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Journal of Virology, February 2003, p. 2330-2337, Vol. 77, No. 4
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.4.2330-2337.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Nuclear Entry of High-Risk Human Papillomavirus Type 16 E6 Oncoprotein Occurs via Several Pathways

Lucia G. Le Roux and Junona Moroianu^*

Biology Department, Boston College, Chestnut Hill, Massachusetts 02467

Received 24 July 2002/ Accepted 20 November 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
The E6 oncoprotein of high-risk human papillomavirus type 16 (HPV16) interacts with several nuclear transcription factors and coactivators in addition to cytoplasmic proteins, suggesting that nuclear import of HPV16 E6 plays a role in the cellular transformation process. In this study we have investigated the nuclear import pathways of HPV16 E6 in digitonin-permeabilized HeLa cells. We found that HPV16 E6 interacted with the karyopherin (Kap) 2 adapter and could enter the nucleus via a classical Kap 2ß1-mediated pathway. Interestingly, HPV16 E6 also interacted, via its basic nuclear localization signal (NLS) located at the C terminus, with both Kap ß1 and Kap ß2 import receptors. Binding of RanGTP to these Kap ßs inhibited their interaction with HPV16 E6 NLS. In agreement with these binding data, nuclear import of the HPV16 E6 oncoprotein in digitonin-permeabilized HeLa cells could be mediated by either Kap ß1 or Kap ß2. Nuclear import via these pathways required RanGDP and was independent of GTP hydrolysis by Ran. Significantly, an E6_R124G mutant had reduced nuclear import activity, and the E6 deletion mutant lacking ₁₂₁KKQR₁₂₄ was not imported into the nucleus. The data reveal that the HPV16 E6 oncoprotein interacts via its C-terminal NLS with several karyopherins and exploits these interactions to enter the nucleus of host cells via multiple pathways.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human papillomaviruses (HPVs) are icosahedral DNA tumor viruses that infect squamous epithelial cells of the skin or of the anogenital and oropharyngeal mucosa. About 85 distinct HPV genotypes have been identified and fully sequenced, and more than 120 types have been partially characterized. Mucosal HPVs have demonstrated various degrees of oncogenic potential, with some classified as high risk, such as types 16, 18, 31, and 45, and others as low risk, such as types 6 and 11 (60). High-risk HPVs are frequently detected in invasive cervical carcinomas, whereas the low-risk types are more often associated with benign exophytic condylomas. HPV infection is associated with more than 90% of all cervical cancers, which is the second leading cause of cancer death among women worldwide (60).

Integration of the viral genome, resulting in deletion of several genes including E2 and maintenance of only the E6 and E7 genes, is a hallmark of malignant progression. Abrogation of E2 expression as a result of integration leads to uncontrolled expression of the E6 and E7 oncoproteins, which is crucial for the establishment and progression of the tumors. High-risk HPV E6 and E7 proteins cooperatively induce cellular immortalization and transformation (32, 40). Studies with transgenic mice suggest that whereas E7 promotes the formation of benign tumors, E6 acts primarily to accelerate progression of these benign tumors to the malignant stage (52).

HPV16 E6 is a basic protein of 151 amino acids that is able to form a complex with the p53 tumor suppressor protein targeting p53 degradation (47). The E6-AP ubiquitin ligase facilitates the formation of the E6-p53 complex and functions together with E6 in the ubiquitination and subsequent degradation of p53 via the ubiquitin-dependent proteolytic system (19, 45). As a consequence, the level and half-life of p53 in E6 immortalized cell lines or in HPV-positive carcinoma cells are decreased (46). High-risk HPV E6 oncoproteins are multifunctional, and several other E6-associated proteins have been identified, including the Ca²⁺-binding protein E6-BP (3), the focal adhesion protein paxilin (55, 56), the transcription factor c-Myc (17), the coactivators CBP and p300 (38), and the transcriptional activator IRF-3 (44). Interaction of HPV16 E6 with cellular proteins results in modification of several cellular activities, including differentiation, signal transduction, cytoskeletal structure, cell polarity, and DNA replication (54).

In the nucleus, high-risk HPV E6 proteins can also bind DNA and activate transcription of several genes (16, 24, 27). HPV16 E6 upregulates the expression of human telomerase reverse transcriptase through a combination of Myc and GC-rich Sp1 binding sites (36). High-risk HPV16 E6 oncoprotein localizes mainly to the nucleus when expressed in COS cells or insect cells (10, 16, 24, 33, 49). In cervical lesions, HPV16 E6 was found in both the nucleus and the cytoplasm (57), suggesting that the HPV16 E6 oncoprotein may shuttle between the two compartments.

The basic paradigm for nuclear import is that the NLS cargo is bound in the cytoplasm directly, or via an adapter, by an import receptor belonging to the karyopherin ß (Kap ß)/importin ß superfamily, translocated through the nuclear pore complex, and released inside the nucleus. Binding of nuclear RanGTP to Kap ßs causes the dissociation of the import complexes with release of the cargoes inside the nucleus (14, 30, 53, 59). Several members of the mammalian Kap ß superfamily have been identified and shown to function in nuclear import of specific cargoes. For example, Kap ß1/importin ß functions together with a Kap /importin adapter in nuclear import of proteins that contain classic monopartite or bipartite NLSs (14, 30). Kap ß1 can also function without adapters in import of different cargoes, including ribosomal proteins L23a, S7, and L5, cyclin B1, Smad, human immunodeficiency virus TAT and Rev proteins, and human T-cell leukemia virus Rex protein (30, 53). Mammalian Kap ß2/transportin mediates the nuclear import of hnRNP A1 and A2 via interaction with their Gly/Asn-rich NLS, termed M9 (2, 39, 50, 51), and of ribosomal proteins via interaction with their arginine-rich NLS (22). Ribosomal proteins L23a, S7, and L5 can interact with Kap ß1, Kap ß2/transportin, Kap ß3/Imp5, and Imp7 and be imported alternatively by these Kaps (22).

Many studies have focused on elucidating the molecular mechanisms of E6-mediated transformation. Nevertheless, there is virtually no information regarding the molecular mechanisms of the nuclear import of E6 oncoproteins. In this study we investigated the nuclear import of the E6 oncoprotein of high-risk HPV16. We found that the HPV16 E6 oncoprotein interacts with Kap 2 and can enter the nuclei of digitonin-permeabilized cells via Kap 2ß1 heterodimers. Interestingly, HPV16 E6 also interacted with Kap ß1 and Kap ß2, and binding of RanGTP to these Kap ßs inhibited their interaction with HPV16 E6 NLS. In agreement with these binding data, nuclear import of the HPV16 E6 oncoprotein could be mediated by either Kap ß1 or Kap ß2. The C terminus of HPV16 E6 (amino acids 121 to 151) interacted with either Kap 2, Kap ß1, or Kap ß2 and mediated nuclear import of a glutathione S-transferase (GST) reporter via either of these pathways. We found that mutation of R₁₂₄ to G reduced the nuclear import of the HPV16 E6 oncoprotein. Moreover, an E6 deletion mutant lacking ₁₂₁KKQR₁₂₄ was not imported into the nucleus, suggesting that the KKQR sequence within the NLS is essential for nuclear import of HPV16 E6 oncoprotein. Together, these data suggest that the HPV16 E6 oncoprotein interacts via its C-terminal NLS with Kap 2, Kap ß1, and Kap ß2 and exploits these interactions to enter the nucleus via Kap 2ß1-, Kap ß1-, and Kap ß2-mediated pathways.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Preparation of recombinant human nuclear import factors. His-tagged Kap 2/hSRP1 (58) and His-tagged Kap ß1/p97 (4) were expressed in Escherichia coli BL21(DE3) (3-h induction with 2 mM isopropyl-ß-D-thiogalactopyranoside [IPTG] at 30°C), and the soluble His-tagged proteins were purified in their native state on Talon beads using a standard procedure. GST-Kap ß1 (5) and GST-Kap ß2 (6) were expressed in E. coli BL21(DE3) (3-h induction with 1 mM IPTG at 30°C), and the soluble GST fusion proteins were purified in their native state on glutathione-Sepharose beads using a standard procedure. To obtain cleaved Kap ß2, the GST-Kap ß2 fusion protein was incubated for 2 h at room temperature with a Tev enzyme that has a His tag (Invitrogen), and after cleavage the GST was removed by binding to glutathione-Sepharose beads and the Tev enzyme was removed by binding to Talon beads, using standard procedures. Human Ran (7) was prepared as described previously (12). All the proteins were checked for purity and lack of proteolytic degradation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. The purified proteins were dialyzed in transport buffer (20 mM HEPES-KOH [pH 7.3], 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM dithiothreitol, plus protease inhibitors) and stored in aliquots at -80°C until use.

Preparation of recombinant HPV16 E6 oncoprotein. The recombinant full-length HPV16 E6 oncoprotein (20, 21) was expressed as a GST fusion protein in E. coli BL21(DE3) and purified in native conditions on glutathione-Sepharose beads using a standard procedure.

Preparation of GST-NLS fusion proteins and GST-E6 mutants. The C-terminal NLS of the HPV16 E6 (₁₂₁KKQRFHNIRGRWTGRCMSCCRSSRTRRETQL₁₅₁) and the E6 R124G NLS mutant were fused to a GST reporter in the following manner. The forward and reverse oligonucleotides consisted of the following base pairs. For GST-HPV16 E6 _121-151: forward, 5'-/5Phos/GGA ATT CCT GGA CAA AAA GCA AAG ATT CCA TAA TAT AAG GGG TCG GTG GAC CGG-3'; reverse, 5'-/5Phos/GCG CGC TCG AGT TAC AGC TGG GTT TCT CTA CGT GTT CTT GAT GAT CTG CAA CAA GAC ATA CAT CGA CCG GTC CAC CGA CCC C-3'). For the GST-HPV16 E6_121-151 mutant (R124 to G): forward, 5'-/5Phos/GGA ATT CCT GGA CAA AAA GCA AGG ATT CCA TAA TAT AAG GGG TCG GTG GAC CGG-3'; reverse, 5'-/5Phos/GCG CGC TCG AGT TAC AGC TGG GTT TCT CTA CGT GTT CTT GAT GAT CTG CAA CAA GAC ATA CAT CGA CCG GTC CAC CGA CCC C-3'. The oligonucleotides were designed with a 15- to 20-bp overlapping GC-rich area and annealed equimolarly. The overhanging areas were filled in using DNA polymerase I, large Klenow fragment (New England Biolabs catalog no. M0210S). The filled-in Klenow fragments were blunt end ligated with T4 DNA ligase. These fragments were double digested with EcoRI and XhoI and inserted into pGEX 4T-1 (Amersham Pharmacia Biotech 27-4580-01) at a vector-to-insert ratio of at least 1:10. To prepare the E6_R124G mutant and the E6_KKQR deletion mutant, the QuikChange site-directed mutagenesis kit from Stratagene was used according to the manual's instructions. All the constructs were transformed into XL1 blue bacteria and confirmed by automated sequence analysis (BioServe Biotechnologies Sequencing Department). GST-mpNLS_HPV16L1 was prepared as previously described (35), and the GST-M9 construct was a gift from Gideon Dreyfuss. For protein expression, the constructs were used to transform E. coli BL21(DE3). After induction of transformed E. coli BL21(DE3) with 1 mM IPTG for 3 h at 37°C, the GST-NLS fusion proteins were purified in their native state on glutathione-Sepharose beads using a standard procedure. The purified proteins were checked by SDS-PAGE and Coomassie blue staining, dialyzed in transport buffer, and stored in aliquots at -80°C until use.

In vitro nuclear import assays. We carried out in vitro nuclear import assays in digitonin-permeabilized HeLa cells, as previously described for HPV L1 major capsid proteins (34, 35). HeLa cells are cervical carcinoma cells that contain HPV18 DNA integrated into the cellular genome with consequent disruption of several viral genes but preservation of the E6 and E7 genes. HeLa cells contain low levels of HPV18 E6, localized by immunofluorescence with specific antibodies (sc-1585 from Santa Cruz Inc., Santa Cruz, Calif.) both in the nucleus and in the cytoplasm (data not shown). Although HeLa cells are HPV18 positive, this does not affect the import properties of their nuclear pore complexes, as demonstrated by the fact that many mammalian nuclear import pathways have been identified and characterized with HeLa cells (1, 4, 15, 25, 37, 39, 41, 42, 48, 58). Briefly, subconfluent HeLa cells, grown on poly-L-lysine (Sigma P-4707)-coated glass coverslips for 1 day, were permeabilized with 70 µg of digitonin/ml for 5 min on ice. Unless otherwise specified, the import reactions contained an energy regenerating system (0.5 mM GTP, 5 mM phosphocreatine, and 0.4 U of creatine phosphokinase) plus various transport factors (1 µM Kap 2; 0.5 µM Kap ß1; 0.5 µM Kap ß2; 3 µM RanGDP), plus the GST-E6 or the GST-NLS fusion protein (0.5 µM). The final import reaction volume was adjusted to 20 µl with transport buffer. At the end of the nuclear import reaction, the cells were fixed with 3.7% formaldehyde on ice followed by methanol for 3 min at -20°C. In order to prevent nonspecific binding, cells were blocked for 1 h in 3% bovine serum albumin with 0.1% Tween in phosphate-buffered saline. For visualization of nuclear import, the GST fusion proteins were detected by immunofluorescence with an anti-GST antibody (1:200 dilution; Amersham Pharmacia 27-4577-01) followed by a fluorescently labeled secondary antibody (1:100 dilution; Sigma F2016). The nuclei were identified by 4',6'-diamidino-2-phenylindole staining. Nuclear import was analyzed with a Nikon Eclipse TE 300 microscope that has a fluorescence attachment and a Sony DKC-5000 charge-coupled device camera.

In-solution binding assays. The interactions between GST-NLS_HPV16E6 and Kap 2, Kap ß1, and Kap ß2 were investigated using in-solution binding assays with the GST fusion proteins immobilized on glutathione-Sepharose (3 µg of protein/10 µl of beads) and the different recombinant Kaps in binding buffer (0.025% Tween and 0.5% glycerol in transport buffer). The final reaction volume was adjusted to 40 µl with binding buffer. In some experiments, the Kap ßs were preincubated for 30 min with RanGTP (3 µg) before incubation with GST-NLS_HPV16E6. As a negative control we used GST, and as positive binding controls for Kap 2 we used GST-mpNLS_HPV16L1, and for Kap ß2 we used GST-M9. After 60 min of incubation at room temperature, the beads were washed three times with binding buffer and the bound proteins were eluted with SDS sample buffer and analyzed by SDS-PAGE and Coomassie blue staining.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The HPV16 E6 oncoprotein can enter the nucleus in complex with either Kap ß1 or Kap 2ß1 heterodimers. E6 oncoproteins interact with several nuclear transcription factors and coactivators in addition to cytoplasmic proteins and consequently have both nuclear and cytoplasmic activities. To identify and characterize the nuclear import pathway(s) exploited by the E6 oncoprotein of high-risk HPV16, we employed in vitro import assays in digitonin-permeabilized HeLa cells. Since recombinant E6 proteins are difficult to obtain in soluble form (43) and since GST fusion proteins have been extensively used in nuclear import studies, we used E6-GST fusion proteins that are readily obtained in soluble native form (26).

We have analyzed the nuclear import of a recombinant HPV16 E6-GST fusion protein and GST (as a control) in digitonin-permeabilized HeLa cells in the presence of only transport buffer or HeLa cytosol. We found that HPV16 E6-GST was transported into the nucleus in the presence of exogenous cytosol (Fig. 1B), but not in its absence (Fig. 1A). As expected, the GST reporter protein lacking an NLS was not imported into the nucleus in the presence of cytosol (data not shown). These data indicate that nuclear import of HPV16 E6 requires transport factors present in the cytosol.

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FIG. 1. Nuclear import of the HPV16 E6 oncoprotein. Digitonin-permeabilized HeLa cells were incubated for 30 min at room temperature with HPV16 E6-GST (A and B) or, as a negative control, the GST reporter protein (C and D) in the presence of either transport buffer alone (A and C) or HeLa cytosol and an energy-regenerating system (B and D). HPV16 E6-GST and GST were detected by immunofluorescence with a primary anti-GST antibody. Note the nuclear import in panel B.

It has been shown in transfection assays that deletion of the C-terminal region of HPV16 E6 (amino acids 121 to 151) abolishes its nuclear import (49), suggesting that this region functions as an NLS. Therefore, we investigated if the C-terminal region (amino acids 121 to 151) of HPV16 E6 is sufficient to target the GST reporter protein to the nucleus. The GST-E6_121-151 fusion protein was imported into the nuclei of digitonin-permeabilized cells in the presence of cytosol (Fig. 2B), whereas the GST control was not (Fig. 2D), confirming that, indeed, the C-terminal region of HPV16 E6 functions as an NLS.

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FIG. 2. Nuclear import of GST-HPV16 E6121-151. Digitonin-permeabilized HeLa cells were incubated for 30 min at room temperature with GST-E6121-151 (A and B), or, as a negative control, the GST reporter protein (C and D) in the presence of either transport buffer alone (A and C) or HeLa cytosol and an energy-regenerating system (B and D). Note the nuclear import in panel B.

Sequence analysis of HPV16 E6 NLS (₁₂₁KKQRFHNIRGRWTGRCMSCCRSSRTRRETQL₁₅₁) shows that it contains two blocks of basic amino acids separated by 19 amino acids. The first block of basic amino acids (₂₁KKQR) resembles a classic monopartite NLS and could interact with Kap 2 adapter, whereas the presence of many arginines throughout the NLS suggests the possibility of interaction directly with some members of the Kap ß family (22).

To analyze the interactions between the NLS of HPV16 E6 with Kap 2 and Kap ß1, we used in-solution binding assays. GST-NLS_HPV16E6, GST-NLS_HPV16L1 (positive control for Kap 2), and GST (negative control) were immobilized on glutathione-Sepharose beads and incubated with either Kap 2, Kap ß1, or Kap 2 plus Kap ß1. GST-NLS_HPV16E6 bound to both Kap 2 and Kap ß1 and most likely formed a trimeric complex with Kap 2ß1 heterodimers (Fig. 3A, lanes 1 to 3). As expected, GST-mpNLS_HPV16L1 bound to Kap 2 and formed a trimeric complex with Kap 2ß1 heterodimers, but did not bind to Kap ß1 (Fig. 3A, lanes 4 to 6). Mutation of R124 to G disrupts the first block of basic amino acids, resembling a monopartite NLS, and the corresponding NLS mutant would be expected to be unable to bind Kap 2. Indeed, the R124G NLS mutant did not bind Kap 2 (Fig. 3A, lane 7). Interestingly, the R124G NLS mutant still bound Kap ß1, although the binding was reduced in comparison with the wild-type NLS (Fig. 3A, compare lanes 8 and 2). Neither Kap 2 nor Kap ß1 bound to the immobilized GST (Fig. 3A, lanes 10 and 11). Significantly, binding of RanGTP to Kap ß1 inhibited the interaction of GST-NLS_HPV16E6 with Kap ß1 (Fig. 3B, compare lanes 1 and 2), suggesting that RanGTP can dissociate the HPV16 E6/Kap ß1 complex.

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FIG. 3. The NLS of the HPV16 E6 oncoprotein interacts with both Kap 2 and Kap ß1. (A) GST-NLSHPV16E6, GST-NLSHPV16L1, GST-NLSR124G, and GST were immobilized on glutathione-Sepharose beads (3 µg of protein/10 µl of beads). The beads containing GST-NLSHPV16E6 (lanes 1 to 3), GST-NLSHPV16L1 (lanes 4 to 6), GST-NLSR124G (lanes 7 to 9), and GST (lanes 10 and 11) were incubated with either Kap 2 (lanes 1, 4, 7, and 10) or Kap ß1 (lanes 2, 5, 8, and 11) or Kap 2 plus Kap ß1 (lanes 3, 6, and 9). Note that the NLS of HPV16 E6 interacts with either the Kap 2 (lane 1), Kap ß1 (lane 2), or Kap 2ß1 heterodimers (lane 3). The monopartite NLS of HPV16 L1 interacts with Kap 2 (lane 4) and forms a trimeric complex with Kap 2ß1 heterodimers (lane 6) but does not interact directly with Kap ß1 (lane 5). The GST-NLSR124G mutant no longer interacts with Kap 2 but still interacts with Kap ß1 (lanes 7 and 8). Neither Kap 2 nor Kap ß1 interacts with GST alone (lanes 10 and 11). The input of Kap 2 is in lane 12, and that of Kap ß1 is in lane 13. Quantitation analysis of two experiments indicates the following percentage of direct binding of Kaps: for GST-NLSHPV16E6, 18% binding of Kap 2 and 29% binding of Kap ß1; for the GST-NLSE6R124G mutant, 12% binding of Kap ß1; for GST-NLSHPV16L1, 41% binding of Kap 2. (B) GST-NLSHPV16E6 immobilized on glutathione-Sepharose beads was incubated with Kap ß1 alone (lane 1) or Kap ß1 plus RanGTP (lane 2). Bound proteins were eluted with sample buffer and analyzed by SDS-PAGE and Coomassie blue staining.

Analysis of the nuclear import of HPV16 E6 in the presence of Kap ß1 plus RanGDP or Kap 2 plus Kap ß1 plus RanGDP revealed that, in agreement with the binding data, HPV16 E6 is imported into the nucleus in both cases (Fig. 4B and C). As expected, the control for the classical pathway, GST-mpNLS_HPV16L1, is imported only in the presence of Kap 2 plus Kap ß1 plus RanGDP (Fig. 4I) and not in the absence of Kap 2 adapter (Fig. 4H). These data suggest that the HPV16 E6 oncoprotein can enter the nucleus via either the Kap ß1-mediated pathway or the classical Kap 2ß1-mediated pathway. Analysis of the nuclear import of the GST-E6_121-151 fusion protein in the presence of either Kap ß1 or Kap 2 plus Kap ß1 revealed that the NLS of HPV16 E6 is sufficient to mediate nuclear import via either the Kap ß1- or the Kap 2ß1-mediated pathway (Fig. 4E and F). Together the binding and nuclear import data suggest that the HPV16 E6 NLS can interact directly with either Kap 2 or Kap ß1 and mediate entry into the nucleus via both Kap 2ß1- and Kap ß1-mediated pathways.

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FIG. 4. Nuclear import of the HPV16 E6 oncoprotein can be mediated by either Kap ß1 or Kap 2ß1 heterodimers. Digitonin-permeabilized HeLa cells were incubated for 30 min at room temperature with either HPV16 E6-GST (A to C), GST-NLSHPV16E6 (D to F), or GST-NLSHPV16L1 (G to I) in the presence of either transport buffer alone (A, D, and G), Kap ß1 plus RanGDP (B, E, and H), or Kap ß1 plus Kap 2 plus RanGDP (C, F, and I). Note the nuclear import in panels B, C, E, F, and I.

The HPV16 E6 oncoprotein interacts with Kap ß2 and can enter the nucleus via a Kap ß2-mediated pathway. The presence of many arginines throughout the NLS of HPV16 E6 suggests the possibility of interaction directly with other members of the Kap ß family (22). Therefore, we tested the ability of Kap ß2 to interact with HPV16 E6 NLS using in-solution binding assays. GST-NLS_HPV16E6, GST-M9 (a positive control for Kap ß2), and GST (negative control) immobilized on glutathione-Sepharose beads were incubated with Kap ß2. We found that Kap ß2 bound to GST-NLS_HPV16E6 and the GST-M9 positive control (Fig. 5A, lanes 1 and 2), but not to GST itself (lane 4). Interestingly, the R124G NLS mutant did not bind to Kap ß2 (lane 3), suggesting that R₁₂₄ is essential for the interaction with Kap ß2.

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FIG. 5. The NLS of the HPV16 E6 oncoprotein interacts with Kap ß2. (A) Immobilized GST-NLSHPV16E6 (lane 1), GST-M9 (lane 2), GST-NLSR124G (lane 3), and GST (lane 4) were incubated with Kap ß2. Note that GST-NLSHPV16E6 and the GST-M9 positive control interact with Kap ß2 (lanes 1 and 2). Binding of the GST-NLSR124G mutant to Kap ß2 is very low (lane 3), at a level similar to that for the GST negative control (lane 4). The input of Kap ß2 is in lane 5. Quantitation analysis of two experiments indicates the following percentages of binding of Kap ß2: 19% for GST-NLSHPV16E6 and 29% for GST-M9. (B) Immobilized GST-NLSHPV16E6 (lanes 1 and 2) was incubated with either Kap ß2 alone (lane 1) or Kap ß2 plus RanGTP (lane 2). Bound proteins were eluted with sample buffer and analyzed by SDS-PAGE and Coomassie blue staining.

Moreover, binding of RanGTP to Kap ß2 reduced the interaction of Kap ß2 with either GST-NLS_HPV16E6 (Fig. 5B, compare lanes 1 and 2) or the GST-M9 control (data not shown). These binding data suggest that the HPV16 E6 oncoprotein could also enter into the nucleus via a Kap ß2-mediated pathway. To test this, digitonin-permeabilized HeLa cells were incubated with either HPV16 E6-GST or M9-GST (as a positive control for Kap ß2) in the presence of either transport buffer alone or RanGDP, or Kap ß2 plus RanGDP. Both HPV16 E6 and M9-GST were imported into the nucleus in the presence of Kap ß2 plus RanGDP (Fig. 6C and I). Moreover, GST-NLS_HPV16E6 was also imported into the nucleus in the presence of Kap ß2 plus RanGDP (Fig. 6F). Together these data indicate that the HPV16 E6 oncoprotein interacts with Kap ß2 via its C-terminal NLS and enters the nucleus via a Kap ß2-mediated pathway.

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FIG. 6. Nuclear import of the HPV16 E6 oncoprotein can be mediated by Kap ß2. Digitonin-permeabilized HeLa cells were incubated for 30 min at room temperature with either HPV16 E6-GST (A to C), GST-NLSHPV16E6 (D to F), or GST-M9 (G to I) in the presence of either transport buffer alone (panels A, D, and G), RanGDP (B, E, and H), or RanGDP plus Kap ß2 (C, F, and I). Note the nuclear import in panels C, F, and I.

An E6_R124G mutant has reduced nuclear import activity, and the E6_KKQR deletion mutant is defective in nuclear import. An HPV16 E6 mutant in which R₁₂₄ is changed to G has less ability to immortalize mammary epithelial cells than wild-type E6 (9). This mutation disrupted the ₁₂₁KKQR sequence, and the corresponding NLS mutant no longer bound Kap 2 and Kap ß2 (Fig. 3A and 5A), suggesting that it would have reduced nuclear import activity. We analyzed the nuclear import of GST-NLS_R124G mutant in comparison with the GST-NLS_E6 wild type in identical conditions in digitonin-permeabilized cells in the presence of cytosol. Quantitation of nuclear import of the GST-NLS_R124G mutant and the GST-NLS_E6 wild type was carried out by measuring the mean nuclear fluorescence using IPLAB software. We found that nuclear import of the GST-NLS_R124G mutant was reduced in comparison with that of the GST-NLS_E6 wild type (Fig. 7, compare panels A and B), with a mean ratio of mutant nuclear import/wild-type nuclear import of 0.58 ± 0.05 (in four experiments).

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FIG. 7. An R124G mutation reduces the nuclear import activity of HPV16 E6 NLS. Digitonin-permeabilized HeLa cells were incubated for 30 min at room temperature with either the GST-NLSHPV16E6 wild type (A) or the GST-NLSR124G mutant (B) in the presence of HeLa cytosol. Note the reduced nuclear fluorescence in panel B in comparison with panel A.

We also compared the nuclear import of wild-type HPV16 E6 with that of two full-length E6 mutant proteins, one containing the R124G mutation and the other containing a deletion of the first block of basic amino acids (KKQR) of the E6 NLS. Nuclear import of the E6_R124G mutant was reduced in comparison with that of wild-type E6 (Fig. 8, compare panels A and B), with a mean ratio of mutant nuclear import/wild-type nuclear import of 0.75 ± 0.02 (in 4 experiments). Significantly, the E6_KKQR deletion mutant was not imported into the nucleus (Fig. 8C). These data suggest that the ₁₂₁KKQR₁₂₄ sequence within the NLS of the HPV16 E6 oncoprotein is essential for E6 nuclear import.

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FIG. 8. The KKQR sequence within the NLS is essential for nuclear import of the HPV16 E6 oncoprotein. Digitonin-permeabilized HeLa cells were incubated for 30 min at room temperature in the presence of cytosol with the GST fusions containing either wild-type HPV16 E6 (A), the E6R124G mutant (B), or the E6KKQR mutant (C). Note the lack of import in panel C.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Interaction of the E6 oncoprotein of high-risk HPV16 with several nuclear proteins, including p53 and c-Myc transcription factors, the coactivators CBP and p300, and the transcriptional activator IRF-3 (28), indicates that E6 has nuclear functions in addition to its cytoplasmic roles.

In this study, we identified and characterized the nuclear import pathways of the HPV16 E6 oncoprotein. We found that the HPV16 E6 oncoprotein interacts with the Kap 2 adapter and can enter the nuclei of digitonin-permeabilized cells via a classical Kap 2ß1-mediated pathway. Interestingly, the HPV16 E6 oncoprotein also interacts directly via its C-terminal arginine-rich NLS with Kap ß1, and binding of RanGTP to Kap ß1 inhibits the interaction. In agreement with these binding data, nuclear import of the HPV16 E6 oncoprotein and of a GST fusion protein containing its C-terminal NLS can be mediated by Kap ß1 without a requirement for the Kap 2 adapter. Significantly, we discovered that the HPV16 E6 oncoprotein also interacts via its C-terminal arginine-rich NLS with Kap ß2, and nuclear import of the HPV16 E6 oncoprotein and of GST-NLS_E6 can be mediated by Kap ß2. Together these data suggest that the HPV16 E6 oncoprotein can enter the nuclei of host cells via Kap 2ß1-, Kap ß1-, and Kap ß2-mediated pathways. The cytosol of HeLa cells (HPV-18-positive cervical carcinoma) contains Kap 2, Kap ß1, and Kap ß2, as detected by immunoblot analysis with specific antibodies (29, 34). This suggests that the HPV16 E6 oncoprotein can exploit the Kap 2ß1-, Kap ß1-, and Kap ß2-mediated pathways for its nuclear entry in cervical cancer cells. It is possible that the HPV16 E6 oncoprotein can interact with additional member(s) of the karyopherin ß superfamily and exploit these interactions for its nuclear entry in a manner similar to that of ribosomal proteins (22). Interestingly, recent studies revealed that Kap ßs/importins fulfill a dual function as nuclear import receptors and cytoplasmic chaperones for exposed basic domains of basic proteins such as ribosomal proteins and histones (23). The interaction of HPV16 E6 with Kap ßs could play a similar dual role in both import of the E6 oncoprotein and prevention of its aggregation in the cytoplasm by shielding its basic C-terminal NLS region.

We determined that the C-terminal region of HPV16 E6, rich in basic amino acids (Table 1), functions as an NLS mediating nuclear import of a GST reporter via Kap 2ß1-, Kap ß1-, or Kap ß2-mediated pathways. Mutation of R124 to G within the NLS reduced the nuclear import activity of the HPV16 E6_R124G mutant in comparison with that of wild-type E6. Interestingly, an HPV16 E6 mutant in which R₁₂₄ is replaced by G has a reduced ability to immortalize mammary epithelial cells in comparison with wild-type E6 (9). Significantly, we determined that a deletion mutant, E6_KKQR (lacking amino acids 121 to 124), was defective in nuclear import, indicating that the KKQR sequence within the NLS is essential for import. Previous mutational analysis of HPV16 E6 established the NLS overlapping regions (amino acids 123 to 127 and 128 to 132) to be essential for the transactivation activity from adenovirus E2, c-fos, and TGF-ß1 promoters, suggesting that these E6 deletion mutants were defective in nuclear import (8, 11, 31).

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TABLE 1. Potential nuclear localization sequences of HPV E6 proteinsa

Interestingly, comparison of the NLS of HPV16 E6 with the corresponding sequences in other HPVs shows that it is partially conserved in high-risk HPV-18 and is not conserved in low-risk HPV-11 and -6 (Table 1). Preliminary data show that a GST fusion protein containing this potential NLS of high-risk HPV18 E6 is indeed actively imported into the nuclei of digitonin-permeabilized cells in the presence of exogenous cytosol (unpublished observations). The lack of homology of the corresponding sequences in low-risk HPV-11 and -6 suggests that there are major differences between the nuclear import activities of high- and low-risk E6 proteins. Future detailed analysis of nuclear import of low-risk E6 proteins in comparison with high-risk E6 proteins will bring new insight into the role of nuclear import of E6 oncoproteins in cellular transformation.

In HPV-positive cancer cells, the E6-dependent pathway of p53 degradation is active and required for the growth of these cancer cells, whereas the Mdm-2-dependent pathway of p53 degradation is inactive (18). Interestingly, inhibition of CRM-1-mediated nuclear export in HPV-positive tumor cells by the drug leptomycin B results in accumulation of p53, suggesting that E6-mediated degradation of p53 is dependent on its nuclear export (13). It remains to be established if nuclear import of E6 oncoproteins in HPV-positive cancer cells may play a role in efficient nuclear export and degradation of p53 in the cytoplasm. Future studies will bring novel insights into the role(s) of nuclear import of the HPV16 E6 oncoprotein in the transformation process that occurs in cervical carcinoma cells.

 
We thank G. Blobel, P. Howley, Y. M. Chook, K. Weis, A. Lamond, S. Adam, and G. Dreyfuss for their generous gifts of expression vectors. We thank L. Heller, M. Garey, P. Felice, and A. Rich for technical assistance in preparation of HPV16 E6-GST, GST-NLSs, and recombinant transport factors. We thank C. Hoffman for help with site-directed mutagenesis of HPV16 E6 oncoprotein. We also thank L. Heller for help in analysis of nuclear import of wild-type and mutant E6 NLS using IPLAB software. We thank A. Annunziato and C. Hoffman for helpful discussions and C. Hoffman for critical reading of the manuscript.

This work was supported by grant RPG-99-210-01-MBC from the American Cancer Society to J.M. and grant 1-RO1 CA94898-01 from NIH to J.M.

 
* Corresponding author. Mailing address: Biology Department, Boston College, 140 Commonwealth Ave., Chestnut Hill, MA 02467-3811. Phone: (617) 552-1713. Fax: (617) 552-2011. E-mail: moroianu{at}bc.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, February 2003, p. 2330-2337, Vol. 77, No. 4
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.4.2330-2337.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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