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).     

Nuclear import and DNA binding of human papillomavirus type 45 L1 capsid protein

During the life cycle of human papillomaviruses (HPVs), the L1 capsid proteins seem to enter the nucleus twice: once after the virions infect the cells, and later during the productive phase when they assemble the replicated HPV genomic DNA into infectious virions. We established for the high-risk HPV45 that when digitonin-permeabilized HeLa cells were incubated with L1 homopentameric capsomers, the HPV45 L1 protein was imported into the nucleus in a receptor-mediated manner. In contrast, intact capsids were not able to enter the nucleus. Immunoisolation assays showed that HPV45 L1 capsomers interact with cytosolic karyopherin alpha 2 beta 1 heterodimers. HPV45 L1 bound strongly to karyopherin alpha 2, and weakly to karyopherin beta 1, as did its nuclear localization signal (NLS). Nuclear import of HPV45 L1, or of a GST-NLS(HPV45L1) fusion protein was efficiently mediated by karyopherin alpha 2 beta 1 heterodimers, and only weakly by karyopherin beta 1. Nuclear import required RanGDP, but was independent of GTP hydrolysis by Ran. Together, these data suggest that the major nuclear import pathway for HPV45 L1 major capsid protein in infected host cells is mediated by karyopherin alpha 2 beta 1 heterodimers and that GTP hydrolysis by Ran is not required for import. Remarkably, HPV45 L1 capsomers can interact nonspecifically with different types of HPV-DNA, and the DNA binding region of HPV45 L1 overlaps with its NLS sequence.     

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.     

人乳头瘤病毒次要衣壳蛋白L2核定位

在人乳头瘤病毒(human papillomavirus,HPV)次要衣壳蛋白L2的N端和C端,有大量带正电荷的氨基酸残基组成核定位信号(nuclear localization signal,NLS)。细胞的核结构域10(nuclear domain 10,ND10)是细胞周期和病毒生活周期的重要调节者。L2定位到ND10的过程不仅会受到早幼粒细胞白血病蛋白(promyleocytic leukaemia protein,PML)、死亡结构域相关蛋白(deathdomain-associated protein,Daxx)、Sp100核抗原(Sp100 nuclear antigen)等细胞蛋白的影响,也会与L1在ND10发生相互作用。在HPV感染和组装过程中,L2的核定位信号有着重要作用。     

The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes

The L1 major capsid protein of human papillomavirus (HPV) type 11, a 55-kDa polypeptide, forms particulate structures resembling native virus with an average particle diameter of 50-60 nm when expressed in the yeast Saccharomyces cerevisiae. We show in this report that these virus-like particles (VLPs) interact with heparin and with cell-surface glycosaminoglycans (GAGs) resembling heparin on keratinocytes and Chinese hamster ovary cells. The binding of VLPs to heparin is shown to exhibit an affinity comparable to that of other identified heparin-binding proteins. Immobilized heparin chromatography and surface plasmon resonance were used to show that this interaction can be specifically inhibited by free heparin and dextran sulfate and that the effectiveness of the inhibitor is related to its molecular weight and charge density. Sequence comparison of nine human L1 types revealed a conserved region of the carboxyl terminus containing clustered basic amino acids that bear resemblance to proposed heparin-binding motifs in unrelated proteins. Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocyte (HaCaT) cells. Removal of heparan sulfate GAGs on keratinocytes by treatment with heparinase or heparitinase resulted in an 80-90% reduction of VLP binding, whereas treatment of cells with laminin, a substrate for alpha6 integrin receptors, provided minimal inhibition. Cells treated with chlorate or substituted beta-D-xylosides, resulting in undersulfation or secretion of GAG chains, also showed a reduced affinity for VLPs. Similarly, binding of VLPs to a Chinese hamster ovary cell mutant deficient in GAG synthesis was shown to be only 10% that observed for wild type cells. This report establishes for the first time that the carboxyl-terminal portion of HPV L1 interacts with heparin, and that this region appears to be crucial for interaction with the cell surface.     

Mitogen-activated protein kinases activate the nuclear localization sequence of human papillomavirus type 11 E1 DNA helicase to promote efficient nuclear import

Human and animal papillomavirus DNA replicates as multicopy nuclear plasmids. Replication requires two viral proteins, the origin-recognition protein E2 and the replicative DNA helicase E1. Using genetic, biochemical, and immunofluorescence assays, we demonstrated that efficient nuclear import of the human papillomavirus (HPV) type 11 E1 protein depends on a codominant bipartite nuclear localization sequence (NLS) and on phosphorylation of the serine residues S89 and S93 by the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase, and c-Jun N-terminal protein kinase. The NLS and the MAPK substrates are located within a 50-amino-acid-long peptide near the amino terminus, previously designated the localization regulatory region (LRR). The downstream NLS overlaps the cyclin-binding motif RRL, which is necessary for phosphorylation by the cyclin-dependent kinases to inactivate a dominant nuclear export sequence, also in the LRR. Alanine mutations of the MAPK substrates significantly impaired nuclear import, whereas phospho-mimetic mutations partially restored nuclear import. We further identified two MAPK docking motifs near the C terminus of E1 that are conserved among E1 proteins of many HPVs and bovine papillomavirus type 1. Mutations of these MAPK docking motifs or addition of specific MAPK inhibitors significantly reduced nuclear import. Interestingly, a fraction of the NLS-minus E1 protein was cotransported with the E2 protein into the nucleus and supported transient viral DNA replication. In contrast, E1 proteins mutated in the MAPK docking motifs were completely inactive in transient replication, an indication that additional properties were adversely affected by those changes.     

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.     

A Ran-binding protein facilitates nuclear import of human papillomavirus type 16

Human papillomaviruses (HPVs) utilize an atypical mode of nuclear import during cell entry. Residing in the Golgi apparatus until mitosis onset, a subviral complex composed of the minor capsid protein L2 and viral DNA (L2/vDNA) is imported into the nucleus after nuclear envelope breakdown by associating with mitotic chromatin. In this complex, L2 plays a crucial role in the interactions with cellular factors that enable delivery and ultimately tethering of the viral genome to mitotic chromatin. To date, the cellular proteins facilitating these steps remain unknown. Here, we addressed which cellular proteins may be required for this process. Using label-free mass spectrometry, biochemical assays, microscopy, and functional virological assays, we discovered that L2 engages a hitherto unknown protein complex of Ran-binding protein 10 (RanBP10), karyopherin alpha2 (KPNA2), and dynein light chain DYNLT3 to facilitate transport towards mitotic chromatin. Thus, our study not only identifies novel cellular interactors and mechanism that facilitate a poorly understood step in HPV entry, but also a novel cellular transport complex.     

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.     

Interaction of Heliothis armigera nuclear polyhedrosis viral capsid protein with its host actin

In order to find the cellular interaction factors of the Heliothis armigera nuclear polyhedrosis virus capsid protein VP39, a Heliothis armigera cell cDNA library was constructed. Then VP39 was used as bait. The host actin gene was isolated from the cDNA library with the yeast two-hybrid system. This demonstrated that VP39 could interact with its host actin in yeast. In order to corroborate this interaction in vivo, the vp39 gene was fused with the green fluorescent protein gene in plasmid pEGFP39. The fusion protein was expressed in the Hz-AM1 cells under the control of the Autographa californica multiple nucleopolyhedrovirus immediate early gene promoter. The host actin was labeled specifically by the red fluorescence substance, tetramethy rhodamine isothicyanete-phalloidin. Observation under a fluorescence microscopy showed that VP39, which was indicated by green fluorescence, began to appear in the cells 6 h after being transfected with pEGFP39. Red actin cables were also formed in the cytoplasm at the same time. Actin was aggregated in the nucleus 9 h after the transfection. The green and red fluorescence always appeared in the same location of the cells, which demonstrated that VP39 could combine with the host actin. Such a combination would result in the actin skeleton rearrangement.     

Karyopherin-alpha2 protein interacts with Chk2 and contributes to its nuclear import

Chk2 is a nuclear protein kinase involved in the DNA damage-induced ataxia telangiectasia mutated-dependent checkpoint arrest at multiple cell cycle phases. Searching for Chk2-binding proteins by a yeast two-hybrid system, we identified a strong interaction with karyopherin-alpha2 (KPNA-2), a gene product involved in active nuclear import of proteins bearing a nuclear localization signal (NLS). This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. Of the three predicted Chk2 NLSs, located at amino acids 179-182 (NLS-1), 240-256 (NLS-2), and 515-522 (NLS-3), only the latter mediated the interaction with KPNA-2 in the yeast two-hybrid system, and in particular with its C terminus. Unlike mutations in NLS-1 or NLS-2, which left the nuclear localization of Chk2 unaffected, mutations in NLS-3 caused a cytoplasmic relocalization, indicating that the NLS-3 motif acts indeed as NLS for Chk2 in vivo. Finally, co-transfection experiments with green fluorescent protein (GFP)-Chk2 and wild type or mutant KPNA-2 confirmed the role of KPNA-2 in nuclear import of Chk2.     

Expression of the human papillomavirus type 11 L1 capsid protein in Escherichia coli: characterization of protein domains involved in DNA binding and capsid assembly.

The L1 major capsid protein of human papillomavirus type 11 (HPV-11) was expressed in Escherichia coli, and the soluble recombinant protein was purified to near homogeneity. The recombinant L1 protein bound DNA as determined by the Southwestern assay method, and recombinant mutant L1 proteins localized the DNA-binding domain to the carboxy-terminal 11 amino acids of L1. Trypsin digestion of the full-length L1 protein yielded a discrete 42-kDa product (trpL1), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting from cleavage at R415, 86 amino acids from the L1 carboxy terminus. Sucrose gradient sedimentation analysis demonstrated that trpL1 sedimented at 11S, while L1 proteins with amino-terminal deletions of 29 and 61 residues sedimented at 4S. Electron microscopy showed that the full-length L1 protein appeared as pentameric capsomeres which self-assembled into capsid-like particles. The trpL1 protein also had a pentameric morphology but was unable to assemble further. In an enzyme-linked immunosorbent assay, the trpL1 and L1 capsids reacted indistinguishably from virus-like particles purified after expression of HPV-11 L1 in insect cells. The carboxy terminus of L1 therefore constitutes the interpentamer linker arm responsible for HPV-11 capsid formation, much like the carboxy-terminal domain of the polyomavirus VP1 protein. The trypsin susceptibility of HPV-11 L1 capsids suggests a possible mechanism for virion disassembly.     

Nuclear translocation of papillomavirus minor capsid protein L2 requires Hsc70

Minor capsid protein L2 of papillomaviruses plays an essential role in virus assembly by recruiting viral components to PML bodies, the proposed sites of virus morphogenesis. We demonstrate here that the function of L2 in virus assembly requires the chaperone Hsc70. Hsc70 was found dispersed in naturally infected keratinocytes and cultured cells. A dramatic relocation of Hsc70 from the cytoplasm to PML bodies was induced in these cells by L2 expression. Hsc70-L2 complex formation was confirmed by coimmunoprecipitation. The complex was modulated by the cochaperones Hip and Bag-1, which stabilize and destabilize Hsc70-substrate complexes, respectively. Cytoplasmic depletion of Hsc70 caused retention of wild-type and N-terminally truncated L2, but not of C-terminally truncated L2, in the cytoplasm. This retention was partially reversed by overexpression of Hsc70 fused to green fluorescent protein but not by ATPase-negative Hsc70. Hsc70 associated with L1-L2 virus-like particles (VLPs) but not with VLPs composed either of L1 alone or of L1 and C-terminally truncated L2. Moreover, displacement of Hsc70 from L1-L2 VLPs by encapsidation of DNA, generating pseudovirions, was found. These data indicate that Hsc70 transiently associates with viral capsids during the integration of L2, possibly via the L2 C terminus. Completion of virus assembly results in displacement of Hsc70 from virions.     

Human papillomavirus type 16 minor capsid protein l2 N-terminal region containing a common neutralization epitope binds to the cell surface and enters the cytoplasm

The first step of papillomavirus infection is believed to be binding of major capsid protein L1 to the cell surface without involvement of minor capsid protein L2, but the viral infectivity can be neutralized either by anti-L1 or anti-L2 antibody. To understand the role of L2 in human papillomavirus (HPV) infection, we examined a segment of HPV type 16 (HPV16) L2, which contains a neutralization epitope common to HPV6, for its involvement in adsorption and penetration of the capsids. Preincubation of monkey COS-1 cells with a synthetic peptide having amino acids (aa) 108 to 120 of HPV16 L2 reduced the susceptibility of COS-1 cells to infection with HPV16 pseudovirions. Confocal microscopy showed that the green fluorescence protein (GFP) fused with the L2 peptide was found to bind to the surface of a HeLa cell, an HPV18-positive human cancer cell line, at 4 degrees C and to enter the cytoplasm after subsequent incubation at 37 degrees C. Flow cytometry showed that fused GFP did not bind to HeLa cells that had been treated with trypsin. Besides COS-1 and HeLa cells, some human and rodent cell lines were detected by flow cytometry to be susceptible to binding with fused GFP, showing a tendency of epithelial cells toward higher susceptibility. Substitutions at aa 108 to 111 inhibited fused GFP from binding to HeLa cells and reduced the infectivity in COS-1 cells of the in vitro-constructed pseudovirions. The results suggest that L2 plays an important role in enhancing HPV infection through interaction between the N-terminal region and a cellular surface protein, facilitating penetration of the virions and determining part of the tropism of HPVs.     

mRNA splicing regulates human papillomavirus type 11 E1 protein production and DNA replication

The papillomavirus replicative helicase E1 and the origin recognition protein E2 are required for efficient viral DNA replication. We fused the green fluorescent protein (GFP) to the human papillomavirus type 11 E1 protein either in a plasmid with the E1 coding region alone (nucleotides [nt] 832 to 2781) (pGFP-11E1) or in a plasmid containing both the E1 and E2 regions (nt 2723 to 3826) and the viral origin of replication (ori) (p11Rc). The former supported transient replication of an ori plasmid, whereas the latter was a self-contained replicon. Unexpectedly, these plasmids produced predominantly a cytoplasmic variant GFP or a GFP-E1 E4 protein, respectively. The majority of the mRNAs had an intragenic or intergenic splice from nt 847 to nt 2622 or from nt 847 to nt 3325, corresponding to the E2 or E1 E4 messages. pGFP-11E1dm and p11Rc-E1dm, mutated at the splice donor site, abolished these splices and increased GFP-E1 protein expression. Three novel, alternatively spliced, putative E2 mRNAs were generated in higher abundance from the mutated replicon than from the wild type. Relative to pGFP-11E1, low levels of pGFP-11E1dm supported more efficient replication, but high levels had a negative effect. In contrast, elevated E2 levels always increased replication. Despite abundant GFP-E1 protein, p11Rc-E1dm replicated less efficiently than the wild type. Collectively, these observations show that the E1/E2 ratio is as important as the E1 and E2 concentrations in determining the replication efficiency. These findings suggest that alternative mRNA splicing could provide a mechanism to regulate E1 and E2 protein expression and DNA replication during different stages of the virus life cycle.     

Interaction of tSNARE syntaxin 18 with the papillomavirus minor capsid protein mediates infection

The papillomavirus capsid mediates binding to the cell surface and passage of the virion to the perinuclear region during infection. To better understand how the virus traffics across the cell, we sought to identify cellular proteins that bind to the minor capsid protein L2. We have identified syntaxin 18 as a protein that interacts with bovine papillomavirus type 1 (BPV1) L2. Syntaxin 18 is a target membrane-associated soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (tSNARE) that resides in the endoplasmic reticulum (ER). The ectopic expression of FLAG-tagged syntaxin 18, which disrupts ER trafficking, blocked BPV1 pseudovirion infection. Furthermore, the expression of FLAG-syntaxin 18 prevented the passage of BPV1 pseudovirions to the perinuclear region that is consistent with the ER. Genetic studies identified a highly conserved L2 domain, DKILK, comprising residues 40 to 44 that mediated BPV1 trafficking through the ER during infection via an interaction with the tSNARE syntaxin 18. Mutations within the DKILK motif of L2 that did not significantly impact virion morphogenesis or binding at the cell surface prevented the L2 interaction with syntaxin 18 and disrupted BPV1 infection.     

Purification of virus-like particles of recombinant human papillomavirus type 11 major capsid protein L1 from Saccharomyces cerevisiae

Recombinant major capsid protein, L1 (M(r) = 55,000), of human papillomavirus type 11 was expressed intracellularly at high levels in a galactose-inducible Saccharomyces cerevisiae expression system by an HPV6/11 hybrid gene. The capsid protein self-assembled into virus-like particles (VLPs) and accounted for 15% of the total soluble protein. A purification process was developed that consisted of two main steps: microfiltration and cation-exchange chromatography. The purified VLPs were 98% homogeneous, and the overall purification yield was 10%. The final product was characterized by several analytical methods and was highly immunogenic in mice.