP-3-mediated apoptosis is inhibited by E7, which binds to IGFBP-3 and triggers its proteolytic cleavage. Two transformation-deficient mutants of E7 failed to inactivate IGFBP-3, suggesting that inactivation of IGFBP-3 may contribute to cell transformation.     

Immune response to E7 protein of human papillomavirus type 16 anchored on the cell surface

To target the E7 protein of human papilloma virus 16 to the cell surface, a fusion gene was constructed. It encodes the signal peptide, part of the immunoglobulin (IgG)-like domain, the transmembrane anchor of vaccinia virus (VV) hemagglutinin (HA), and the complete E7-coding sequence. The fusion gene was expressed under the HA late promoter by a recombinant VV, designated VV-E7-HA. The E7-HA protein was displayed on the surface of cells infected with the recombinant virus and was more stable than unmodified E7. The biological properties of the VV-E7-HA virus were compared with those of a VV-E7 virus that expressed the unmodified E7 and with a VV expressing the Sig-E7-LAMP fusion protein. While the first two of these recombinants were based on VV strain Praha, the third was derived from the WR strain of VV. Infection of mice with the VV-E7-HA virus induced the formation of E7-specific antibodies with the predominance of the IgG2a isotype, whereas the other two viruses did not induce the formation of E7-specific antibodies. Unlike the other two viruses, VV-E7-HA did not induce a response of cytotoxic T lymphocytes or Th1 cells and did not protect mice against the growth of E7-expressing tumors. Thus, VV-E7-HA induced a differently polarized immune response to the E7 protein than the other two viruses.     

Human papillomavirus type 16 E6 promotes retinoblastoma protein phosphorylation and cell cycle progression

We show that E6 proteins from benign human papillomavirus type 1 (HPV1) and oncogenic HPV16 have the ability to alter the regulation of the G(1)/S transition of the cell cycle in primary human fibroblasts. Overexpression of both viral proteins induces cellular proliferation, retinoblastoma (pRb) phosphorylation, and accumulation of products of genes that are negatively regulated by pRb, such as p16(INK4a), CDC2, E2F-1, and cyclin A. Hyperphosphorylated forms of pRb are present in E6-expressing cells even in the presence of ectopic levels of p16(INK4a). The E6 proteins strongly increased the cyclin A/cyclin-dependent kinase 2 (CDK2) activity, which is involved in pRb phosphorylation. In addition, mRNA and protein levels of the CDK2 inhibitor p21(WAF1/CIP1) were strongly down-regulated in cells expressing E6 proteins. The down-regulation of the p21(WAF1/CIP1) gene appears to be independent of p53 inactivation, since HPV1 E6 and an HPV16 E6 mutant unable to target p53 were fully competent in decreasing p21(WAF1/CIP1) levels. E6 from HPV1 and HPV16 also enabled cells to overcome the G(1) arrest imposed by oncogenic ras. Immunofluorescence staining of cells coexpressing ras and E6 from either HPV16 or HPV1 revealed that antiproliferative (p16(INK4a)) and proliferative (Ki67) markers were coexpressed in the same cells. Together, these data underline a novel activity of E6 that is not mediated by inactivation of p53.     

Human coronavirus 229E binds to CD13 in rafts and enters the cell through caveolae

CD13, a receptor for human coronavirus 229E (HCoV-229E), was identified as a major component of the Triton X-100-resistant membrane microdomain in human fibroblasts. The incubation of living fibroblasts with an anti-CD13 antibody on ice gave punctate labeling that was evenly distributed on the cell surface, but raising the temperature to 37 degrees C before fixation caused aggregation of the labeling. The aggregated labeling of CD13 colocalized with caveolin-1 in most cells. The HCoV-229E virus particle showed a binding and redistribution pattern that was similar to that caused by the anti-CD13 antibody: the virus bound to the cell evenly when incubated on ice but became colocalized with caveolin-1 at 37 degrees C; importantly, the virus also caused sequestration of CD13 to the caveolin-1-positive area. Electron microscopy confirmed that HCoV-229E was localized near or at the orifice of caveolae after incubation at 37 degrees C. The depletion of plasmalemmal cholesterol with methyl beta-cyclodextrin significantly reduced the HCoV-229E redistribution and subsequent infection. A caveolin-1 knockdown by RNA interference also reduced the HCoV-229E infection considerably. The results indicate that HCoV-229E first binds to CD13 in the Triton X-100-resistant microdomain, then clusters CD13 by cross-linking, and thereby reaches the caveolar region before entering cells.     

Protection of rabbits against challenge with rabbit papillomaviruses by immunization with the N terminus of human papillomavirus type 16 minor capsid antigen L2

Current L1 virus-like particle (VLP) vaccines provide type-restricted protection against a small subset of the human papillomavirus (HPV) genotypes associated with cervical cancer, necessitating continued cytologic screening of vaccinees. Cervical cancer is most problematic in countries that lack the resources for screening or highly multivalent HPV VLP vaccines, suggesting the need for a low-cost, broadly protective vaccinogen. Here, N-terminal L2 polypeptides comprising residues 1 to 88 or 11 to 200 derived from HPV16, bovine papillomavirus type 1 (BPV1), or cottontail rabbit papillomavirus (CRPV) were produced in bacteria. Rabbits were immunized with these N-terminal L2 polypeptides and concurrently challenged with CRPV and rabbit oral papillomavirus (ROPV). Vaccination with either N-terminal L2 polypeptides of CRPV effectively protected rabbits from CRPV challenge but not from papillomas induced by cutaneous challenge with CRPV genomic DNA. Furthermore, papillomas induced by CRPV genomic DNA deficient for L2 expression grew at the same rate as those induced by wild-type CRPV genomic DNA, further suggesting that the L2 polypeptide vaccines lack therapeutic activity. Neutralizing serum antibody titers of >15 correlated with protection (P < 0.001), a finding consistent with neutralizing antibody-mediated protection. Surprisingly, a remarkable degree of protection against heterologous papillomavirus types was observed after vaccination with N-terminal L2 polypeptides. Notably, vaccination with HPV16 L2 11-200 protected against cutaneous and mucosal challenge with CRPV and ROPV, respectively, papillomaviruses that are evolutionarily divergent from HPV16. Further, vaccination with HPV16 L2 11-200 generates broadly cross-neutralizing serum antibody, suggesting the potential of L2 as a second-generation preventive HPV vaccine antigen.     

Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection

The previously characterized monoclonal antibodies (MAbs) A1, A69, B1, and A20 are directed against assembled or nonassembled adeno-associated virus type 2 (AAV-2) capsid proteins (A. Wistuba, A. Kern, S. Weger, D. Grimm, and J. A. Kleinschmidt, J. Virol. 71:1341-1352, 1997). Here we describe the linear epitopes of A1, A69, and B1 which reside in VP1, VP2, and VP3, respectively, using gene fragment phage display library, peptide scan, and peptide competition experiments. In addition, MAbs A20, C24-B, C37-B, and D3 directed against conformational epitopes on AAV-2 capsids were characterized. Epitope sequences on the capsid surface were identified by enzyme-linked immunoabsorbent assay using AAV-2 mutants and AAV serotypes, peptide scan, and peptide competition experiments. A20 neutralizes infection following receptor attachment by binding an epitope formed during AAV-2 capsid assembly. The newly isolated antibodies C24-B and C37-B inhibit AAV-2 binding to cells, probably by recognizing a loop region involved in binding of AAV-2 to the cellular receptor. In contrast, binding of D3 to a loop near the predicted threefold spike does not neutralize AAV-2 infection. The identified antigenic regions on the AAV-2 capsid surface are discussed with respect to their possible roles in different steps of the viral life cycle.     

The isolated major homology region of the HIV capsid protein is mainly unfolded in solution and binds to the intact protein

Assembly of the mature human immunodeficiency virus type 1 (HIV-1) capsid involves the oligomerization of the capsid protein, CA. During retroviral maturation, the CA protein undergoes structural changes and forms exclusive intermolecular interfaces in the mature capsid shell, different from those in the immature precursor. The most conserved region of CA, the major homology region (MHR), is located in the C-terminal domain of CA (CTD). The MHR is involved in both immature and mature virus assembly; however, its exact function during both assembly stages is unknown. To test its conformational preferences and to provide clues on its role during CA assembly, we have used a minimalist approach by designing a peptide comprising the whole MHR (MHRpep, residues Asp152 to Ala174). Isolated MHRpep is mainly unfolded in aqueous solution, with residual structure at its C terminus. MHRpep binds to monomeric CTD with an affinity of ~30μM (as shown by fluorescence and ITC); the CTD binding region comprises residues belonging to α-helices 10 and 11. In the immature virus capsid, the MHR and α-helix 11 regions of two CTD dimers also interact [Briggs JAG, Riches JD, Glass B, Baratonova V, Zanetti G and Kr?usslich H-G (2009) Proc. Natl. Acad. Sci. USA 106, 11090-11095]. These results can be considered a proof-of-concept that the conformational preferences and binding features of isolated peptides derived from virus proteins could be used to mimic early stages of virus assembly.