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Journal of Virology, October 2008, p. 10042-10051, Vol. 82, No. 20
0022-538X/08/$08.00+0     doi:10.1128/JVI.01240-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Bap31 Is a Novel Target of the Human Papillomavirus E5 Protein{triangledown}

Jennifer A. Regan and Laimonis A. Laimins*

Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611

Received 14 June 2008/ Accepted 31 July 2008


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ABSTRACT
 
The E5 proteins of human papillomaviruses (HPVs) are small hydrophobic proteins that are expressed in the early and late stages of the viral life cycle; however, their role in HPV pathogenesis is not clearly understood. In this study, a split-ubiquitin yeast (Saccharomyces cerevisiae) two-hybrid system was used to identify B-cell-associated protein 31 (Bap31) as a binding partner of HPV E5 proteins. The association of these proteins was confirmed by coimmunoprecipitation of complexes of Bap31 with either HPV type 16 (HPV16) or HPV31 E5. In addition, Bap31 and E5 were found to colocalize in perinuclear patterns consistent with localization to the endoplasmic reticulum. Mutational analysis of E5 identified amino acids in the extreme C terminus as important for stabilizing the interaction with Bap31. Deletion of these C-terminal amino acids of E5 in the context of complete HPV31 genomes resulted in impaired proliferative capacity of HPV-positive keratinocytes following differentiation. When small interfering RNAs were used to reduce the levels of Bap31, the proliferative ability of HPV-positive keratinocytes upon differentiation was also reduced, implicating Bap31 as a regulator of this process. These studies identify a novel binding partner of the high-risk HPV E5 proteins and provide insight into how the E5 proteins may modulate the life cycle in differentiating cells.


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INTRODUCTION
 
Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that are the causative agents of cervical cancer, with over 99.7% of cases positive for viral sequences (49). Over 100 different types of HPVs have been identified, and approximately one-third target the genital tract for infection. These genital papillomaviruses are sexually transmitted and can be further divided into low- and high-risk types. The high-risk types, including HPV types 16 (HPV16), -18, and -31, are associated with the development of genital cancers, while the low-risk types, such as HPV6 and -11, induce benign hyperproliferative lesions (55).

The HPV productive life cycle is closely linked with the differentiation status of the host cell, the keratinocyte. HPV infection occurs in cells in the basal layer of stratified squamous epithelia following exposure through microabrasions. Upon entry, the genomes are established as nuclear episomes at approximately 50 to 100 copies per cell and are coordinately replicated along with the cellular DNA (24). In HPV-infected basal cells, early transcripts encoding E6, E7, E1, E2, E4, and E5 are expressed. Due to the polycistronic nature of HPV transcripts, only low levels of factors encoded at the 3' end of these messsages, such as E4 and E5, are translated into proteins. Upon cell division, viral DNA is partitioned to daughter cells, one of which migrates away from the basement membrane and differentiates in the suprabasal epithelium. Expression of the viral oncoproteins enables these differentiating cells to remain active in the cell cycle and allows for amplification of viral genomes to several thousand copies per cell in the differentiated layers (41). Synthesis of HPV capsid proteins and production of progeny virus are then induced in the uppermost layers.

The development and maintenance of genital cancers are dependent on the expression of the early viral oncoproteins E6 and E7. The E6 protein interacts with the tumor suppressor p53, resulting in its ubiquitination and consequent degradation through the recruitment of the cellular ubiquitin ligase E6-AP (23, 43, 44, 51). The E7 protein inactivates and induces the degradation of the retinoblastoma protein (pRB), a cell cycle regulatory protein required for the G1/S transition and DNA synthesis (6, 14, 32, 34).

Upon epithelial differentiation, the late proteins L1, L2, E1{wedge}E4, and E5 are expressed. The first two open reading frames found on the majority of late transcripts are E1Ê4 and E5, which lead to their high-level expression in differentiated cells. The E1{wedge}E4 proteins are generated as a result of splicing such that the first 5 amino acids of E1 are joined to E4 coding sequences. These E1{wedge}E4 proteins can induce the collapse of the cytokeratin network as well as facilitate late viral functions in differentiating cells (12, 52). The HPV E5 proteins are small hydrophobic proteins located predominately in the endoplasmic reticulum (ER), but very little is known about how they function in HPV pathogenesis in keratinocytes (7, 10, 11).

In contrast to the HPV E5 proteins, the bovine papillomavirus type 1 (BPV1) E5 protein has been extensively characterized, and it has been shown to be the main oncoprotein of the fibrosarcoma-inducing virus. The BPV1 E5 protein induces transformation of mouse cells by binding and constitutively activating the platelet-derived growth factor (PDGF) β receptor (38). The BPV E5 gene encodes a 44-amino-acid protein which is about half the size of the HPV E5 proteins and has little sequence homology to its human counterpart. Specifically, the amino acids shown to be necessary for BPV E5 transforming activity are not conserved in the HPV E5 proteins.

Much less is known about how the HPV E5 proteins function, and the majority of this information comes from the use of heterologous assay systems. Expression of HPV E5 can stimulate cell proliferation, and it exhibits weak transforming ability in heterologous cell types (4, 48). Several reports have implicated the epidermal growth factor receptor (EGFR), rather than the PDGF receptor, as a key player in the action of HPV E5. In the presence of EGF, E5 has been shown to increase cellular proliferation in mouse fibroblasts (31). One study reported that HPV16 E5 can bind to the EGFR in COS cells; however, these complexes have not been seen in other studies (8, 25). Additional reports suggested that E5 might affect EGFR recycling by inhibiting or delaying the internalization of the EGFR (47). Furthermore, transgenic mice expressing HPV16 E5 displayed increased frequency of epithelial tumors, and this was dependent upon EGFR expression (18).

A second reported target of E5 proteins is the vacuolar H+-ATPase. Both HPV16 E5 and BPV1 E5 can bind the 16-kDa subunit of the vacuolar H+-ATPase, which is a proton pump responsible for acidifying cellular organelles such as the Golgi apparatus, lysosomes, and endosomes (7, 19, 20). The amino acids in BPV1 E5 that are necessary for transformation, however, are not necessary for binding to the 16-kDa subunit (21). HPV16 E5 has been shown to inhibit the acidification of endosomes (10, 46), and it has been speculated that this might facilitate increased levels of receptors at the cell surface resulting in constitutive signaling.

Studies examining the role of E5 in the productive life cycle of HPV31 and HPV16 have demonstrated that E5 can modulate late viral functions following differentiation (15, 17). Keratinocytes that maintain wild-type HPV31 genomes remain active in the cell cycle following differentiation and retain proliferation ability, as measured by colony-forming ability. In contrast, cells harboring the HPV31 E5 mutant genomes showed a significant reduction in colony-forming ability following methylcellulose-induced differentiation (15). This suggests that E5 acts to keep cells in a proliferation-competent state upon differentiation. In addition, HPV31 and HPV16 genomes containing mutations that disrupted E5 expression exhibited reduced ability to amplify viral genomes and activate late gene expression. Interestingly, the level and phosphorylation state of the EGFR were not affected in differentiating cells that maintained complete HPV31 genomes, indicating there may be other targets of E5. In the present study, we used yeast (Saccharomyces cerevisiae) two-hybrid analysis to identify B-cell-associated protein 31 (Bap31) as a target of HPV16 as well as HPV31 E5 and show that these proteins form complexes in vivo. Furthermore, we demonstrate that binding correlates with the ability to retain the proliferative capacity of keratinocytes following differentiation.


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MATERIALS AND METHODS
 
Cell culture. Human foreskin keratinocytes (HFKs) were isolated from human neonatal foreskins as previously described (42) and grown in serum-free keratinocyte growth medium (Lonza). HPV31-positive cell lines, including CIN612 cells and LKP-1 cells, were grown in the presence of mitomycin C-treated J2 3T3 fibroblasts and maintained in serum-containing medium (E medium) containing mouse EGF (5 ng/ml; B.D. Bioscience). 293TT cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone). To induce differentiation, keratinocytes were grown in raft cultures as previously described (53). Briefly, cells were plated onto a solidified collagen matrix containing J2 3T3 fibroblasts. Upon confluence, the cells were transferred to a metal grid to induce differentiation. After 14 days, the raft cultures were harvested, fixed in 4% paraformaldehyde, paraffin embedded, and sectioned.

Plasmids. The Bap31 open reading frame was isolated from the yeast two-hybrid positive clones and cloned into the pSG5 vector to generate pSG5-Bap31. A codon-optimized HPV16 E5* with an N-terminal AU1 tag was a gift from R. Schlegel and was expressed in a pSG5 vector (11). To construct recombinant retroviral vectors expressing E5, the HPV16 E5* gene with an N-terminal AU1 tag was cloned into the LXSN vector, generating LXSN-16E5*. The wild-type HPV31 E5 gene with an N-terminal AU1 tag was cloned into the pSG5 vector to generate pSG5-31WTE5. Mutations in the E5 gene were introduced using the Quikchange-XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). The C-terminal deletion mutants of E5 included pSG5-16E5*-5AA and pSG5-16E5*-10AA. For the genomic studies, the HPV31 genome was subcloned into a pBR322-derived plasmid. E5 mutant genomes were constructed via site-directed mutagenesis and sequenced. The fragment containing the mutant E5 gene was then exchanged with the wild-type sequence in pBRmin-HPV31 by using the single-cutting restriction endonucleases EcoRI (nucleotide 3361) and XbaI (nucleotide 4998). HPV31E5-1 contains a deletion of amino acids 41 to 45 (TLLLL). HPV31E5-2 contains two alanine amino acids in place of arginine 58 and cysteine 59. HPV31E5-3 contains two stop codons in place of serine 79 and phenylalanine 80.

Transfection. For the transient transfection experiments, pSG5 constructs were transfected into 293TT or CIN612 cells using polyethylenimine reagent at a final concentration of 0.2 mg/ml (Polyscience, Inc.) at 30% confluence. The cells were harvested for protein or immunofluorescence analysis at 48 h posttransfection. Stable transfection of the HPV31 genome into normal human keratinocytes was performed as previously described (16). Briefly, the genomes were first released from the pBR322-derived plasmid following digestion with HindIII at 37°C overnight. Following heat inactivation of the restriction enzyme at 65°C for 10 min, the genomes were unimolecularly religated using T4 DNA ligase at 16°C overnight (New England Biolabs). The DNA was then precipitated with isopropyl ethanol and 5 M NaCl overnight at –20°C. The following day, the DNA was isolated by centrifugation, washed with 70% ethanol, and resuspended in 10 mM Tris-1 mM EDTA (pH 7.5). Three micrograms of DNA was cotransfected with 1 µg of pSV2neo into 50% confluent HFKs using Lipofectamine (Roche). The next day, the keratinocytes were plated onto mitomycin C-treated fibroblasts in E medium. Cells were selected with G418 (Invitrogen) at a final concentration of 200 mg/ml for the first 4 days followed by 100 mg/ml for the final 4 days. The selected colonies were then pooled and expanded.

Differentiation of keratinocytes in semisolid media. Keratinocyte differentiation was induced through suspension in 1.5% methylcellulose (Sigma), as previously described (16). The methylcellulose solution was prepared by combining dry autoclaved methylcellulose with E medium and heating the solution to 60°C for 20 min. An equal volume of E medium was then added, and the solution was stirred at 4°C overnight. At 80% confluence, the keratinocytes were harvested and suspended in a 10 cm-diameter petri dish with 25 ml of methylcellulose for either 24 or 48 h at 37°C in a CO2-humidified incubator. The cells were scraped into two 50-ml conical tubes, centrifuged, and washed four times with phosphate-buffered saline (PBS) at 4°C. DNA, RNA, and protein were then extracted from the cell pellets as previously described (53).

Immunohistochemistry. CIN612 cells were grown on coverslips and transiently transfected using polyethylenimine reagent (Polyscience, Inc.) with the various pSG5 constructs at 30% confluence. Forty-eight hours posttransfection, the coverslips were washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Following permeabilization with cold 0.2% Triton-X in PBS, the coverslips were blocked at room temperature for 1 h in 0.5% NP-40 and 1% bovine serum albumin (BSA) in PBS. The coverslips were subsequently incubated with the primary antibody at 4°C overnight. The slides were then incubated with the corresponding secondary antibody at room temperature for 1 h. For colocalization studies, the slides were incubated with a second primary antibody at room temperature for 1 h followed by the secondary antibody at room temperature for 1 h. After staining with 4',6'-diamidino-2-phenylindole-2HCl (DAPI) for 5 min, the slides were mounted and sealed. The primary antibodies used included an AU1 antibody (1:100 [Covance]) and a Bap31 antibody (1:100 [Abcam]). The secondary antibodies that were used were conjugated to fluorescein isothiocyanate (FITC) or Texas Red, and both were diluted in the blocking buffer at a 1:50 ratio (GE Healthcare).

Immunoprecipitation analysis. 293TT cells were transfected with various expression vectors. Forty-eight hours posttransfection, the cells were harvested, washed once with PBS, and lysed in a CHAPS {15 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate} buffer solution (CHAPS, 30 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl [pH 7.4]) (Sigma). The lysis buffer was supplemented with 1 mM phenylmethylsulfonyl fluoride, 2 mM sodium orthovanadate (Na3VO4) (Sigma), and a cocktail of protease inhibitors (Complete Mini; Roche). Lysates were then clarified by centrifugation at 4°C for 10 min and stored at –80°C. Immunoprecipitation analysis was performed using 1 mg of protein lysate in a total volume of 1 ml. The samples were precleared for 3 h with 50 µl of protein G agarose (Roche). The beads were then removed by centrifugation at 12,000 x g for 20 s at 4°C, and the protein was incubated overnight with various primary antibodies. E5 was immunoprecipitated using 5 µl of mouse or rabbit anti-AU1 antibody (Covance). For Bap31 immunoprecipitation, 5 µl of rat anti-BAP31 antibody was used (Abcam). The following day, 50 µl of protein G agarose was added and the mixture was incubated at 4°C for 3 h. The beads were isolated by centrifugation at 12,000 x g for 20 s at 4°C and subsequently washed with CHAPS lysis buffer and reisolated by centrifugation for a total of five washes. After the final centrifugation, the protein complexes were resuspended in a sodium dodecyl sulfate loading buffer and boiled at 95°C for 15 min. The beads were removed by centrifugation at 12,000 x g for 20 s, and the supernatant was analyzed by Western analysis.

Western analysis. Whole-cell extracts were prepared using the CHAPS lysis buffer supplemented with phenylmethylsulfonyl fluoride, Na3VO4, and a cocktail of protease inhibitors. Protein concentration was assessed with a Bradford assay (Bio-Rad), and equal amounts of protein were separated by electrophoresis on a 15% sodium dodecyl sulfate-polyacrylamide gel. Fifty micrograms of protein was analyzed in the experiments which did not involve immunoprecipitation. The protein was transferred to a polyvinylidene difluoride membrane (Immobolin-P; Millipore) which was blocked in a 5% nonfat milk solution (0.1% Tween 20 in PBS). The following primary antibodies were used: anti-AU1 (1:1,000 [Covance]), anti-Bap31 (1:1,500 [Abcam]), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam). Enhanced chemiluminescence was used to visualize the proteins (Amersham Pharmacia).

Yeast two-hybrid screen. A split ubiquitin-based yeast two-hybrid screen was performed to identify possible E5 binding partners. This screen (DUALmembrane system) is based on an adaptation of the ubiquitin-based split protein sensor (USPS) developed by Johnsson and Varshavsky (26). HPV16 E5* was expressed as a fusion to the C-terminal half of ubiquitin (Cub) along with a transcription factor (LexA-VP16). Prey proteins from a HeLa cDNA library were expressed as a fusion to the N-terminal half of ubiquitin (NubG). Upon interaction of a prey protein with the bait protein, the C-terminal half and N-terminal half of ubiquitin formed a functional enzyme with consequent translocation of the transcription factor LexA-VP16 into the nucleus. Transcription of LexA-VP16 was assessed using a quantitative LacZ assay, and the strongest positive clones were identified and subsequently isolated. The sequences were then used for further studies. These experiments were performed in collaboration with Dualsystems Biotech.

Replating assay. Keratinocytes were grown in monolayer cultures in the presence of fibroblast feeder cells to a confluence of approximately 80%. The keratinocytes were harvested and suspended in 1.5% methylcellulose for 24 h to induce differentiation. The cells were then collected, washed with PBS, and replated as monolayer cultures onto fibroblasts. Colonies were counted from at least 10 different random fields after 5 days in culture.

siRNA experiments. Two hundred thousand HPV31-positive LKP-1 keratinocytes per well were seeded onto J2 fibroblast feeders in a six-well plate. The following day, the cells were treated with small interfering RNA (siRNA) at a confluence of approximately 50 to 60% as recommended by the manufacturer. Briefly, 6 µl of siRNA duplex was diluted in 100 µl siRNA transfection medium (Santa Cruz). Human Bap31 siRNA (10 µM) and control siRNA-A (10 µM) were buffered in a solution containing 10 µM Tris-HCl (pH 8.0), 20 mM NaCl, and 1 mM EDTA and used for these experiments (Santa Cruz). In a separate tube, 6 µl of siRNA transfection reagent (Santa Cruz) was diluted in 100 µl of siRNA transfection medium. The siRNA duplex solution was added directly to the transfection reagent solution, mixed gently, and incubated at room temperature for 45 min in the dark. Prior to siRNA treatment, the cells were washed with 2 ml of siRNA transfection medium. For each transfection, 0.8 ml of siRNA transfection medium was added to each tube containing the siRNA and gently mixed. The 1-ml solution was overlaid onto the washed cells and incubated for 5 to 7 h at 37°C in a CO2 incubator. After the incubation, 3 ml of E medium was added to each well without removing the transfection mixture for a final volume of 4 ml/well and incubated for an additional 24 h. The following day, the medium was replaced with fresh E medium and cells were grown for an additional 24 h. The cells were then assayed using the replating assay, and protein was harvested at various time points to confirm knockdown of Bap31.

Generation of cell lines using recombinant retroviruses. PT67 cells (Clontech) were transfected with the various LXSN retroviral constructs using FuGENE transfection reagent (Roche) and selected using 1,000 µg/ml G418 for 4 days followed by an additional 4 days of selection with 500 µg/ml G418. Filtered viral supernatants were used to infect normal human keratinocytes in the presence of 8 µg/ml Polybrene (Sigma) for 6 h. Cells were then selected with G418 as previously described (22).

FACS analysis. To determine the level of surface expression, fluorescence-activated cell sorting (FACS) analysis was performed using antibodies to CD9 (BD Biosciences), CD81 (BD Biosciences), and major histocompatibility complex (MHC) class I (AbD [Serotec]). Briefly, monolayer keratinocytes were trypsinized, washed with PBS, and resuspended in PBS-1% BSA at a concentration of 1 x 107 cells/ml. One hundred microliters of cells was incubated at 4°C with the primary antibody, washed with PBS-1% BSA, and incubated for 30 min with the secondary antibody at 4°C in the dark. Cells were washed with PBS-1% BSA and analyzed by FACS analysis.


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RESULTS
 
Bap31 is identified as a novel target using a yeast two-hybrid screen. To identify potential interacting partners of the high-risk HPV E5 proteins, we performed a yeast two-hybrid analysis. Since HPV E5 is a membrane protein, we used a membrane-associated yeast two-hybrid system for our studies (26). A codon-modified HPV16 E5* was expressed as a fusion to the C-terminal half of ubiquitin (Cub) linked to the transcription factor LexA-VP16. Prey proteins from a HeLa cDNA library were expressed as fusions to the N-terminal half of ubiquitin (NubG). Positive clones were identified based on quantitative LacZ assays, and the clones with the highest expression were isolated. As a result of this screen, Bap31, a ubiquitously expressed polytopic integral membrane protein of the ER, was identified as a viable candidate binding partner of E5. Bap31 was initially identified as a B-cell-associated protein, but further studies have shown it to be expressed in a broad range of cell types. Additional studies have shown Bap31 to be an important mediator of the trafficking of membrane proteins and a regulator of apoptosis.

Bap31 is expressed in monolayer and differentiated HPV-positive cells. To determine if Bap31 could be a physiologically relevant binding partner of HPV E5, it was first important to determine if Bap31 was expressed in HFKs as well as HPV-positive keratinocytes. For this analysis, HFKs and HPV31-positive CIN612 kerantinocytes were grown in monolayer cultures and screened for Bap31 expression by immunohistochemistry using antibodies to Bap31. Both normal and HPV-positive cells were found to exhibit a perinuclear localization pattern for Bap31, and this is similar to what has been reported in other cell types (Fig. 1A) (5, 35). To determine whether the level of expression or the cellular localization pattern of Bap31 was altered by differentiation, histological cross-sections of organotypic raft cultures of HFKs and stably transfected HPV-positive keratinocytes were examined by immunofluorescence for endogenous Bap31. As seen in undifferentiated cells, a perinuclear localization pattern was observed for Bap31 in HPV31 differentiated cells and was similar to that seen in HFKs (Fig. 1B). To investigate more closely if HPV proteins altered the expression of Bap31, we examined the protein levels in monolayer and methylcellulose-differentiated keratinocytes by Western blot analysis and found no differences between HFKs and HPV-positive keratinocytes (data not shown). These studies indicate that Bap31 is expressed in both undifferentiated and differentiated HPV-positive keratinocytes. In addition, we found that HPV proteins do not significantly alter the levels of Bap31.


Figure 1
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  		FIG. 1. Bap31 and HPV16 E5 colocalize in a perinuclear localization pattern. (A) Monolayer cultures of HFKs and HPV31-positive CIN612 cells were examined by immunofluorescence for endogenous Bap31. (B) Cross sections of organotypic raft cultures of normal human keratinocytes (NHK) and HPV-positive cells. The keratinocytes were induced to differentiate by growth in raft cultures, and histological cross-sections were examined by immunofluorescence for endogenous Bap31 expression.

Bap31 and HPV E5 proteins colocalize in a perinuclear localization pattern. We next investigated if HPV E5 and endogenous Bap31 colocalized to the same cellular compartment. Both Bap31 and the HPV E5 proteins have been independently reported to be localized in the ER and to exhibit a perinuclear localization pattern (7, 10, 35). To examine if Bap31 and E5 colocalized, CIN612 cells were transiently transfected with a vector expressing AU1-tagged HPV16 E5 proteins. After 48 h, immunofluorescence analysis was performed using antibodies to the AU1 tag to detect E5 as well as with antibodies to Bap31. Our analysis indicates that both proteins localize in a perinuclear pattern that is consistent with the ER (Fig. 2). In addition, a comparison of staining patterns indicated that the two proteins colocalized. Interestingly, initial studies suggest E5 may alter the localization of endogenous Bap31 into a more punctate pattern in keratinocytes. It is, however, unclear if this relocalization is significant for E5 interactions with Bap31.


Figure 2
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  		FIG. 2. Colocalization of Bap31 and HPV16 E5. HPV31-positive CIN612 cells were transiently transfected with an N-terminal AU1-tagged HPV16 E5 and examined by immunohistochemistry using an antibody to AU1 (Texas Red) and endogenous Bap31 (FITC). Colocalization of Bap31 and E5 is indicated in yellow in the merged images. Nuclear staining with DAPI is shown in blue.

Bap31 coimmunoprecipates in complexes with HPV16 and -31 E5 proteins. Since HPV E5 and Bap31 were found to colocalize, it was important to determine if they were associated in complexes. For these studies, a series of immunoprecipitation experiments were performed using 293TT cells transiently transfected with expression vectors for HPV E5 and Bap31. Forty-eight hours after cotransfection, cells were harvested and solubilized in a CHAPS lysis buffer followed by immunoprecipitation with antibodies to Bap31. As shown in Fig. 3A, immunoprecipitation with an antibody to Bap31 and Western blot analysis for the AU1 tag of E5 indicated the two proteins formed a complex in vivo. We next examined whether HPV16 E5 could also form a complex with the endogenous Bap31 proteins. HPV16 E5 expression vectors were transfected into 293TT cells, and complexes immunoprecipitated with an antibody to Bap31 were examined by Western blot analysis with antibodies to AU1. Consistent with the previous observations using expression vectors for Bap31, we observed complex formation between HPV16 E5 and the endogenous proteins (Fig. 3A). These results provide further support for the existence of a physiologically relevant complex between Bap31 and the E5 protein.


Figure 3
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  		FIG. 3. HPV16 and -31 E5 form a complex with Bap31 in vivo. (A) 293TT cells were transiently transfected with a vector-only control or plasmids expressing Bap31 (pSG5-Bap31) and/or HPV16 E5 (pSG5-HPV16 E5*). Cells were solubilized in CHAPS buffer, and protein complexes were immunoprecipitated using a Bap31 antibody (Ab). The presence of coprecipitating AU1-E5 was detected by Western blotting (WB) using an AU1 antibody. (B) Reverse immunoprecipitation (IP) experiments were performed in which E5 was immunoprecipitated using antibodies to AU1 and Western analysis using antibodies to Bap31. Cells were transiently transfected with a vector-only control or plasmids expressing Bap31 (pSG5-Bap31) and/or HPV16 E5 (pSG5-HPV16 E5*). Both the full-length protein and the p20 fragment of Bap31 were shown to form a complex with HPV16 E5. The lanes in which AU1 antibody was used are indicated. (C) 293TT cells were transfected with expression vectors for AU1-tagged wild-type HPV31 E5 and Bap31. Immunoprecipitations were performed with AU1 antibodies followed by Western blot analysis using an antibody to Bap31. The middle lane shows the coimmunoprecipitation of endogenous Bap31 with HPV31 E5.

In the next set of studies, we investigated if Bap31 could be immunoprecipitated using antibodies to the AU1 tag on E5. For this analysis, 293TT cells were transiently transfected with HPV16 E5 expression vectors and cell lysates were isolated. E5-associated proteins were then immunoprecipitated with antibodies to AU1 followed by Western analysis using antibodies to Bap31. In addition to the full-length form, Bap31 can be proteolytically processed to generate a peptide of approximately 20 kDa in size (36, 50). As shown in Fig. 3B, both the full-length Bap31 protein as well as the p20 fragment were found to coimmunoprecipitate with the HPV16 E5 protein. It has been previously reported that Bap31 can form dimers with the cleaved peptide, and so it is unclear whether the E5 protein directly binds the p20 fragment or if this occurs through complex formation with the full-length protein (1).

If the interaction between HPV16 E5 and Bap31 is physiologically significant, it is likely that other high-risk HPV E5 proteins form similar complexes. To test whether other high-risk E5 proteins also bound Bap31, we performed transient transfections with expression vectors for wild-type HPV31 E5 that contained an N-terminal AU1 tag along with vectors for Bap31. At 48 h posttransfection, protein lysates were isolated and immunoprecipitations performed using antibodies to AU1. The proteins that coprecipitated with E5 were then screened by Western blotting with antibodies to Bap31. As shown in Fig. 3C, we observed an interaction between HPV31 E5 and Bap31, indicating that this interaction is conserved among the two high-risk HPV E5 proteins that were tested.

The C terminus of E5 stabilizes the interaction with Bap31. The C terminus of the E5 proteins has been suggested to be a prime candidate as a binding domain for E5-interacting proteins as it is a highly hydrophilic portion of the protein. We investigated if this domain could mediate the binding of HPV E5 to Bap31 by constructing two C-terminal deletion mutations of AU1-tagged HPV16 E5* which removed 5 and 10 amino acids, respectively (Fig. 4A). We first examined the localization pattern of the E5 C-terminal mutants to confirm that they still localized to the ER. Following transfection of mutant E5 proteins into CIN612 cells, we observed no change in the perinuclear localization pattern of the mutant E5 proteins from that of the full-length wild-type E5 protein (Fig. 4B). In addition, these mutant proteins were present in the same compartment as Bap31 (Fig. 4B). In order to demonstrate that the C-terminal deletions of HPV16 E5* did not affect the stability of the protein, 293TT cells were transfected with the E5 expression vectors and screened by Western analysis. These studies confirmed that the mutant proteins were expressed at levels similar to those of wild-type E5 (Fig. 4C).


Figure 4
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  		FIG. 4. The C terminus of E5 stabilizes the interaction with Bap31. (A) Schematic representation of the HPV16 E5* C-terminal mutants that were constructed using site-directed mutagenesis. The shaded regions indicate the three hydrophobic domains of the protein. (B) CIN612 cells were transiently transfected with various E5 constructs and stained with antibodies to AU1 and Bap31, as previously described in the legend to Fig. 1. Immunofluorescence analysis was performed to examine the localization pattern of E5 (Texas Red) and/or endogenous Bap31 (FITC). Nuclear staining with DAPI is indicated in blue in the merged images. WT, wild type. (C) 293TT cells were transiently transfected with pSG5-16E5*, pSG5-16E5*5AA, and pSG5-16E5*10AA, and Western analysis was performed using an AU1 antibody. (D) The C terminus of E5 stabilizes interaction with Bap31. 293TT cells were transiently transfected with expression vectors for HPV16E5* and the HPV16E5* deletion mutants with 5 amino acids (5AA) or 10 amino acids (10AA) removed. Protein complexes were immunoprecipitated with an antibody (Ab) to endogenous Bap31, and the levels of bound E5 were examined by Western analysis using an antibody to AU1.

In order to determine if the C-terminal domain was important for E5 binding to Bap31, expression vectors for the full-length E5 as well as C-terminal deletion mutants were transfected into 293TT cells. After 48 h, cell lysates were isolated and immunoprecipitations were performed with Bap31 antibodies followed by Western blot analysis for the AU1 tag on E5. Our studies indicate that both the 5- and 10-amino-acid deletions of HPV16 E5 exhibited significantly reduced binding to Bap31 (Fig. 4D). Interestingly, a low level of binding of Bap31 to the mutant E5 proteins was still observed, suggesting that other sequences in E5 may provide a weak interaction domain. Similar results were seen in which expression vectors for Bap31 were cotransfected with E5 expression vectors (data not shown). These results identify an important role of the C terminus of HPV16 E5 in providing stability for the interaction with Bap31.

HPV E5 targets Bap31 to support proliferative competence following differentiation. Since our studies indicate that HPV E5 and Bap31 form a complex in cells, we next sought to determine how this interaction influences HPV pathogenesis. For these studies, three different mutations in E5 were made in the context of the entire HPV31 genome (Fig. 5A). These mutations consisted of changes in conserved regions based on a comparison of the amino acid sequences of E5 proteins for different HPV types. Mutant HPV31E5-1 contains a 5-amino-acid deletion in the putative second hydrophobic domain of the protein. The second mutant (HPV31E5-2) contains two alanine substitutions in place of the arginine 58 and cysteine 59 in the E5 open reading frame. The third mutation (HPV31E5-3) introduced two stop codons at serine 79 and phenylalanine 80 in the C terminus of the E5 protein that resulted in a truncation of 6 amino acids. The wild-type, E5KO, and three mutant HPV31 genomes were transfected into HFKs. Drug-resistant colonies were isolated, and stable cell lines were generated. All cell lines stably maintained HPV episomes at similar copy numbers in undifferentiated cells (data not shown).


Figure 5
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  		FIG. 5. The C terminus of E5 is necessary for HPV-positive keratinocytes to retain cellular proliferative capacity following differentiation. (A) Diagram showing the E5 mutations that were used in this study. (B) Replating of keratinocytes that maintain either wild-type HPV31 or genomes with mutations in E5 following methylcellulose-induced differentiation. Cells were first grown as monolayer cultures, trypsinized, and induced to differentiate by suspension in 1.5% methylcellulose. After 24 h, cells were harvested, washed, and replated as monolayer cultures onto fibroblasts. Proliferative competence was quantified by counting the number of colonies from 10 random fields 5 days after replating. The data shown are the average of four experiments using two different transfected cell lines.

We previously reported that keratinocytes harboring the E5KO mutant genomes showed a significant reduction in colony-forming ability compared to cells with wild-type genomes following methylcellulose-induced differentiation (15). This indicated that E5 played a role in controlling proliferative competence in differentiating cells. We next investigated how cells containing the three HPV31 E5 mutant genomes functioned in this assay. Keratinocytes that stably maintained wild-type and E5 mutant genomes were grown as monolayer cultures, trypsinized, and suspended in 1.5% methylcellulose. Twenty-four hours after suspension in methylcellulose, the five sets of keratinocyte cell lines were washed and replated onto fibroblast feeders. After 5 additional days in culture, the colonies were counted to quantify proliferative competence. Using this assay, no significant difference was observed for the HPV31E5-1 and HPV31E5-2 mutant genomes from that of the wild-type cells. Interestingly, the HPV31E5-3 mutant in which the last 6 amino acids were disrupted exhibited the same phenotype as the keratinocytes which maintained genomes with a mutated AUG codon for E5 (Fig. 5B). A second property associated with E5 is the ability to enhance the activation of genome amplification following differentiation. Our studies indicate that cells with genomes containing a deletion of the C-terminal amino acids of E5, however, exhibited no differences with respect to wild-type genomes in the ability to amplify viral DNA (data not shown). We conclude that amino acids at the extreme C terminus of E5 are necessary for HPV-positive cells to retain proliferative competence following differentiation and play an important role in the stabilization of an interaction with the Bap31 protein.

While our studies demonstrate that the C-terminal amino acids of E5 are important for retaining proliferative capacity upon differentiation, it was possible that another factor other than Bap31 bound this region of E5 and mediated this activity. We therefore investigated if Bap31 was important for this function through the use of siRNAs targeted against Bap31. To knock down Bap31 expression, a pooled set of three siRNAs that specifically target Bap31 was used. Keratinocytes that stably maintained the HPV31 genomes were first transfected with siRNAs against Bap31 as well as control scrambled siRNAs and screened for effects on Bap31 levels. At 48 and 72 h posttransfection, cell lysates were harvested and screened for Bap31 by Western blot analysis. As shown in Fig. 6A, the levels of Bap31 were reduced following transfection with siRNAs. Using Scion Image analysis, we determined that Bap31 levels were reduced by approximately 60% and 69% at 48 and 72 h posttransfection respectively. We next examined the effect of reducing Bap31 levels on proliferative capability following differentiation. HPV31-positive cells were transfected with siRNAs and after 48 h suspended in methylcellulose for an additional 24 h. The cells were then harvested and replated in monolayer cultures, and colonies were counted from 10 different random fields after 5 additional days. Our studies indicate that knockdown of Bap31 significantly reduced the number of colonies that formed in this replating assay. Similar data were derived from three separate experiments and are summarized in Fig. 6B. These results confirm a role for Bap31 in the maintenance of proliferative competence following differentiation and demonstrate that association with E5 is important for this activity.


Figure 6
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  		FIG. 6. Expression of Bap31 supports cellular proliferative capacity following differentiation. (A) Western analysis confirmed knockdown of Bap31 in siRNA-treated HPV31-positive LKP-1 keratinocytes. Using Scion Image analysis, a 60% decrease and 69% decrease in Bap31 levels were seen at 48 and 72 h posttransfection, respectively. (B) To evaluate the role of Bap31 in the replating assay, cells were transfected with siRNA to Bap31 and 48 h later trypsinized and resuspended in methylcellulose. After an additional 24 h, cells were harvested, washed, and replated in monolayer cultures. After 5 days of growth, colonies were counted from 10 random fields. The data shown represent three separate experiments.

Bap31 has been reported to play a number of roles in the cell, including regulating the trafficking of MHC class I molecules to the cell surface (29, 37) as well as modulating the levels of the integrin regulatory proteins tetraspanins CD9 and CD81 (45). We investigated if E5 expression altered these two activities of Bap31 by first constructing recombinant retroviruses expressing HPV16 E5 and infecting normal keratinocytes. Following selection and expansion of these cells, we screened for the surface levels of MHC class I molecules and found no significant differences from control cells. Similarly, we examined the surface levels of CD9 and CD81 by FACS analysis and found no effect of E5 expression (data not shown). It is also possible that by association with E5, Bap31 acquires new functions, and this is suggested by our studies showing this interaction is necessary for maintaining cell proliferative capability in HPV-positive cells following differentiation.


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DISCUSSION
 
In this study, Bap31 has been identified as a novel target of the high-risk HPV E5 proteins. Bap31 is a ubiquitously expressed integral membrane protein that was originally reported to contribute to B-cell receptor activation. It is localized, like high-risk E5 proteins, to the ER and has been reported to regulate the trafficking of a number of membrane-integrated complexes through the ER. These Bap31-regulated complexes include tetraspanins, MHC class I proteins, and the cystic fibrosis transmembrane conductance regulator (CFTR) (2, 30, 37, 45). Bap31 has also been shown to be part of a large heteromeric complex that contains components of the actomyosin complex (13). Our identification of Bap31 using a membrane-associated yeast two-hybrid analysis indicates that E5 binds directly to Bap31 rather than through intermediary proteins. It is, however, possible that E5 is part of a larger complex of proteins that associate with Bap31. Our studies indicate that the HPV16 and -31 E5 proteins bind to Bap31, but it is unclear at this time whether this activity extends to low-risk types. We have also shown that HPV E5 binds to Bap31 through amino acids located at the extreme C terminus. Mutation of this C-terminal domain in E5 in the context of the complete HPV31 genome significantly impaired the proliferative capability of cells following differentiation. The interaction of E5 with Bap31 thus appears to be important for maintaining proliferative capability in differentiating cells, and this is consistent with the results from siRNA experiments targeting Bap31.

It has previously been reported that E7 acts to maintain differentiating cells active in the cell cycle by targeting Rb (6, 32), and our present study suggests that E5 may contribute to this activity. One way this could occur is through Bap31's ability to modify apoptotic responses such as those mediated through caspase 8 whose activity Bap31 can modulate. In addition, the HPV16 E5 protein has been shown to protect HaCaT keratinocytes from both Fas- and TRAIL-mediated apoptosis, a process also influenced by Bap31 (27, 28). Recent studies from our laboratory have demonstrated that HPV E6 and E7 proteins activate caspases upon differentiation, and it is possible that E5 may affect this activity (33). Our initial studies suggest that E6 and E7 activate the extrinsic pathway, and whether Bap31 has any effects on this pathway remains unclear. Bap31 has also been reported to control the trafficking of MHC proteins (29, 37). In addition, HPV E5 has been implicated in the regulation of either the expression or activity of MHC antigens (3, 54). Our initial studies indicate that E5 expression alone is not sufficient to alter the surface levels of MHC class I molecules or the levels of the integrin regulatory proteins, tetraspanins CD9 and CD81 (45). Bap31 has a large number of other activities that could be altered by E5, and determining if any of these functions are altered is an area of active study. It is also possible that E5 binding induces new activities for Bap31 similar to the effect of E6 binding to E6AP. Our studies are consistent with the latter possibility, as we have demonstrated that E5 binding to Bap31 is necessary to maintain HPV-positive cells in a proliferation-competent state following differentiation and this is not seen in normal keratinocytes.

Several other candidate binding partners of HPV E5 proteins have been proposed. One study reported that expression of HPV16 E5 increases the activation of the EGFR in the presence of ligand, without altering the total number of receptors present (9, 39). Further studies have reported that the last 5 amino acids are necessary for the E5-mediated EGFR overactivation (40). It has also been suggested that E5 regulates EGFR through manipulation of receptor recycling (47). Previously, we examined the total levels of EGFR in cells that contained either wild-type HPV31 or HPV31 E5 mutant genomes and found no differences, but these studies did not look at surface expression of EGFR (15). It is possible that Bap31 plays a role in regulating the recycling of EGFR, and this is an area of active study. A second potential binding partner of HPV E5 proteins is the 16-kDa subunit of the vacuolar H+-ATPase. Rodriguez et al. mapped the binding domain on E5 to amino acids 54 to 78 and demonstrated that the last C-terminal 5 amino acids (79 to 83) were not involved in this interaction (40). These results demonstrate that the binding capacity of E5 to the vacuolar H+-ATPase can be dissociated from the ability of E5 to bind Bap31. In addition, the vacuolar H+-ATPase is localized to endosomes while high-risk HPV E5 proteins are found predominantly in the ER. It is thus unlikely that significant amounts of these factors associate in vivo, although it is possible that this is an auxiliary function of HPV E5 proteins.

HPVs infect stratified epithelia and induce virion production in differentiating cells. While it has not been possible to detect the levels of E5 proteins in the various stratified layers of infected epithelia, transcript analyses suggest that E5 is expressed at low levels in undifferentiated cells and at high levels in differentiated suprabasal cells. Studies are currently under way to tag E5 in the context of the HPV genome to study the interaction with Bap31 in the correct physiological context. Our genetic data indicate that E5 modulates proliferation ability in differentiated cells (14), and we have shown this correlates with the binding of Bap31. Our previous studies also suggested that E5 expression could enhance amplification of viral genomes in differentiating cells; however, in our current study mutation of the Bap31 binding domain had a minimal effect on this activity. This indicates that the two properties are separable and may act through different interacting partners. It is possible that the binding of Bap31 to E5 and maintenance of proliferation ability act to modulate late functions other than amplification, such as mediating posttranscriptional effects or facilitating virion assembly. In undifferentiated cells, our previous studies indicated E5 had no effect on early viral functions but only a limited number of parameters have been examined. It is also possible that the interaction of E5 with Bap31 has a role in undifferentiated cells that is important for the viral life cycle that has yet to be elucidated. In conclusion, our study has identified Bap31 as a binding partner of the HPV E5 proteins and we have demonstrated that this association correlates with the ability of HPV-positive cells to remain competent for proliferation in differentiated cells. Further characterization of this complex will help clarify the role of the E5 proteins in HPV pathogenesis.


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ACKNOWLEDGMENTS
 
We gratefully acknowledge members of the laboratory of L.A.L., especially Melanie Beglin, for thoughtful discussions and ongoing collaborative efforts. In addition, we thank Kathy Rundell for helpful advice and a critical review of the manuscript.

This study was supported by a grant from the National Cancer Institute (R37CA74202) to L.A.L.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, 320 E. Superior St., Chicago, IL 60611. Phone: (312) 503-0648. Fax: (312) 503-1339. E-mail: l-laimins{at}northwestern.edu Back

{triangledown} Published ahead of print on 6 August 2008. Back


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REFERENCES
 
    1
  1. Adachi, T., W. W. A. Schamel, K.-M. Kim, T. Watanabe, B. Becker, P. J. Nielsen, and M. Reth. 1996. The specificity of association of the IgD molecule with the accessory proteins BAP31/BAP29 lies in the IgD transmembrane sequence. EMBO J. 15:1534-1541.[Medline]
    2. 2
  2. Annaert, W. G., B. Becker, U. Kistner, M. Reth, and R. Jahn. 1997. Export of cellubrevin from the endoplasmic reticulum is controlled by BAP31. J. Cell Biol. 139:1397-1410.[Abstract/Free Full Text]
    3. 3
  3. Ashrafi, G., D. R. Brown, K. H. Fife, and M. S. Campo. 2006. Down-regulation of MHC class I is a property common to papillomavirus E5 proteins. Virus Res. 120:208-211.[CrossRef][Medline]
    4. 4
  4. Bouvard, V. G., G. Matlashewski, Z. M. Gu, A. Storey, and L. Banks. 1994. The human papillomavirus type 16 E5 gene cooperates with the E7 gene to stimulate proliferation of primary cells and increases viral gene expression. Virology 203:73-80.[CrossRef][Medline]
    5. 5
  5. Breckenridge, D. G., M. Nguyen, S. Kuppig, M. Reth, and G. C. Shore. 2002. The procaspase-8 isoform, procaspase-8L, recruited to the BAP31 complex at the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA 99:4331-4336.[Abstract/Free Full Text]
    6. 6
  6. Cheng, S., D. C. Schmidt-Grimminger, T. Murant, T. R. Broker, and L. T. Chow. 1995. Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes. Genes Dev. 9:2335-2349.[Abstract/Free Full Text]
    7. 7
  7. Conrad, M., V. J. Bubb, and R. Schlegel. 1993. The human papillomavirus type 6 and 16 E5 proteins are membrane-associated proteins which associate with the 16-kilodalton pore-forming protein. J. Virol. 67:6170-6178.[Abstract/Free Full Text]
    8. 8
  8. Conrad, M., D. Goldstein, T. Andresson, and R. Schlegel. 1994. The E5 protein of HPV-6, but not HPV-16, associates efficiently with cellular growth factor receptors. Virology 200:796-800.[CrossRef][Medline]
    9. 9
  9. Crusius, K., E. Auvinen, B. Steuer, H. Gaissert, and A. Alonso. 1998. The human papillomavirus type 16 E5 protein modulates ligand-dependent activation of the EGF receptor family in the human epithelial cell line HaCaT. Exp. Cell Res. 241:76-83.[CrossRef][Medline]
    10. 10
  10. Disbrow, G. L., J. A. Hanover, and R. Schlegel. 2005. Endoplasmic reticulum-localized human papillomavirus type 16 E5 protein alters endosomal pH but not trans-Golgi pH. J. Virol. 79:5839-5846.[Abstract/Free Full Text]
    11. 11
  11. Disbrow, G. L., I. Sunitha, C. C. Baker, J. Hanover, and R. Schlegel. 2003. Codon optimization of the HPV-16 E5 gene enhances protein expression. Virology 311:105-114.[CrossRef][Medline]
    12. 12
  12. Doorbar, J., S. Ely, J. Sterling, C. McLean, and L. Crawford. 1991. Specific interaction between HPV-16 E1{wedge}E4 and cytokeratins results in collapse of the epithelial cell intermediate filament network. Nature 352:824-827.[CrossRef][Medline]
    13. 13
  13. Ducret, A., M. Nguyen, D. G. Breckenridge, and G. C. Shore. 2003. The resident endoplasmic reticulum protein, BAP31, associates with gamma-actin and myosin B heavy chain. Eur. J. Biochem. 270:342-349.[Medline]
    14. 14
  14. Dyson, N., P. M. Howley, K. Munger, and E. Harlow. 1989. The human papillomavirus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243:934-937.[Abstract/Free Full Text]
    15. 15
  15. Fehrmann, F., D. J. Klumpp, and L. A. Laimins. 2003. Human papillomavirus type 31 E5 protein supports cell cycle progression and activates late viral functions upon epithelial differentiation. J. Virol. 77:2819-2831.[Abstract/Free Full Text]
    16. 16
  16. Fehrmann, F., and L. A. Laimins. 2005. Human papillomavirus type 31 life cycle: methods for study using tissue culture models. Methods Mol. Biol. 292:317-330.[Medline]
    17. 17
  17. Genther, S. M., S. Sterling, S. Duensing, K. Münger, C. Sattler, and P. F. Lambert. 2003. Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle. J. Virol. 77:2832-2842.[Abstract/Free Full Text]
    18. 18
  18. Genther Williams, S. M., G. L. Disbrow, R. Schlegel, D. Lee, D. W. Threadgill, and P. F. Lambert. 2005. Requirement of epidermal growth factor receptor for hyperplasia induced by E5, a high-risk human papillomavirus oncogene. Cancer Res. 65:6534-6542.[Abstract/Free Full Text]
    19. 19
  19. Goldstein, D., and R. Schlegel. 1990. The E5 oncoprotein of bovine papillomavirus binds to a 16 kd cellular protein. EMBO J. 9:137-145.[Medline]
    20. 20
  20. Goldstein, D. J., M. E. Fmbow, T. Andresson, P. McLean, K. Smith, V. Bubb, and R. Schlegel. 1991. Bovine papillomavirus E5 oncoproteins binds to the 16K component of vacuolar H+-ATPases. Nature 352:347-349.[CrossRef][Medline]
    21. 21
  21. Goldstein, D. J., R. Kulke, D. Dimaio, and R. Schlegel. 1992. A glutamine residue in the membrane-associating domain of the bovine papillomavirus type 1 E5 oncoprotein mediates its binding to a transmembrane component of the vacuolar H+-ATPase. J. Virol. 66:405-413.[Abstract/Free Full Text]
    22. 22
  22. Hebner, C. M., R. Wilson, J. Rader, M. Bidder, and L. A. Laimins. 2006. Human papillomaviruses target the double-stranded RNA protein kinase pathway. J. Gen. Virol. 87:3183-3193.[Abstract/Free Full Text]
    23. 23
  23. Huibregtse, J. M., M. Scheffner, and P. M. Howley. 1991. A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18. EMBO J. 10:4129-4135.[Medline]
    24. 24
  24. Hummel, M., J. B. Hudson, and L. A. Laimins. 1992. Differentiation-induced and constitutive transcription of human papillomavirus type 31b in cell lines containing viral episomes. J. Virol. 66:6070-6080.[Abstract/Free Full Text]
    25. 25
  25. Hwang, E. S., T. Nottoli, and D. DiMaio. 1995. The HPV16 E5 protein: expression, detection, and stable complex formation with transmembrane proteins in COS cells. Virology 211:227-233.[CrossRef][Medline]
    26. 26
  26. Johnsson, N., and A. Varshavsky. 1994. Split ubiquitin as a sensor of protein interactions in vivo. Proc. Natl. Acad. Sci. USA 91:10340-10344.[Abstract/Free Full Text]
    27. 27
  27. Kabsch, K., and A. Alonso. 2002. The human papillomavirus type 16 E5 protein impairs TRAIL- and FasL-mediated apoptosis in HaCaT cells by different mechanisms. J. Virol. 76:12162-12172.[Abstract/Free Full Text]
    28. 28
  28. Kabsch, K., N. Mossadegh, A. Kohl, G. Komposch, J. Schenkel, A. Alonso, and P. Tomakidi. 2004. The HPV-16 E5 protein inhibits TRAIL- and FasL-mediated apoptosis in human keratinocyte raft cultures. Intervirology 47:48-56.[CrossRef][Medline]
    29. 29
  29. Ladasky, J. J., S. Boyle, M. Seth, H. Li, T. Pentcheva, F. Abe, S. J. Steinberg, and M. Edidin. 2006. Bap31 enhances the endoplasmic reticulum export and quality control of human class I MHC molecules. J. Immunol. 177:6172-6181.[Abstract/Free Full Text]
    30. 30
  30. Lambert, G., B. Becker, R. Schreiber, A. Boucherot, M. Reth, and K. Kunzelmann. 2001. Control of cystic fibrosis transmembrane conductance regulator expression by BAP31. J. Biol. Chem. 276:20340-20345.[Abstract/Free Full Text]
    31. 31
  31. Leechanachai, P., L. Banks, F. Moreau, and G. Matlashewski. 1992. The E5 gene from human papillomavirus type 16 is an oncogene which enhances growth factor-mediated signal transduction to the nucleus. Oncogene 7:19-25.[Medline]
    32. 32
  32. Martin, L. G., G. W. Demers, and D. A. Galloway. 1998. Disruption of the G1/S transition in human papillomavirus type 16 E7-expressing human cells is associated with altered regulation of cyclin E. J. Virol. 72:975-985.[Abstract/Free Full Text]
    33. 33
  33. Moody, C. A., A. Fradet-Turcotte, J. Archambault, and L. A. Laimins. 2007. Human papillomaviruses activate caspases upon epithelial differentiation to induce viral genome amplification. Proc. Natl. Acad. Sci. USA 104:19541-19546.[Abstract/Free Full Text]
    34. 34
  34. Munger, K., B. A. Werness, N. Dyson, W. C. Phelps, E. Harlow, and P. M. Howley. 1989. Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J. 8:4099-4105.[Medline]
    35. 35
  35. Ng, F. W. H., M. Nguyen, T. Kwan, P. E. Branton, D. W. Nicholson, J. A. Cromlish, and G. C. Shore. 1997. p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum. J. Cell Biol. 139:327-338.[Abstract/Free Full Text]
    36. 36
  36. Nguyen, M., D. G. Breckenridge, A. Ducret, and G. C. Shore. 2000. Caspase-resistant BAP31 inhibits Fas-mediated apoptotic membrane fragmentation and release of cytochrome c from mitochondria. Mol. Cell. Biol. 20:6731-6740.[Abstract/Free Full Text]
    37. 37
  37. Paquet, M.-E., M. Cohen-Doyle, G. C. Shore, and D. B. Williams. 2004. Bap29/31 influences the intracellular traffic of MHC class I molecules. J. Immunol. 172:7548-7555.[Abstract/Free Full Text]
    38. 38
  38. Petti, L., L. Nilson, and D. DiMaio. 1991. Activation of the platelet-derived growth factor receptor by the bovine papillomavirus E5 transformin protein. EMBO J. 10:845-855.[Medline]
    39. 39
  39. Pim, D., M. Collins, and L. Banks. 1992. Human papillomavirus type 16 E5 gene stimulates the transforming activity of the epidermal growth factor receptor. Oncogene 7:27-32.[Medline]
    40. 40
  40. Rodriguez, M. I., M. E. Fmbow, and A. Alonso. 2000. Binding of human papillomavirus 16 E5 to the 16 kDa subunit c (proteolipid) of the vacuolar H+-ATPase can be dissociated from the E5-mediated epidermal growth factor receptor overactivation. Oncogene 19:3727-3732.[CrossRef][Medline]
    41. 41
  41. Ruesch, M. N., and L. A. Laimins. 1998. Human papillomavirus oncoproteins alter differentiation-dependent cell cycle exit on suspension in semisolid medium. Virology 250:19-29.[CrossRef][Medline]
    42. 42
  42. Ruesch, M. N., F. Stubenrauch, and L. A. Laimins. 1998. Activation of papillomavirus late gene transcription and genome amplification upon differentiation in semisolid medium is coincident with expression of involucrin and transglutaminase but not keratin-10. J. Virol. 72:5016-5024.[Abstract/Free Full Text]
    43. 43
  43. Scheffner, M., J. M. Huibregtse, R. D. Vierstra, and P. M. Howley. 1993. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75:495-505.[CrossRef][Medline]
    44. 44
  44. Scheffner, M., B. A. Werness, J. M. Huibregtse, A. J. Levine, and P. M. Howley. 1990. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63:1129-1136.[CrossRef][Medline]
    45. 45
  45. Stojanovic, M., M. Germain, M. Nguyen, and G. C. Shore. 2005. BAP31 and its caspase cleavage product regulate cell surface expression of tetraspanins and integrin-mediated cell survival. J. Biol. Chem. 280:30018-30024.[Abstract/Free Full Text]
    46. 46
  46. Straight, S. W., B. Herman, and D. J. McCance. 1995. The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes. J. Virol. 69:3185-3192.[Abstract]
    47. 47
  47. Straight, S. W., P. M. Hinkle, R. J. Jewers, and D. J. McCance. 1993. The E5 oncoprotein of human papillomavirus type 16 transforms fibroblasts and effects the downregulation of the epidermal growth factor receptor in keratinocytes. J. Virol. 67:4521-4532.[Abstract/Free Full Text]
    48. 48
  48. Valle, G. F., and L. Banks. 1995. The human papillomavirus (HPV)-6 and HPV-16 E5 proteins co-operate with HPV-16 E7 in the transformation of primary rodent cells. J. Gen. Virol. 76:1239-1245.[Abstract/Free Full Text]
    49. 49
  49. Walboomers, J. M. M., M. V. Jacobs, M. M. Manos, F. X. Bosch, J. A. Kummer, K. Shah, V., P. J. F. Snijders, J. Peto, C. J. L. M. Meijer, and N. Munoz. 1999. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J. Pathol. 189:12-19.[CrossRef][Medline]
    50. 50
  50. Wang, B., M. Nguyen, D. G. Breckenridge, M. Stojanovic, P. A. Clemons, S. Kuppig, and G. C. Shore. 2003. Uncleaved BAP31 in association with A4 protein at the endoplasmic reticulum is an inhibitor of Fas-initiated release of cytochrome c from mitochondria. J. Biol. Chem. 278:14461-14468.[Abstract/Free Full Text]
    51. 51
  51. Werness, B. A., A. J. Levine, and P. M. Howley. 1990. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248:76-79.[Abstract/Free Full Text]
    52. 52
  52. Wilson, R., F. Fehrmann, and L. A. Laimins. 2005. Role of the E1{wedge}E4 protein in the differentiation-dependent life cycle of human papillomavirus type 31. J. Virol. 79:6732-6740.[Abstract/Free Full Text]
    53. 53
  53. Wilson, R., and L. A. Laimins. 2005. Differentiation of HPV-containing cells using organotypic "raft" culture or methylcellulose. Methods Mol. Med. 119:157-169.[Medline]
    54. 54
  54. Zhang, B., P. Li, E. Wang, Z. Brahmi, K. W. Dunn, J. S. Blum, and A. Roman. 2003. The E5 protein of human papillomavirus type 16 perturbs MHC class II antigen maturation in human foreskin keratinocytes treated with interferon-{gamma}. Virology 310:100-108.[CrossRef][Medline]
    55. 55
  55. zur Hausen, H. 2002. Papillomaviruses and cancer: from basic studies to clinical application. Nat. Rev. Cancer 2:342-350.[CrossRef][Medline]

Journal of Virology, October 2008, p. 10042-10051, Vol. 82, No. 20
0022-538X/08/$08.00+0     doi:10.1128/JVI.01240-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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