Nonconserved Lysine Residues Attenuate the Biological Function of the Low-Risk Human Papillomavirus E7 Protein

Abstract

The mucosotrophic human papillomaviruses (HPVs) are classified as high-risk (HR) or low-risk (LR) genotypes based on their neoplastic properties. We have demonstrated previously that the E7 protein destabilizes p130, a pRb-related pocket protein, thereby promoting S-phase reentry in postmitotic, differentiated keratinocytes of squamous epithelia, and that HR HPV E7 does so more efficiently than LR HPV E7. The E7 proteins of LR HPV-11 and -6b uniquely possess lysine residues following a casein kinase II phosphorylation motif which is critical for the biological function of E7. We now show that mutations of these lysine residues elevated the efficiency of S-phase reentry, independent of their charge. An 11E7 K39,42R mutation moderately increased the association with and the destabilization of p130. Unexpectedly, polyubiquitination on these lysine residues did not attenuate E7 activity, as their mutation caused elevated proteasomal degradation and decreased protein stability. In this regard, the biologically more potent HR HPV E7 proteins were also less stable than the LR HPV E7 proteins. We infer that these lysine residues impede functional protein-protein interactions. A G22D mutation of 11E7 at the pocket protein binding motif possessed augmented efficiency in promoting S-phase reentry and strongly enhanced association with p130 and pRb. The combined effects of these two classes of 11E7 mutations exhibited an efficiency of S-phase reentry comparable to that of HR HPV E7. Thus, these nonconserved residues are primarily responsible for the differential abilities of LR and HR HPV E7 proteins to promote unscheduled DNA replication in organotypic raft cultures.

INTRODUCTION

The human papillomavirus (HPV) genome amplifies in suprabasal, differentiated keratinocytes as extrachromosomal plasmids. Viral DNA replication requires the host DNA replication proteins. The normal squamous epithelium is, however, postmitotic and nonsupportive of viral DNA amplification. After asymmetrical cell division from the proliferative basal and transit amplifying compartments, cell cycle reentry by differentiated keratinocytes is permanently repressed by retinoblastoma family pocket proteins. Pocket proteins p130 and pRb repress cell cycle entry by regulation of the E2F family of transcription factors (for a review, see reference 54). During the keratinocyte differentiation process, the pRb expression level decreases while that of p130 increases, implying a dominant role for p130 in repressing cell cycle entry in the postmitotic epithelium (19, 43, 65). The HPV E7 protein inactivates p130, thereby evoking cell cycle entry permissive for amplification of the viral genome. Our recent study demonstrated that viral genome amplification occurs in spinous cells arrested in a prolonged G₂ state in the absence of competition from host DNA replication (3a, 59).

Mucosotrophic HPV types of the Alphapapillomavirus genus are classified as low risk (LR; e.g., types 6b and 11) or high risk (HR; e.g., types 16 and 18), according to the potential of the infections to progress to high-grade intraepithelial neoplasias and cancers (16). The oncogenicity of the HR HPVs is conferred by the properties of their E6 and E7 oncoproteins, which are highly expressed in anogenital cancers and have the ability to immortalize cells in vitro (for reviews, see references 37, 38, and 66). In contrast, LR HPV infections are only rarely associated with cancers, and LR HPV E6 and E7 immortalize cells inefficiently, if at all (23). Despite the differences in their neoplastic potentials, both the HR and LR E6 and E7 proteins provide common functions during the viral infection cycle: maintenance and replicative amplification of the viral DNA genome (42, 55, 59).

Unscheduled host DNA replication in the stratum spinosum occurs during active infections with both HR and LR HPV types (11, 59). Using retrovirus-mediated gene transfer into primary human keratinocytes (PHKs), we and others demonstrated previously that cell cycle reentry by suprabasal keratinocytes in organotypic cultures is attributable to E7 and that HR HPV E7 orthologs promote this activity more efficiently than do the LR orthologs (3, 11, 19, 23). The phenotypic differences between the LR and HR HPV E7 proteins correlate with their relative abilities to inactivate the pocket protein repressors of S-phase gene expression. HR HPV E7 binds with a high affinity and destabilizes both pRb and p130 (5, 22, 24, 65). Their targeted degradation is mediated through proteasome-dependent (5, 6, 22, 28, 40, 60, 65), as well as proteasome-independent (15), mechanisms. Additionally, interaction of the HR HPV E7 protein with pRb can directly disrupt the pRb-E2F complex (9, 10, 27, 33, 63). In contrast, LR HPV E7 binds the pocket proteins with a lower affinity (10, 24, 49) and has been demonstrated to destabilize only p130 (19, 56, 65).

The critical pathological divergence between the E7 proteins of HR and LR HPV types can be attributed to peptide sequence differences. Both HR and LR HPV E7 orthologs bind pocket proteins through an LxCxE motif within conserved region 2 (CR2), which is present in families of DNA tumor viruses (Fig. 1A). Closely downstream of this motif, nearly all HPV E7 orthologs possess a casein kinase II (CKII) binding motif and serine substrates. Phosphorylation of E7 on the CKII substrates enhances association with and destabilization of p130 (19) and is essential for the induction of S phase in differentiated keratinocytes (13, 19). The affinity of E7 for the pocket protein is also influenced by the particular amino acid which precedes the LxCxE motif (Fig. 1A). A glycine (G) residue present in LR HPV E7 orthologs correlates with weak association with pRb, whereas an aspartic acid (D) residue conserved among HR HPV E7 orthologs confers strong association (24, 49, 65). A D→G mutation of HR HPV-18E7 expressed in organotypic raft cultures leads to impaired hyperplasia relative to that of the wild type (56). Whether the reciprocal G→D mutation of LR HPV E7 is sufficient to recapitulate the HR phenotype in epithelial cultures has not been examined. Moreover, unique among the E7 orthologs, LR HPV-11 and HPV-6b E7 proteins possess a nonconserved lysine residue(s) following the negatively charged CKII motif (Fig. 1A). In this study, we identified these lysines as critical residues that limit the efficiency with which LR E7 induces S-phase reentry in the squamous epithelium. The basis of the augmented phenotypes elicited by the LR HPV-11E7 lysine mutations was characterized through comparative assessment with a G→D mutation in pocket protein binding and destabilization in keratinocytes. We further examined the role of the 11E7 lysine residues in the polyubiquitination and proteolytic turnover of the protein.

Fig. 1.

Unscheduled DNA replication by HPV E7 or mutations thereof in organotypic raft cultures of PHKs. (A) Amino acid sequence alignment of the pocket protein binding regions of E7 proteins encoded by LR HPV types 6b, and 11 and HR HPV types 16 and 18. HPV sequences are aligned with conserved region 2 (CR2) of the human adenovirus type 5 (hAd-5) E1A proteins. The bracket at the EIA G residue corresponds to the downstream boundary of CR2, as defined by the mRNA donor splice site shared between the 12S mRNA (243-amino-acid protein) and the 13S mRNA (289-amino-acid protein). Numbers represent the positions of the first residue displayed. The mutated residues are indicated by a larger font size, and the amino acid substitutions are listed below the vertical line. The underlined sequence indicates the conserved LxCxE pocket protein binding domain. The lowercase letter p identifies consensus casein kinase II phosphorylation sites. The charges of residues are provided above each sequence. Posttranslationally linked phosphate moieties add additional negative charges to the domain. Raft cultures of PHKs were transduced with the empty-vector-only retrovirus (pBabepuro), the retrovirus expressing wild-type HPV-11 E7, or mutations thereof (B) or with wild-type HR or LR HPV E7 orthologs and mutations of 6bE7 (C). BrdU was detected by immunohistochemistry (dark nuclei) and counterstained with hematoxylin to reveal tissue morphology.

MATERIALS AND METHODS

Retrovirus construction and transduction of primary human keratinocyte cultures.

All wild-type or mutant forms of E7 proteins were expressed from a puromycin-selectable pBabe retroviral vector (39). E7 open reading frames were amplified by PCR from genomic clones as the wild type or mutations and cloned between the BamHI and SalI restriction sites downstream of the retroviral long terminal repeat promoter. Retroviral vector plasmids expressing the ligand binding domain of human estrogen receptor α (ER) or E7-ER fusions were constructed as described previously (3, 19). PHKs for retroviral transduction were isolated from neonatal foreskins obtained from the University of Alabama at Birmingham Well Baby Nursery following elective circumcision and cultured in KFSM (Invitrogen, Carlsbad, CA) or LLKSFM (Lifeline Cell Technology, Walkersville, MD) as described previously (62). The murine fibroblast GP+envAM12 packaging line (36) was used to prepare retroviruses for transducing PHKs as described previously (39). PHKs were infected with supernatant for 2.5 h in the presence of hexadimethrine bromide (5 μg/ml) for 2.5 h, followed by addition of an equal volume of KSFM for another 2.5 h of incubation. The medium was changed to KSFM, and cells were cultured for 24 h prior to selection with puromycin (1 μg/ml for 5 days). Selected PHKs were grown either as submerged monolayer cultures in KSFM or as organotypic raft cultures as previously described (3). BrdU (10 μg/ml) was added for 12 h prior to harvest on day 10.

Bright-field microscopy.

Formalin-fixed, paraffin-embedded organotypic raft cultures were cut into 4-μm sections and mounted onto glass slides. The tissues were deparaffinized, hydrated, and blocked with 25% goat serum. Slides were incubated with a mouse anti-bromodeoxyuridine (BrdU) monoclonal antibody (MAb; clone ZBU; Invitrogen) overnight at 4°C, followed by AEC chromogenic detection using the LSAB immunohistochemistry kit (K0680; Dako North America, Carpinteria, CA). Bright-field images were captured with an Olympus BH-2 microscope with a SPOT digital camera at ×20 magnification (Diagnostic Instruments, Sterling Heights, MI). Images were processed using Adobe Photoshop CS2 (Adobe Systems, San Jose, CA).

Coimmunoprecipitation and Western blot analysis of keratinocyte culture lysates.

Organotypic raft cultures expressing ER fusions of wild-type E7 or mutations therein were induced with 5 μM β-estradiol for 72 h prior to harvest. The epidermal strata were manually separated from the collagen bed and lysed on ice using a Dounce homogenizer in 500 μl mammalian cell lysis buffer (MCLB). The MCLB contained 75 mM NaCl, 50 mM Tris-Cl (pH 8.0), 5 mm Na-EDTA, 10 mM NaF, 0.5% NP-40, 1 mM NaVO₄, protease inhibitor cocktail (P8340; Sigma-Aldrich, St. Louis, MO), and 2 mM dithiothreitol. Cellular debris was pelleted at 4°C and discarded. The protein concentrations of the supernatants were determined using a BCA assay kit (Pierce, Rockford, IL) and adjusted to 1.25 mg/400 μl per immunoprecipitation reaction. A 125-μg sample (1/10 load) of lysate protein was reserved for protein expression analysis. A 100-μl volume of sterile 50% glycerol solution and 10 μg of rabbit anti-ER Ab-16 pAb (Lab Vision, Fremont, CA) were added, and the samples were incubated overnight at 4°C on a rotating rack. PureProteome protein G magnetic beads were prepared and added to the reaction tubes for immunoprecipitation according to the manufacturer's instructions (LSKMAGG10; Millipore Corp., Billerica, MA). The precipitates were washed three times using 200 μl MCLB buffer. Immune complexes were denatured using standard electrophoretic sample buffer. Total cell lysates and immunoprecipitates were resolved by 5 to 12% SDS-PAGE and transferred to nitrocellulose membranes. Antigens were detected using mouse anti-p130 MAb Rb2 (clone 10; BD Biosciences, Franklin Lakes, NJ), mouse anti-Rb MAb Ab-11 (clone IF8; EMD Chemicals, Gibbstown, NJ), mouse anti-ER MAb (clone F-10; Santa Cruz Biotechnology, Santa Cruz, CA), and anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) MAb (clone V-18; Santa Cruz Biotechnology). Western blot detection was conducted using the ECL plus kit (RPN2132; GE Healthcare, Chalfont St. Giles, United Kingdom) for the Western blot assays of raft lysates or the ECL kit (RPN2108; GE Healthcare) for all other experiments. Densitometric analyses of bands were conducted using ImageJ software (ImageJ 1.40g; National Institutes of Health, Bethesda, MD).

Ubiquitination of E7-ER in transfected cells.

COS-7 cells (5.0 × 10⁶) were transfected by electroporation with 5 μg pBabe plasmid expressing ER, wild-type 11E7-ER, or 11E7 K39E,42A-ER together with 5 μg of pCW7-his-myc-ubiquitin (58). β-Estradiol was added at 5 μM for 48 h of incubation. Dimethyl sulfoxide (DMSO) or 50 μM MG132 was added 24 h prior to harvest by lysis in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 0.5% NP-40, 0.5 mM phenylmethylsulfonyl fluoride, 0.5% Sigma protease inhibitor cocktail). After removal of the debris, total protein contents of the supernatants were determined by the BCA method and the concentrations were each adjusted to 115 μg/ml. Ten micrograms of rabbit anti-ER Ab-16 pAb (Lab Vision) was mixed with each lysate before overnight incubation at 4°C using a rotating rack. The immunoprecipitation conjugation medium and protein A–Sepharose CL-4B were prepared according to the manufacturer's instructions (17-0780-01; GE Healthcare). Twenty microliters of protein A-Sepharose slurry was added to each reaction mixture before overnight incubation at 4°C for secondary conjugation and immunoprecipitation. An additional 20 μl of carrier Sepharose was added to each tube, and conjugates were centrifuged at 4°C. The immunoprecipitates were washed three times with 1 ml RIPA buffer, denatured, resolved by 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes. The immunoblots were probed using anti-ER MAb (clone F-10; Santa Cruz) or anti-myc MAb Ab-2 (clone 9E10.3; Lab Vision). Blots were developed and processed as described above.

RESULTS

Functional characterization of critical nonconserved residues of the LR HPV E7 protein.

A glycine residue precedes the LxCxE pocket protein binding motif and is common to LR HPV E7 proteins (Fig. 1A). To assess the functional influence of the HPV-11E7 G22 residue on the ability of the protein to promote unscheduled DNA replication, an 11E7 G22D mutation was transduced via a retrovirus into PHKs that were then developed into organotypic raft cultures. This mutant form of E7 induced a hyperplastic morphology with an increased number of cells in S phase in the spinous strata compared to raft cultures expressing wild-type 11E7 (Fig. 1B; Table 1). However, the suprabasal S-phase phenotype was considerably inferior to that of HPV-16 E7, indicating the presence of other residues that limit the LR HPV E7 activity. The amino acids proximal to the CKII domain among E7 orthologs are not highly conserved; aspartate, glutamate, isoleucine, valine, glycine, and alanine constitute the prevalent residues. The E7 protein of 11E7 uniquely possesses two lysine residues in this region (Fig. 1A). Thus, we prepared 11E7 K39E,42G or A (G/A) mutations to elucidate the functional influence of these residues. Similar to the cultures expressing the 11E7 G22D mutation, both 11E7 K39E,42G/A-transduced raft culture tissues exhibited hyperplastic morphology with unscheduled DNA replication occurring with greater frequency than with wild-type 11E7 (Fig. 1B; Table 1). HPV-6b E7 possesses a nonconserved lysine residue further downstream, where threonine is prevalent among other E7 orthologs. As did the 11E7 lysine mutation, a 6bE7 K49T mutation produced an augmented ability to promote S-phase reentry (Fig. 1C; Table 1).

Table 1.

Relative efficiencies of unscheduled S-phase induction in raft cultures by wild-type E7 orthologs and LR HPV E7 mutations^a

Expt no. and E7 ortholog/mutation(s)	No. of suprabasal S-phase nuclei	Fold increaseb
1
11E7	119 ± 7	1.0
11E7 G22D	422 ± 25	3.6
11E7 K39E, 42G	410 ± 34	3.5
2
11E7	315 ± 17	1.0
11E7 K39E, 42A	599 ± 22	1.9
11E7 K39, 42R	597 ± 17	1.9
11E7 G22D, K39, 42R	1,119 ± 10	3.2
3
11E7	181 ± 4	1.0
11E7 G22D	658 ± 13	3.6
11E7 K39R, 42R	447 ± 14	2.5
11E7 G22D, K39, 42R	985 ± 26	5.4
16E7	1,114 ± 36	NAc
4
18E7	1,324 ± 201	NA
16E7	1,251 ± 143	NA
11E7	210 ± 7	NA
6bE7	40 ± 16	1.0
6bE7 K49T	157 ± 28	3.9
6bE7 K49R	187 ± 30	4.7

^aQuantification of suprabasal BrdU-positive S-phase nuclei in PHK raft cultures expressing the wild-type or mutant forms of LR HPV 11E7 and 6bE7 relative to HR HPV-16 and -18 E7. The comparative assessments for experiments 1, 2, and 3, represent the enumeration of the mean number and the standard deviation of S-phase nuclei from two tissue sections per organotypic culture. The comparative assessment of experiment 4 represents the mean number and standard deviation of S-phase nuclei from individual tissue sections of three raft cultures.

^bFold increase in S-phase induction by the LR HPV E7 mutations relative to wild-type LR HPV E7 (set to 1.0) in the same experiment.

^cNA, not applicable.

The lysine residues impart several attributes to the LR HPV E7 protein, including a positive charge, potential substrates for posttranslational modification, and potentially increased surface entropy (21, 35). To distinguish among these possibilities, LR HPV E7 lysine-to-arginine (K→R) mutations were prepared wherein the charge was conserved while the predicted surface entropy of the proteins was reduced. Furthermore, arginine residues are not substrates for certain posttranslational modifications, such as polyubiquitination. Both 11E7 and 6bE7 K→R mutations produced phenotypes similar those produced by the 11K39E,42G/A mutations or the 6bK49T mutation, respectively (Fig. 1B and C; Table 1). Thus, the influence of the lysine residues was charge independent, but polyubiquitination could not be ruled out as having contributed to the relatively low activity of LR HPV E7.

To establish whether G22D and the lysine mutations contribute additively to the phenotype, triple mutations of 11E7 were expressed in raft cultures. G22D K39,42R or G22D K39E,42G/A mutations elicited more suprabasal S-phase nuclei than either of the individual mutations, irrespective of the charges present at residues 39 and 42 (Fig. 1B; Table 1). A quantification of suprabasal S-phase cells showed that the contributions from the individual class mutations were indeed additive and the S-phase reentry phenotype was comparable to that due to HR HPV 16E7 (Table 1). We conclude that the phenotypic difference between the 16E7 and 11E7 proteins with regard to the efficiencies in promoting S-phase reentry is largely attributable to these three critical residues.

HPV-11 E7 lysine residues 39 and 42 modulate p130 association and destabilization.

To establish the molecular basis of the augmented phenotypes imparted by the 11E7 K39,42 mutations relative to that of the phenotypically similar G22D mutation, we sought to determine the influence of these mutations on the degradation of p130, a repressor of cell cycle entry. Submerged PHK cultures were transduced with retroviruses that expressed the wild-type or mutant forms of 11E7. To follow p130 destabilization, the cultures were treated with cycloheximide prior to harvest. Western blot analysis of lysates revealed that p130 destabilization in cultures expressing the 11E7 K39,42R mutation was greater than that in cultures expressing wild-type 11E7 and was comparable to that in cultures expressing 16E7 (Fig. 2A, upper panel). Unexpectedly, the G22D mutation reproducibly possessed a moderately impaired ability to destabilize p130 in comparison to wild-type 11E7, despite sharing a pocket protein binding motif homologous to 16E7 (Fig. 2A, lower panel, see also panel D).

Fig. 2.

Influence of the 11E7 K39,42 and G22 residues on targeting of p130 and pRb. (A) Western blot analysis of p130 destabilization in submerged PHK cultures expressing the native E7 protein. (B) Coimmunoprecipitation of pocket proteins by E7-ER in organotypic raft cultures. Coimmunoprecipitation was conducted with an antibody to the ER moiety from raft culture lysates. The immunoprecipitates were analyzed for the presence of p130, pRb, and ER by Western blot assay. (C) Western blot analyses of the same raft culture lysates for steady-state levels of p130, pRb, and E7-ER. The amount of protein loaded was 1/10 of that used in the coimmunoprecipitation shown in panel B. Destabilization of p130 (D) and pRb (E) steady-state levels in raft cultures expressing the native wild-type or mutant E7 proteins. Cultures prepared for panels A and D were treated with 250 μM cycloheximide for 6 h prior to harvest to resolve p130 destabilization. Arrows mark the bands of interest, whereas asterisks indicate nonspecific bands. GAPDH and the nonspecific bands served as internal controls for protein loading.

The divergent influences of the 11E7 K39,42R and G22D mutations on p130 destabilization connote possibly distinct mechanisms in the induction of S-phase reentry in the differentiated epithelium. To investigate the nature of these mechanisms, we systematically examined pocket protein association and destabilization induced in organotypic raft cultures by these mutant proteins. We have previously shown that E7 fused to the ligand binding domain of the human estrogen receptor (ER) maintains its ability to bind and destabilize the p130 pocket protein, stimulating S-phase reentry after induction of nuclear import upon exposure to β-estradiol. The ER moiety alone is ineffective in either of these assays (3, 19). The subcellular localization of the β-estradiol-induced ER fusion is predominantly in the nucleus (3, 52), as was also demonstrated in recent studies for the native E7 proteins of LR and HR HPVs (17, 30, 45). Thus, we utilized β-estradiol-induced E7-ER fusion proteins to investigate how the 11E7 mutations might have influenced the association with and the steady-state levels of the pocket proteins.

The ER fusions of wild-type 11E7 or mutant forms thereof were immunoprecipitated from the lysates of raft cultures with an antibody to ER. The precipitates were then Western blotted and probed for p130, pRb, and ER (Fig. 2B). Steady-state levels of the proteins examined in the immunoprecipitation reactions are shown in Fig. 2C. The wild-type 11E7 fusion bound low levels of p130 and pRb. Comparatively, the 16E7 fusion demonstrated higher association with both p130 and pRb, notwithstanding the disproportionately lower steady-state level of 16E7-ER (Fig. 2C). The K39,42R fusion exhibited a small increased association with p130 but not with pRb. The G22D mutation fusion proteins exhibited a highly elevated association with both pRb and p130.

E7 destabilization of pocket proteins requires direct binding (13, 19, 22, 25). However, this interaction alone does not necessarily result in pocket protein destabilization. For instance, the nononcogenic E7 protein of HPV-1a binds both p130 and pRb with an affinity similar to that of HR E7 orthologs yet destabilizes p130 selectively (7, 14, 20, 22, 50). To establish whether increased levels of pocket proteins bound by the mutant forms of 11E7 corresponded to their elevated degradation in raft cultures, levels of p130 and pRb were examined in the lysates of raft cultures expressing the native proteins with or without prior exposure to cycloheximide (Fig. 2D and E). Expression of the 11E7 K39,42R mutation resulted in augmented destabilization of p130 relative to wild-type 11E7 but did not alter the steady-state pRb levels. In contrast, cultures expressing the 11E7 G22D mutation exhibited impaired p130 destabilization, as in submerged PHK cultures (Fig. 2A), but levels of pRb were significantly reduced relative to those of cultures expressing wild-type E7. These results suggest that E7 can inactivate p130 by mechanisms other than destabilization, as has been reported for pRb.

Polyubiquitination and proteasomal degradation of HPV-11 E7.

Thus far, we have demonstrated a charge-independent influence of K39,42 on the 11E7 phenotype. One interpretation of this result is that K39,42 might be substrates of posttranslational modification such as polyubiquitination. Polyubiquitination of proteins, either at the N-terminal α-amino group or at internal lysine ε-amino groups, is a well-characterized process by which proteins are labeled for proteasomal degradation, rendering the modified species highly unstable (for a review, see reference 26). To determine whether 11E7 is polyubiquitinated on K39 and K42 in vivo, we conducted a polyubiquitination assay using an epitope-tagged ubiquitin as described previously (8, 29, 44, 51, 58, 61). The ER moiety or ER fusions of wild-type 11E7 or a 11E7 K39E,42A mutation were each transiently expressed in COS-7 cells together with an expression vector of myc-tagged ubiquitin. Prior to harvest, the cells were exposed to either the proteasome inhibitor MG132 or the solvent DMSO. The E7-ER fusion proteins were immunoprecipitated from the lysates and probed with an antibody to ER to detect the fusion proteins or with an antibody to myc to detect polyubiquitinated E7-ER.

In DMSO-treated cultures, polyubiquitinated E7-ER fusions were not detected. However, in the MG132-treated cultures, there was an increased abundance of the E7-ER fusion proteins and slowly migrating smears typical of polyubiquitinated proteins. In sharp contrast, there was no accumulation of polyubiquitinated ER protein (Fig. 3A). Thus, the ER moiety did not contribute to the polyubiquitination status of the fusion proteins.

Fig. 3.

Polyubiquitination and proteasomal degradation of HPV-11 E7-ER in situ. (A) COS-7 cultures coexpressing the ER moiety or E7-ER fusions and myc-tagged ubiquitin were treated with DMSO or MG132. Culture lysates were immunoprecipitated via the ER moiety. Immunoprecipitates were analyzed by Western blot assay for the myc tag (upper panel) or ER (lower panel). The ratios of myc-ubiquitin to E7-ER were determined by densitometry and are presented below their respective lanes. pUb designates the polyubiquitin smear. (B) Submerged PHK cultures expressing the ER moiety alone or E7-ER were exposed to DMSO or MG132. The Western blot of resolved lysates was probed for either ER or actin. Arrows mark the E7-ER band (upper) or the ER band (lower). The asterisk indicates nonspecific bands. Actin and the lower nonspecific band within the ER Western blot serve as internal controls for protein loading.

The major polyubiquitination substrate of HPV-16 E7 is the N-terminal residue, with minor levels of ubiquitination occurring on two internal lysine residues (4, 46). The ubiquitination substrates of HPV-11 E7 have not been characterized previously. If polyubiquitination does occur at K39,42, then the K39E,42A mutant protein should exhibit reduced polyubiquitination relative to that of wild-type 11E7. This appears to be the case. To quantify the relative polyubiquitination of the E7-ER fusions, the ratios of the smears of polyubiquitinated E7-ER to the nonubiquitinated E7-ER bands were determined by densitometry. The ratio of the 11E7 K39E,42A-ER mutant protein was lower than that of wild-type 11E7-ER. These findings indicate that polyubiquitination on the K39,42 residues accounts for a portion of the total polyubiquitination of 11E7.

The above data suggest that the lysine residues at positions 39 and 42 of 11E7 can serve as substrates of polyubiquitination. The abundance of wild-type 11E7-ER present in the immunoprecipitates was, however, greater than that of the K39E, 42A-ER mutant protein in both the DMSO- and MG132-treated cells. This observation conflicted with the reasoning that polyubiquitination on these lysine residues had rendered the wild-type protein more susceptible to proteasomal degradation than the mutant protein. Thus, we further assessed the susceptibility of these proteins to proteasomal degradation in submerged cultures of PHKs. The retrovirus-transduced PHK cultures were induced with β-estradiol for 48 h and treated for 6 h with MG132 or DMSO prior to harvest. The lysates were then analyzed for E7-ER or ER accumulation by Western blot assays (Fig. 3B). In DMSO-treated cultures, the steady-state level of K39E,42A-ER was reduced in comparison to that of wild-type 11E7-ER, consistent with relative levels of the fusion proteins immunoprecipitated from COS-7 cultures in Fig. 3A. MG132 exposure resulted in acute accumulation of K39E,42A-ER, whereas the levels of 11E7-ER were not comparably altered. Taken together, these data demonstrate that, contrary to the original expectation, polyubiquitination of 11E7 on lysine residues 39 and 42 is not a rate-limiting event during proteasomal degradation of the 11E7 protein and that polyubiquitination of the lysine residues is not responsible for the low 11E7 activities.

An inverse correlation between E7 stability and its ability to promote unscheduled DNA replication.

Despite the heightened S-phase phenotypes of 16E7 relative to 11E7 in raft cultures (Fig. 1C; Table 1), we noted that the steady-state expression level of the 16E7-ER fusion was much reduced compared to that of the 11E7-ER fusion (Fig. 2C). This low level could suggest reduced protein stability relative to that of 11E7. Thus, we assessed the relative stabilities of E7-ER fusions of the LR and HR HPV types in PHKs. Translational inhibition by cycloheximide is a standard method used previously to determine E7 stability (4, 31, 40, 41). Thus, PHKs retrovirally transduced with the E7-ER fusions were treated with cycloheximide for various durations prior to lysis. The lysates were Western blotted and then probed with the ER antibody. The remaining E7-ER bands were quantified by densitometry and plotted as a percentage of the steady-state levels detected in untreated cultures (Fig. 4A). The ER was stable for the duration of the cycloheximide treatment and thus did not confer instability on the E7 fusion proteins. The native 16E7 protein induced S-phase reentry in raft cultures more efficiently than the LR HPV E7 proteins (Table 1). The 16E7-ER fusion exhibited the highest turnover rate. The half-life of about 50 min of 16E7-ER expressed in PHKs falls within the range of native 16E7 reported previously (31, 41, 53). The half-lives of the ER fusions of 18E7, 11E7, and 6bE7 were approximately 125, 165, and 200 min, respectively. Though nonlinear, an inverse relationship between the relative stabilities of the E7 proteins and their efficiencies in promoting S-phase reentry (Fig. 1C; Table 1) is apparent.

Fig. 4.

Stability of E7. PHKs were transduced with retroviruses, each expressing E7-ER fusions. (A) HR and LR HPV E7 orthologs. (B) HPV-11 E7 or mutant proteins. The cells were cultured to 40% confluence, induced with 5 μM β-estradiol, and cultured for 48 h until harvest. Prior to harvest, the cells were additionally treated with 250 μM cycloheximide (CHX) for the durations indicated. Total E7-ER levels were determined by Western blot analysis with a rabbit MAb to ER (clone SP1; Lab Vision). Actin was used as an internal control for protein loading (left panels). The percentages of remaining ER or E7-ER are represented as fractions of that detected in the untreated culture (right panels).

Mutation of the 11E7 lysine residues at positions 39 and 42 resulted in increased efficiency of promoting S-phase reentry (Fig. 1B; Table 1) and reduced steady-state levels of the ER fusion (Fig. 3B) relative to the respective wild-type 11E7 native and fusion proteins. To assess whether the relationship between stability and S-phase induction was maintained in the lysine mutation, the proteolytic turnover rates of the ER fusions of native 11E7 and the 11E7 K39,42R mutant protein were analyzed in submerged cultures of PHKs as just described. In addition, a lysineless 11E7 (K9, 39, 42, 97R)-ER fusion was examined in parallel to determine whether any potential influence on stability imparted by the K39,42 residues was specific to these residues (Fig. 4B). The K39,42R mutant protein and the lysineless mutant protein both exhibited a higher proteolytic turnover rate than the wild-type 11E7-ER fusion. These observations support the notion that lysine residues internal to 11E7 limit the proteolytic turnover of the protein independent of the potential for polyubiquitination on these residues.

DISCUSSION

In this study, we identify nonconserved lysine residues of LR HPV-11 E7 and 6bE7 that limit the functional capacity of their respective proteins to promote unscheduled DNA replication in differentiated keratinocytes of organotypic raft cultures. These previously uncharacterized residues account in part for the functional divergence among the E7 proteins of the most clinically prevalent LR and HR HPV types. In combination with the mutation of G22 to D, mutation of the K39 and 42 residues of 11E7 was sufficient to rectify the overall disparity in the efficiency of S-phase reentry induced by the 11E7 protein relative to that induced by the 16E7 protein (Fig. 1B; Table 1). Mutations of the K39 and 42 residues exhibited enhanced targeting of p130, but not of pRb (Fig. 2B and E), in submerged (Fig. 2A) and raft (Fig. 2B and D) cultures of PHKs, underscoring the role of p130 as a critical repressor of cell cycle entry in the differentiated strata of the squamous epithelium. This observation corroborates our previous study demonstrating that binding to and degradation of p130 by the E7 protein is sufficient for this activity (19). An ER fusion of the 11E7 G22D mutant protein possessed an augmented ability to bind pRb in raft culture lysates (Fig. 2B). The native 11E7 G22D mutant protein reduced steady-state pRb levels (Fig. 2E), consistent with a G22D mutation of the related HPV-6b E7 protein characterized previously (24, 49, 65). However, unlike a glutathione S-transferase fusion of the 6bE7 G22D mutant protein which did not exhibit altered association with p130 in Jurkat nuclear extracts (65), the 11E7 G22D-ER fusion possessed a strongly increased ability to bind p130 in PHKs (Fig. 2B). Intriguingly, the capacity of the native 11E7 G22D mutant protein to destabilize p130 in submerged and raft PHK cultures was diminished (Fig. 2A and D).

The diametric influences of the K39,42R and G22D mutations on p130 and pRb targeting suggest alternative mechanisms of S-phase activation in the suprabasal epithelium by the E7 protein in pocket protein inactivation. Augmented p130 degradation by the K39,42R mutation can account for the increased S-phase induction. Though the G22D mutant protein was less efficient in p130 degradation than the wild type, the enhanced S-phase phenotype could be attributable to an elevated capacity for p130 interaction, thereby disrupting the p130-E2F complexes in a manner analogous to that of pRb (9, 10, 27, 33, 63). Alternately, the enhanced binding and degradation of pRb by the G22D mutant protein may allude to an ancillary role for pRb in maintaining the cell cycle homeostasis of differentiated keratinocytes. In this regard, it is interesting that epidermal pRb knockout models also exhibit epithelial hyperplasia and a suprabasal S phase (2, 47). However, there is a major difference between our PHK raft cultures and these pRb knockout mice in that squamous differentiation was not altered in our raft cultures which expressed the HR HPV E7 protein (12; data not shown), but it is significantly retarded in the knockout mice (2, 47).

Due to the proximity of the position to the LxCxE motif, the G22D mutation is thought to stabilize directly the HR HPV E7 interaction with the small groove conserved among retinoblastoma family pocket proteins. What might be the reason for the limiting effect of the nonconserved lysine residues of LR HPV E7? Our data suggest that the lysine residues at positions 39 and 42 of 11E7 can serve as substrates for polyubiquitination because polyubiquitination of the K39E,42A mutant protein was reduced relative to that of wild-type 11E7 (Fig. 3A). However, the biological activity-limiting effect of the 11E7 K39,42 residues cannot be attributed to polyubiquitination-dependent destabilization imparted by these residues, as their mutation leads to decreased, rather than increased, protein stability (Fig. 4B) and a subsequent reduction in the steady-state protein level (Fig. 3B). Similar relationships between stability and functional activity have been reported previously for transcriptional activators (32, 48).

Interestingly, the relative proteolytic turnover rates among the ER fusion proteins directly correlated with the N-terminal fold index previously reported for the E7 proteins of HPV types 6b, 11, 16, and 18 (18). The HR 16E7 protein, the ER fusion of which exhibited the highest turnover rate, contains a highly disordered N-terminal domain. The N-terminal disorder of the HR 18E7 protein is intermediate between the LR E7 orthologs and type 16E7, a relationship recapitulated by the intermediate stability of the 18E7 fusion (57). Implication of the N-terminal region of HPV E7 orthologs as a target of phosphorylation or polyubiquitination in combination with the N-terminal disorder may, in addition to the presence of internal lysine residues, offer explanation to differences in their stabilities and functional activities (1, 4, 28, 31, 46).

Our mutational analyses clearly indicated that the positive charge(s) of the nonconserved lysine residues of 11E7 or 6bE7 does not limit their activities, as their K→R mutations possessed an equally elevated activity in raft cultures as their respective K→E/G/A/T mutations relative to the wild-type protein (Fig. 1; Table 1). Even though we could not rule out the possibility that phosphorylation at the 11E7 CKII motif was hindered by these downstream lysine residues, we considered it unlikely. This is because we have demonstrated previously that, in COS-7 cells, the intensity of 11E7-ER metabolically labeled with ³²P was not distinguishable from that of the 18E7-ER fusion which lacks lysine residues downstream (19) (Fig. 1). We are inclined to suggest that the lysine side chain confers high conformational surface entropy, which is thought to interfere with protein-protein interaction. Thus, lysine residues are infrequently incorporated within the interface domains of proteins (34, 64). In a corresponding manner, an analysis of the 11E7 or 6bE7 secondary structure, conformational entropy, and amino acid sequence conservation by the Surface Entropy Reduction Prediction Server identified 11E7 lysine residues 39 and 42 and 6bE7 lysine residue 49 as nonconserved, high-entropy residues which may impede the formation of protein complexes (21) (http://www.doe-mbi.ucla.edu/Services/SER). Therefore, we propose that these nonconserved LR HPV E7 residues attenuate host protein interactions necessary for both E7 proteolysis and cell cycle deregulation in keratinocytes during epidermal differentiation.