A novel recombinant protein vaccine containing the different E7 proteins of the HPV16, 18, 6, 11 E7 linked to the HIV-1 Tat (47-57) improve cytotoxic immune responses.

OBJECTIVES: Human papillomavirus infection (HPV) is the most common viral infection which is causes of cervical, penal, vulvar, anal and, oropharyngeal cancer. E7 protein of HPV is a suitable target for induction of T cell responses and controlling HPV-related cancer. The aim of the current study was to designed and evaluated a novel fusion protein containing the different E7 proteins of the HPV 16, 18, 6 and 11, linked to the cell-penetrating peptide HIV-1 Tat 49-57, in order to improve cytotoxic immune responses in in-vitro and in-vivo. RESULTS: In this study whole sequence of HPV16,18,6,11 E7-Tat (47-57) and HPV16,18,6,11 E7 cloned into the vector and expressed in E. coli (BL21). The purified protein was confirmed by SDS page and western blotting and then injected into the C57BL/6 mice. The efficiency of the fusion protein vaccine was assessed by antibody response assay, cytokine assay (IL-4 and IFN-γ), CD + 8 cytotoxicity assay and tumor challenge experiment. Result showed that fusion proteins containing Adjuvant (IFA,CFA) could express higher titer of antibody. Also, we showed that vaccination with E7-Tat and, E7-Tat-ADJ induced high frequencies of E7-specific CD8 + T cells and CD107a expression as well as IFN-γ level and enhanced long-term survival in the therapeutic animal models. CONCLUSION: Our finding suggested that this novel fusion protein vaccine was able to induce therapeutic efficacy and immunogenicity by improving CD8 + T cell in TC-1 tumor bearing mice; so this vaccine may be appreciated for research against HPV and tumor immunotherapies.

A ubiquitin variant-based affinity approach selectively identifies substrates of the ubiquitin ligase E6AP in complex with HPV-11 E6 or HPV-16 E6.

The E6 protein of both mucosal high-risk human papillomaviruses (HPVs) such as HPV-16, which have been causally associated with malignant tumors, and low-risk HPVs such as HPV-11, which cause the development of benign tumors, interacts with the cellular E3 ubiquitin ligase E6-associated protein (E6AP). This indicates that both HPV types employ E6AP to organize the cellular proteome to viral needs. However, whereas several substrate proteins of the high-risk E6-E6AP complex are known, e.g. the tumor suppressor p53, potential substrates of the low-risk E6-E6AP complex remain largely elusive. Here, we report on an affinity-based enrichment approach that enables the targeted identification of potential substrate proteins of the different E6-E6AP complexes by a combination of E3-selective ubiquitination in whole-cell extracts and high-resolution MS. The basis for the selectivity of this approach is the use of a ubiquitin variant that is efficiently used by the E6-E6AP complexes for ubiquitination but not by E6AP alone. By this approach, we identified ∼190 potential substrate proteins for low-risk HPV-11 E6 and high-risk HPV-16 E6. Moreover, subsequent validation experiments in vitro and within cells with selected substrate proteins demonstrate the potential of our approach. In conclusion, our data represent a reliable repository for potential substrates of the HPV-16 and HPV-11 E6 proteins in complex with E6AP.

HPV11E7 inhibits IMQ-induced chemokine and colony-stimulating factor production in keratinocytes.

Imiquimod (IMQ) is approved as a first-line treatment for genital warts caused by human papillomavirus (HPV) infection. However, the recurrence rate is very high. HPV E7 protein plays a critical role in HPV immune escape. However, the role of HPV11 E7 protein in genital warts recurrence during IMQ treatment is not clear. Here, we found that the expression profile of NHEK cells was obviously changed after IMQ treatment, and a large number of genes encoding cytokines and genes involved in cytokine-mediated signaling pathways and cellular metabolic signaling pathways were up- or downregulated. HPV11E7 overexpression inhibited the IMQ-induced production of of multiple chemokines and colony-stimulating factors in NHEK cells. Furthermore, we found that HPV11E7 could impair the activation of mitogen-activated protein kinase (MAPK) signaling pathway. Therefore, our results suggested that HPV11 E7 diminishes the production of chemokines, colony-stimulating factors and other cytokines via inhibition of the MAPK signaling pathway, which suppresses the therapeutic effect of IMQ and promotes the recurrence of diseases, such as condyloma acuminatum.

Induction of co-inhibitory molecule CTLA-4 by human papillomavirus E7 protein through downregulation of histone methyltransferase JHDM1B expression.

Human papillomavirus causes various skin diseases and even cancer. Unfortunately, the host immune system often fails to generate effective responses against HPV infection due to the ability of HPV to evade immune-mediated eradication, although the detailed mechanisms by which HPV inhibits host antiviral immunity are not fully understood. In this study, we reported a novel role of HPV E7 oncoprotein in inducing the expression of co-inhibitory molecule cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) in cells of epithelial origin. Mechanistically, HPV E7 protein downregulated the cellular abundance of Jumonji C histone demethylase 1B (JHDM1B), increasing the levels of H3K36 methylation within the promoter region of CTLA-4. Our findings expand the current understanding of HPV-mediated immune evasion mechanisms and may be helpful in developing optimal anti-HPV therapeutic strategies and relevant drugs.

Human Papillomavirus 11 Early Protein E6 Activates Autophagy by Repressing AKT/mTOR and Erk/mTOR.

Low-risk human papillomaviruses (LR-HPVs) are the causative agents of genital warts, which are a widespread sexually transmitted disease. How LR-HPVs affect autophagy and the specific proteins involved are unknown. In the current study, we investigated the impact of LR-HPV11 early protein 6 (E6) on the activity of the autophagy pathway. We transfected an HPV11 E6 (11E6) plasmid into HaCaT cells, H8 cells, and NHEK cells and established a stable cell line expressing the HPV11 E6 protein. The differences in autophagy activity and upstream regulatory pathways compared with those in the parent cell lines were investigated using a Western blot analysis of the total and phosphorylated protein levels and confocal microscopy of immunostained cells and cells transfected with an mCherry-green fluorescent protein-LC3 expression plasmid. We used short hairpin RNA (shRNA) to knock down 11E6 and showed that these effects require continued 11E6 expression. Compared with its expression in the control cells, the expression of HPV11 E6 in the cells activated the autophagy pathway. The increased autophagy activity was the result of the decreased phosphorylation levels of the canonical autophagy repressor mammalian target of rapamycin (mTOR) at its Ser2448 position (the mTOR complex 1 [mTORC1] phosphorylation site) and decreased AKT and Erk phosphorylation. Therefore, these results indicate that HPV11 E6 activates autophagy through the AKT/mTOR and Erk/mTOR pathways. Our findings provide novel insight into the relationship between LR-HPV infections and autophagy and could help elucidate the pathogenic mechanisms of LR-HPV.IMPORTANCE We transfected an HPV11 E6 plasmid into HaCaT cells, H8 cells, and NHEK cells and established a stable cell line expressing the HPV11 E6 protein. Then, we confirmed that HPV11 E6 induces autophagy by suppressing the AKT/mTOR and Erk/mTOR pathways. In contrast to the high-risk HPV E6 genes, HPV11 E6 did not affect the expression of p53. To the best of our knowledge, this study represents the first direct in-depth investigation of the relationship between the LR-HPV E6 gene and autophagy, which may help to reveal the pathogenesis of LR-HPV infection.

E6 proteins from low-risk human papillomavirus types 6 and 11 are able to protect keratinocytes from apoptosis via Bak degradation.

Infection of epithelial surfaces with low-risk human papillomavirus (HPV) types 6 and 11 causes troublesome clinical diseases, such as recurrent respiratory papillomatosis, that carry a significant cost burden to the healthcare system. Despite this, less has been studied at the molecular level for the low-risk HPV types when compared with their high-risk counterparts. Recent studies have shown the ability of the HPV E6 protein to degrade the pro-apoptotic family member Bak in high-risk and betapapillomavirus HPV types, which confers a cytoprotective advantage on E6-expressing cells. It is unknown whether low-risk E6 expression disrupts the apoptosis pathway and confers a cytoprotective advantage as a result of Bak degradation. We tested the abilities of 6E6 and 11E6 to degrade Bak and protect keratinocytes from UV-initiated apoptosis. Both low-risk 6E6 and 11E6 proteins were able to degrade activated Bak following UV treatment of keratinocytes. The degradation of Bak in 6E6- and 11E6-expressing cells occurred through the proteasomal pathway, and protected them from apoptosis, specifically through the intrinsic pathway to the same extent as their high-risk HPV16 E6 counterpart. In conclusion, we have found a new, critical and conserved function of low-risk HPV E6 proteins, i.e. the ability to degrade Bak, which gives them a cytoprotective advantage over normal, uninfected cells by specifically disrupting the intrinsic pathway of apoptosis.

[Expression and polyclonal antibody preparation of HPV-11E7 protein].

OBJECTIVE: To express human papillomavirus type 11, E7 protein (HPV11E7) via a prokaryotic expression vector and produce anti-HPV11E7 polyclonal antibody. METHODS: A prokaryotic expression vector pGEX-4T2-HPV11E7 was constructed and soluble GST-HPV11E7 fusion protein was expressed in E.coli by IPTG induction and purified. The purified HPV11E7 protein was used to immunize New Zealand rabbits to prepare the anti-HPV11E7 polyclonal antibody followed by protein G agarose purification to obtain the IgG type polyclonal antibody. Western blotting and immunofluorescence analysis were used to test the specificity and titer of the antibody. RESULTS: SDS-PAGE analysis demonstrated that large amounts of soluble GST-HPV11E7 fusion protein was expressed in E.coli after 6 hours of IPTG induction. Western blotting and immunofluorescence confirmed that the purified anti-HPV11E7 polyclonal IgG antibody obtained from immunized rabbits had a high titer and specificity. CONCLUSION: The prokaryotic expression system could express a great deal of soluble HPV11E7 protein, and anti-HPV11E7 polyclonal IgG antibody from HPV11E7-immunized rabbits was proved to have a high titer and specificity.

Association between septal deviation and sinonasal papilloma.

Sinonasal papilloma is a common benign epithelial tumor of the sinonasal tract and accounts for 0.5% to 4% of all nasal tumors. The etiology of sinonasal papilloma remains unclear, although human papilloma virus has been proposed as a major risk factor. Other etiological factors, such as anatomical variations of the nasal cavity, may be related to the pathogenesis of sinonasal papilloma, because deviated nasal septum is seen in patients with chronic rhinosinusitis. We, therefore, investigated the involvement of deviated nasal septum in the development of sinonasal papilloma. Preoperative computed tomography or magnetic resonance imaging findings of 83 patients with sinonasal papilloma were evaluated retrospectively. The side of papilloma and the direction of septal deviation showed a significant correlation. Septum deviated to the intact side in 51 of 83 patients (61.4%) and to the affected side in 18 of 83 patients (21.7%). Straight or S-shaped septum was observed in 14 of 83 patients (16.9%). Even after excluding 27 patients who underwent revision surgery and 15 patients in whom the papilloma touched the concave portion of the nasal septum, the concave side of septal deviation was associated with the development of sinonasal papilloma (p = 0.040). The high incidence of sinonasal papilloma in the concave side may reflect the consequences of the traumatic effects caused by wall shear stress of the high-velocity airflow and the increased chance of inhaling viruses and pollutants. The present study supports the causative role of human papilloma virus and toxic chemicals in the occurrence of sinonasal papilloma.

Activation of cap-dependent translation by mucosal human papillomavirus E6 proteins is dependent on the integrity of the LXXLL binding motif.

The human papillomavirus (HPV) type 16 (HPV16) E6 protein can stimulate mechanistic target of rapamycin complex 1 (mTORC1) signaling and cap-dependent translation through activation of the PDK1 and mTORC2 kinases. Here we report that HPV18 E6 also enhances cap-dependent translation. The integrity of LXXLL and PDZ protein binding domains is important for activation of cap-dependent translation by high-risk mucosal HPV E6 proteins. Consistent with this model, low-risk mucosal HPV6b and HPV11 E6 proteins, which do not contain a PDZ protein binding motif, also activate cap-dependent translation and mTORC1, albeit at a lower efficiency than high-risk HPV E6 proteins. In contrast, cutaneous HPV5 and HPV8 E6 proteins, which lack LXXLL and PDZ motif protein binding, do not enhance cap-dependent translation. Mutagenic analyses of low-risk HPV E6 proteins revealed that association with the LXXLL motif containing ubiquitin ligase E6AP (UBE3A) correlates with activation of cap-dependent translation. Hence, activation of mTORC1 and cap-dependent translation may be important for the viral life cycle in specific epithelial tissue types and contribute to cellular transformation in cooperation with other biological activities of high-risk HPV E6-containing proteins.

Identification and analysis of papillomavirus E2 protein binding sites in the human genome.

Papillomavirus E2 protein is required for the replication and maintenance of viral genomes and transcriptional regulation of viral genes. E2 functions through sequence-specific binding to 12-bp DNA motifs-E2 binding sites (E2BS)-in the virus genome. Papillomaviruses are able to establish persistent infection in their host and have developed a long-term relationship with the host cell in order to guarantee the propagation of the virus. In this study, we have analyzed the occurrence and functionality of E2BSs in the human genome. Our computational analysis indicates that most E2BSs in the human genome are found in repetitive DNA regions and have G/C-rich spacer sequences. Using a chromatin immunoprecipitation approach, we show that human papillomavirus type 11 (HPV11) E2 interacts with a subset of cellular E2BSs located in active chromatin regions. Two E2 activities, sequence-specific DNA binding and interaction with cellular Brd4 protein, are important for E2 binding to consensus sites. E2 binding to cellular E2BSs has a moderate or no effect on cellular transcription. We suggest that the preference of HPV E2 proteins for E2BSs with A/T-rich spacers, which are present in the viral genomes and underrepresented in the human genome, ensures E2 binding to specific binding sites in the virus genome and may help to prevent extensive and possibly detrimental changes in cellular transcription in response to the viral protein.

Identification of key amino acid residues that determine the ability of high risk HPV16-E7 to dysregulate major histocompatibility complex class I expression.

High risk human Papillomavirus (HPV) types are the major causative agents of cervical cancer. Reduced expression of major histocompatibility complex class I (MHC I) on HPV-infected cells might be responsible for insufficient T cell response and contribute to HPV-associated malignancy. The viral gene product required for subversion of MHC I synthesis is the E7 oncoprotein. Although it has been suggested that high and low risk HPVs diverge in their ability to dysregulate MHC I expression, it is not known what sequence determinants of HPV-E7 are responsible for this important functional difference. To investigate this, we analyzed the capability to affect MHC I of a set of chimeric E7 variants containing sequence elements from either high risk HPV16 or low risk HPV11. HPV16-E7, but not HPV11-E7, causes significant diminution of mRNA synthesis and surface presentation of MHC I, which depend on histone deacetylase activity. Our experiments demonstrate that the C-terminal region within the zinc finger domain of HPV-E7 is responsible for the contrasting effects of HPV11- and HPV16-E7 on MHC I. By using different loss- and gain-of-function mutants of HPV11- and HPV16-E7, we identify for the first time a residue variation at position 88 that is highly critical for HPV16-E7-mediated suppression of MHC I. Furthermore, our studies suggest that residues at position 78, 80, and 88 build a minimal functional unit within HPV16-E7 required for binding and histone deacetylase recruitment to the MHC I promoter. Taken together, our data provide new insights into how high risk HPV16-E7 dysregulates MHC I for immune evasion.

Tax1BP1 interacts with papillomavirus E2 and regulates E2-dependent transcription and stability.

The papillomavirus E2 proteins regulate viral replication, gene transcription, and genome maintenance by interacting with other viral and host proteins. From a yeast two-hybrid screen, we identified the cellular protein Tax1BP1 as a novel binding partner of human papillomavirus type 18 (HPV18) E2. Tax1BP1 also interacts with the HPV16 and bovine papillomavirus type 1 (BPV1) E2 proteins, with the C-terminal region of Tax1BP1 interacting with the N-terminal transactivation domain of BPV1 E2. Tax1BP1 complexes with p300 and acts synergistically as a coactivator with p300 to enhance E2-dependent transcription. Using chromatin immunoprecipitation assays, we show that Tax1BP1 and E2 localize to the long control region on the BPV1 genome. Tax1BP1 was recently reported to bind ubiquitin and to function as an essential component of an A20 ubiquitin-editing complex. We demonstrate that Tax1BP1 plays a role in the regulation of the steady-state level of E2 by preventing its proteasomal degradation. These studies provide new insights into the regulation of E2 functions.

Possible role for cellular karyopherins in regulating polyomavirus and papillomavirus capsid assembly.

Polyomavirus and papillomavirus (papovavirus) capsids are composed of 72 capsomeres of their major capsid proteins, VP1 and L1, respectively. After translation in the cytoplasm, L1 and VP1 pentamerize into capsomeres and are then imported into the nucleus using the cellular alpha and beta karyopherins. Virion assembly only occurs in the nucleus, and cellular mechanisms exist to prevent premature capsid assembly in the cytosol. We have identified the karyopherin family of nuclear import factors as possible "chaperones" in preventing the cytoplasmic assembly of papovavirus capsomeres. Recombinant murine polyomavirus (mPy) VP1 and human papillomavirus type 11 (HPV11) L1 capsomeres bound the karyopherin heterodimer alpha2beta1 in vitro in a nuclear localization signal (NLS)-dependent manner. Because the amino acid sequence comprising the NLS of VP1 and L1 overlaps the previously identified DNA binding domain, we examined the relationship between karyopherin and DNA binding of both mPy VP1 and HPV11 L1. Capsomeres of L1, but not VP1, bound by karyopherin alpha2beta1 or beta1 alone were unable to bind DNA. VP1 and L1 capsomeres could bind both karyopherin alpha2 and DNA simultaneously. Both VP1 and L1 capsomeres bound by karyopherin alpha2beta1 were unable to assemble into capsids, as shown by in vitro assembly reactions. These results support a role for karyopherins as chaperones in the in vivo regulation of viral capsid assembly.

Do G protein-coupled receptors expressed in human lingual epithelium interact with HPV11?

Human papillomaviruses infect epithelia but little is known about the nature of cell surface receptors interacting with the viral particles. It has been proposed that glycosaminoglycans and integrins may be involved in the attachment process. In the present study, the putative interactions of virus-like particles of human papillomavirus type 11 (HPV11), which present a tropism for nasopharyngeal epithelia, with olfactory and taste receptors expressed in the human lingual epithelium were studied. The L1 protein of HPV11 was produced in insect cells. The presence of L1 virus-like particles was analyzed by ELISA using monoclonal antibodies specific for full-size particles and by electron microscopy. Using immunofluorescence, it was observed that virus-like particles interacted with taste buds from murine tongue, with the tagged human olfactory receptor hJCG5 expressed in HEK-293 but not with the tagged taste receptor hT2R4. This therefore suggests that hJCG5 may be involved in the adsorption process of HPV11 to lingual epithelium serving as a so-called "adsorption-adhesive molecule."

Optimization and determination of the absolute configuration of a series of potent inhibitors of human papillomavirus type-11 E1-E2 protein-protein interaction: a combined medicinal chemistry, NMR and computational chemistry approach.

We have previously reported the discovery and initial SAR optimization of the first series of inhibitors of the human papillomavirus type-11 (HPV11) E1-E2 protein-protein interaction. These inhibitors featured an indandione system spiro-fused onto an all syn substituted tetrahydrofuran ring. In this paper, we report new SAR efforts which have led to the identification of the first low nanomolar inhibitor of the HPV11 E1-E2 protein-protein interaction. In addition, we report a combined NMR and computational chemistry approach which allowed the successful determination of the absolute stereochemistry of the active species originating from the initial racemic lead.

The large and small isoforms of human papillomavirus type 16 E6 bind to and differentially affect procaspase 8 stability and activity.

Human papillomavirus type 16 (HPV-16) has developed numerous ways to modulate host-initiated immune mechanisms. The HPV-16 E6 oncoprotein, for example, can modulate the cellular level, and consequently the activity, of procaspase 8, thus modifying the cellular response to cytokines of the tumor necrosis factor family. E6 from HPV-16, but not E6 from the low-risk types 6b and 11, alters the cellular level of procaspase 8 in a dose-dependent manner. Both the large and small (E6*) isoforms of E6, which originate by way of alternate splicing, can modulate procaspase 8 stability. Intriguingly, although both isoforms bind to procaspase 8, the large isoform accelerates the degradation of procaspase 8 while the small isoform stabilizes it. Binding leads to a change in the ability of procaspase 8 to bind either to itself or to FADD (Fas-associated death domain), with the large version of E6 able to inhibit this binding while the small isoform does not. Consistent with this model, knockdown of the large version of E6 by small interfering RNA leads to increases in the levels of procaspase 8 and its binding to both itself and FADD. Thus, these alternatively spliced isoforms can modulate both the level and the activity of procaspase 8 in opposite directions.

Association of E6AP (UBE3A) with human papillomavirus type 11 E6 protein.

The cellular E3 ubiquitin ligase E6AP (UBE3A) interacts with the cancer-associated HPV E6 oncoproteins, where together with the viral E6 oncoprotein it binds and targets the degradation of the p53 tumor suppressor. We find that the HPV-11E6 protein also associates with E6AP in vivo, and thereby can target the degradation of an E6-associated protein. Mutation of an E6-binding LXXLL peptide motif on E6AP eliminated the association, revealing a common mode of interaction between high- and low-risk E6 proteins and E6AP. E6AP was required for the in vivo degradation of DLG1 by both HVP-18 E6 and a chimeric HPV-11E6. The common functional interaction of both cancer-associated and non-cancer-associated E6 proteins with E6AP establishes a common mechanism for E6 proteins trophic to mucosal squamous epithelium.

Keratinocyte-secreted laminin 5 can function as a transient receptor for human papillomaviruses by binding virions and transferring them to adjacent cells.

Human papillomaviruses (HPVs) replicate only in the terminally differentiating epithelium of the skin and mucosa. While infection of basal keratinocytes is considered a requirement for permissive infection, it remains unclear whether virions can specifically target basal cells for adsorption and uptake following epithelial wounding. We present evidence that HPV binds specifically to laminin 5 (LN5), a component of the extracellular matrix (ECM) secreted by migrating and basal keratinocytes. HPV type 11 capsids colocalized with LN5 in the ECM secreted by vaginal keratinocytes. Binding of both virions and virus-like particles to purified LN5 and to the LN5-rich ECM secreted by cultured keratinocytes was effectively blocked by pretreatment with anti-LN5 antibodies. HPV capsid binding to human cervical mucosa sections included the basement membrane which contains LN5. Cultured keratinocytes expressing alpha6 integrin, a transmembrane protein known to bind LN5, were readily infected by virions preadsorbed to LN5-containing substrates, whereas mutant keratinocytes lacking alpha6 integrin were relatively resistant to infection via this route. These findings suggest a model of natural HPV infection in which proliferating keratinocytes expressing alpha6 integrin at the site of epithelial wounding might be targeted by virions adsorbed transiently to LN5 secreted by migrating keratinocytes.

Brd4 links chromatin targeting to HPV transcriptional silencing.

The E2 protein encoded by human papillomaviruses (HPVs) inhibits expression of the viral E6 oncoprotein, which, in turn, regulates p53 target gene transcription. To identify cellular proteins involved in E2-mediated transcriptional repression, we isolated an E2 complex from human cells conditionally expressing HPV-11 E2. Surprisingly, the double bromodomain-containing protein Brd4, which is implicated in cell cycle control and viral genome segregation, was found associated with E2 and conferred on E2 the ability to inhibit AP-1-dependent HPV chromatin transcription in an E2-binding site-specific manner as illustrated by in vitro reconstituted chromatin transcription experiments. Knockdown of Brd4 in human cells alleviates E2-mediated repression of HPV transcription. The E2-interacting domain at the extreme C terminus and the chromatin targeting activity of a bromodomain-containing region are both essential for the corepressor activity of Brd4. Interestingly, E2-Brd4 blocks the recruitment of TFIID and RNA polymerase II to the HPV E6 promoter region without inhibiting acetylation of nucleosomal histones H3 and H4, indicating an acetylation-dependent role of Brd4 in the recruitment of E2 for transcriptional silencing of HPV gene activity. Our finding that Brd4 is a component of the virus-assembled transcriptional silencing complex uncovers a novel function of Brd4 as a cellular cofactor modulating viral gene expression.

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.