A Synthetic E7 Gene of Human Papillomavirus Type 16 That Yields Enhanced Expression of the Protein in Mammalian Cells and Is Useful for DNA Immunization Studies

A synthetic E7 gene of human papillomavirus (HPV) type 16 wasgenerated that consists entirely of preferred human codons.Expression analysis of the synthetic E7 gene in human and animalcells showed levels of E7 protein 20- to 100-fold higher thanthose obtained with wild-type E7. Enhanced expression of E7protein resulted from highly efficient translation, as wellas increased stability of the E7 mRNA due to its codon optimization.Higher levels of E7 protein in cells transfected with syntheticE7 correlated with significant loss of cell viability in varioushuman cell lines. In contrast, lower E7 protein expression drivenby the wild-type gene resulted in a slight induction of cellproliferation. Furthermore, mice inoculated with plasmids expressingthe synthetic E7 gene produced significantly higher levels ofE7 antibodies than littermates injected with wild-type E7, suggestingthat synthetic E7 may be useful for DNA immunization studiesand the development of genetic vaccines against HPV-16. In viewof these results, we hypothesize that HPVs may have retaineda pattern of G + C content and codon usage distinct from thatof their host cells in response to selective pressure. Thus,the nonhuman codon bias may have been conserved by HPVs to preventcompromising viability of the host cells by excessive viralearly protein expression, as well as to evade the immune system.

INTRODUCTION

Top

Introduction

Human papillomaviruses (HPVs) are small double-stranded-DNAviruses that infect stratified epithelia and cause benign andmalignant proliferative lesions. A subset of HPVs with mucosaltropism, the so-called "high-risk" HPVs, has been linked tocancers of the uterine cervix (55), which account for about11% of the global cancer incidence in women. Of these, morethan 90% contain HPV DNA, most notably HPV type 16 (HPV-16)and HPV-18, integrated into the host cell genome, where theyexpress two viral early genes, E6 and E7, whose products blockp53 and retinoblastoma protein-mediated cell cycle control pathways(38, 56). Indeed, the tumorigenic phenotype of HPV relies oncontinuous expression of the E6 and E7 genes (9). E7 is an oncoproteinwhich can transform rodent fibroblasts (24), cooperate withactivated ras to transform primary cells (34), and, in associationwith E6, immortalize keratinocytes (10, 19, 30).

Progression from detectable HPV infection to invasive canceroccurs in less than 1% of cases and usually takes more than2 decades, indicating that additional factors are involved inthe process of carcinogenesis. Several lines of evidence suggestthat such factors may act by intensifying HPV early gene expression,as illustrated in the following examples. First, disruptionor mutation of the viral E2 gene, which is known to repressthe HPV early promoter and hence expression of E6 and E7, isobserved in cervical carcinogenesis (7, 36, 42, 47, 52). Moreover,the site of chromosomal integration of the viral DNA influencesexpression of viral early genes (48). Second, viral load appearsto be a determinant for the development of cervical carcinoma(23), and viral amplification is a frequent phenomenon in cervicaltumors and tumor cell lines (3, 5, 42). Third, tumor formationin nude mice by cervical cancer cells is inhibited by antisenseE6-E7 RNA (49). Fourth, repression of HPV gene expression bytumor necrosis factor alpha (TNF- ${alpha}$ ) is observed in nontumorigeniccervical cancer cell lines, while tumorigenic lines are notsensitive to TNF- ${alpha}$ (41), suggesting a correlation between tumorigenicityand deregulation of HPV expression. Fifth, constitutive expressionof E6 and E7 driven by keratin promoters causes epidermal hyperplasiaand skin cancer in transgenic mice (2, 28). Altogether, theseobservations suggest that progressive intensification of viralgene expression is required for malignant transformation.

At the transcriptional level, besides the viral E2 gene producta number of cellular proteins, including AP-1 family members,SP-1, YY1 glucocorticoid, and progesterone receptors, regulatethe HPV early promoter, located in the viral long control region,in cervical cancer cells as well as in transgenic mice. Sinceefforts to identify tissue-specific transcription factors havefailed, it is conceivable that an interplay between those factorsmay account for the cell type specificity of the HPV-16 earlypromoter-enhancer, which is strictly epithelial in vivo (8),and for the continuous expression of E6 and E7 in cancer tissues.

Transcriptional analysis of HPV-infected cervical cancer tissuesand cell lines has revealed that viral transcription is restrictedto the early region and that the most abundant mRNA speciesencodes the E7 protein (11, 40, 43). Intriguingly, in contrastwith the high levels of E7 mRNA that can be demonstrated byNorthern blotting and in situ hybridization in carcinoma celllines and tissues, the E7 protein is hardly detectable by immunostaining,immunoprecipitation, or Western blot analysis (15). Possibleexplanations for this discrepancy may be that the protein isexpressed at concentrations too low for immunodetection andthe fact that it is rapidly degraded by the ubiquitin-proteasomepathway (35). Additional evidence in support of a restrictedpresence of E7 in infected cells comes from patients with HPV-associatedgenital dysplasia, who, in most cases, generate a poor antibodyresponse to E7, suggesting that this protein is expressed insmall amounts and hence remains inaccessible to the immune system.Moreover, it has been proposed that epitopes of E7 could remainmasked due to interaction with host cell proteins in the nucleus,where E7 is mainly localized (15, 25).

While the molecular mechanisms underlying the oncogenic functionsof E7 through its interaction with various cellular regulatoryproteins have been studied extensively, very little is knownabout factors that limit E7 expression. As the transcriptionalrate from the HPV LCR is maintained at relatively high levels(40), it is likely that posttranscriptional mechanisms accountfor the low expression of E7. One such mechanism may resultfrom the different codon usage of HPV and human genes. The extensivethird-position degeneracy of the genetic code permits amplevariation in the set of codons preferred by viruses with respectto that prevalent in human cells (45). This may result, however,in less efficient synthesis of viral protein due to insufficientamount of the tRNA species necessary to translate the correspondingmRNAs, as has been observed in other organisms (14, 21, 50).Indeed, limitations in protein expression due to codon usagedivergence from human genes have been postulated for the latentgenes of Epstein-Barr virus (26) and have been demonstratedfor the env gene of human immunodeficiency virus type 1 (17),and the L1 and L2 capsid genes of bovine papillomavirus type1 (54). In both cases, synthetic genes designed to have codonusage resembling that of human genes yielded enhanced expressionof the corresponding proteins. It seems likely that a nonhumancodon bias leading to inefficient expression of viral proteinsresulted from selective pressure to escape the defense mechanismsof the host. Should this apply to HPV, it would help explainthe need for a high transcriptional activity to overcome thelimitation in viral translation observed in cancer tissues.

In view of these observations, we decided to examine the roleof the divergent codon bias of HPV-16 and human genes in therestricted expression of E7 in mammalian cells. We report asynthetic E7 gene engineered to contain exclusively codons preferredby highly expressed human genes, thereby yielding expressionof remarkably high levels of E7 protein compared with the wild-typeE7 gene. Enhanced expression of E7 from the synthetic gene appearedto result from both increased translation and higher stabilityof its mRNA. High expression levels of E7 protein had a negativeeffect on cell viability, which was not observed at the lowerlevels of protein expressed by the wild-type gene. Furthermore,DNA immunization experiments showed higher immunogenicity toE7 elicited by the synthetic gene, suggesting that it may beuseful for the development of new DNA vaccines against HPV-16.In addition, our data suggest that selective pressure may haveforced HPVs to adopt a suboptimal codon usage in order to persistas commensal agents and carry out their slow transforming effectsin the cells.

MATERIALS AND METHODS

Top

Materials and Methods

Synthetic genes and plasmid constructions. A synthetic HPV-16 E7 gene was engineered in vitro by introducing64 silent mutations (22% of the E7 sequence), which createdan open reading frame consisting entirely of preferred humancodons. In addition, a sequence encoding a Flag tag was fusedto its 3' end. The synthetic E7 gene was generated by two consecutivePCRs. Platinum Pfx polymerase (Invitrogen) was used in all PCRamplifications. The following overlapping oligonucleotides wereused (numbers in parentheses correspond to nucleotide positionsin the sequence given in Fig. 1C): A, 5'-GACAGCTAGC-(1 to 89)-3'(forward primer); B, 5'-(156 to 65)-3' (reverse primer); C,5'-(138 to 232)-3' (forward primer); D, 5'-ACACGAATTC-(318 to211)-3' (reverse primer); E, 5'-AACAGCTAGC-ATG-(106 to 131)-3'(forward primer); and F, 5'-ACACGAATTC-AAT-(291 to 268)-3' (reverseprimer). In a first PCR, primers A and B and primers C and Dwere used to generate two fragments, AB and CD, by chain extensionusing no template. In a second reaction, the overlapping productsAB and CD were mixed together and used as a template to producea synthetic E7-Flag fusion gene by using primers A and D. Furthermore,the resulting E7-Flag gene was used as a template to amplifya synthetic E7 gene with no Flag tag (with primers A and F)and a truncated E7-Flag fusion gene (with primers E and D) lackingthe first 35 codons of E7. The synthetic E7 genes were isolatedfrom the PCR mixtures by agarose gel electrophoresis, purifiedwith QiaexII (Qiagen), double digested with NheI and EcoRI,and cloned into the polylinker of pIRES-neo2 (Clontech), whichwas digested with the same restriction enzymes. Single cloneswere identified by transformation of DH5 ${alpha}$ bacteria and sequenced.A green fluorescent protein (GFP)-expressing plasmid (pQBI-25-fPA;Q-BIOgene) was used to monitor efficiency of transfections.Plasmids were purified with commercial kits (DNA Maxiprep, endotoxinfree; Qiagen).

View larger version (45K):
[in this window]
[in a new window]
FIG. 1. (A) Comparison of codon usage frequencies in highly expressed human genes (Hu_*), according to Haas et al. (17), and HPV-16 E6 and E7 (transforming genes) and L1 and L2 (capsid genes). (B) (Top) Comparison of the G+C content of average (Hu) and highly expressed (Hu_*) human genes (17, 31) with the coding regions of the most prevalent genital HPVs: HPV-16 and -18 (high risk, oncogenic) and HPV-6 and -11 (low risk, nononcogenic). The frequencies of A and T in the third position in human and HPV codons (middle) and among HPV-16 genes are also compared (bottom). (C) Nucleotide sequence of the synthetic HPV-16 E7 gene (eE7) generated in this work. Silent mutations introduced to create an open reading frame of preferred human codons are in capitals. The sequence encoding a Flag tag is underlined, and an asterisk indicates the stop codon. The encoded amino acid sequence is given below the nucleotide sequence.

Mice, cell lines, and transfections. BALB/c mice (WiGa-Charles River, Hamburg, Germany) 6 to 8 weeksold were used for DNA immunization experiments with endotoxin-freeplasmid complexed to polyethylenimine (in vivo jet-PEI; Q-Biogene)as suggested by the manufacturer. Equal amounts of plasmid (4µg) were injected subcutaneously three times at 2-weekintervals. Vero, HeLa, SiHa, and C-33 cells were grown in Dulbecco'sModified Eagle Medium with Glutamax (Invitrogen-Life Technologies)supplemented with 10% fetal calf serum (heat inactivated; Invitrogen-LifeTechnologies) and penicillin-streptomycin. Cells were transfectedat 50 to 70% confluence by using Effectene (Qiagen) or FuGENE(Roche) by following the manufacturer's instructions.

Western blotting. Cells growing on 6-cm plates were transfected with 2 µgof plasmid and allowed to express the protein for 24 h. Thencells were washed twice with phosphate-buffered saline (PBS)and lysed in sodium dodecyl sulfate (SDS) loading buffer containing1 mM dithiothreitol. Cellular proteins were separated on 15%polyacrylamide gels by SDS-polyacrylamide gel electrophoresis(PAGE), blotted onto polyvinylidene difluoride membranes, andhybridized with the corresponding specific antibodies, followedby detection with horseradish peroxidase (HRP)-conjugated anti-mouseor -rabbit immunoglobulin G antibodies (Molecular Probes). Antibodiesused were anti-Flag M2 conjugated to HRP (Sigma), anti-HPV-16E7 antibody (Oncogene), anti-neomycin phosphotransferase II(anti-NPT-II) (Upstate), antiactin (Santa Cruz), and anti-GFP(Clontech). Proteins were visualized after reaction with enhanced-chemiluminiscencereagents (Renaissance; NEN Life Science Products).

Immunofluorescence. Cells growing on glass coverslips were transfected in separatewells of 24-well plates with 0.4 ml of growth medium. At varioustimes after transfection, the medium was aspirated and the cellswere washed once with PBS, fixed with 4% paraformaldehyde for15 min at room temperature, and washed once with PBS. Backgroundfluorescence was reduced by quenching free aldehydes in 50 mMNH₄Cl-PBS for 10 min. After a brief washing with PBS, the cellswere permeabilized by immersing coverslips in PBS-0.2% TritonX-100 for 5 min at room temperature, washed again with PBS,and blocked in blocking solution (2% fetal calf serum, 2% bovineserum albumin, and 0.2% gelatin in PBS) for 30 min. Primaryand secondary antibodies were diluted in a 1:10 dilution ofblocking solution in PBS. Incubation times were 1 h for eachantibody. Coverslips were washed six times in PBS after incubationwith each antibody. Finally, nuclei were stained with DAPI (4',6-diamidino-2-phenylindole)or propidium iodide (Molecular Probes). Primary antibodies wereanti-Flag M2 (Sigma) and anti-HPV-16 E7 (Oncogene). Secondaryantibodies were Cy2- or Cy3-conjugated anti-mouse and anti-rabbitantibodies (Jackson ImmunoResearch). Cells were analyzed witha confocal laser scanning microscope, Leica TCS SP, with thedetector pinhole set at 1 Airy disk unit.

Expression of E7 protein. The complete HPV-16 E7 protein with an N-terminal six-His tagwas expressed by using a T7 expression plasmid [pET28a(+); Novagen]and purified over nickel-agarose columns (Qiagen). The purifiedprotein was quantified by using the Bradford assay (Bio-Rad).

ELISA. Enzyme-linked immunosorbent assay (ELISA) plates were coatedovernight with 1 µg of protein per ml in carbonate buffer,pH 9.3, at 4°C and blocked in 5% fetal calf serum in PBSfor 2 h at room temperature. Plates were subsequently washed,and 100 µl of serum (diluted in 0.5% milk-0.1% Tween 20in PBS) was added to each well and incubated for 1 h at 37°C.Bound antibody was detected with goat anti- mouse immunoglobulinG conjugated to HRP (Molecular Probes) for 1 h at 37°C.Plates were developed by adding 100 µl of O-phenylenediamine(Sigma). After 30 min at room temperature in a darkened area,the reaction was stopped by the addition of 50 µl of 1M sulfuric acid per well. Plates were read at 450 nm.

Northern blotting. Northern blotting was performed essentially as described previously(8). Briefly, total RNA was extracted from HeLa cells 24 h aftertransfection with plasmids expressing either synthetic or wild-typeHPV-16 E7, by using an RNA extraction kit (RNeasy; Qiagen) asdirected by the manufacturer. Purified RNA samples were separatedon 1% agarose-formaldehyde gels, blotted onto nylon membranes(Genescreen; NEN), and hybridized with mixed (1:1 molar ratio)E7 and enhanced E7 (eE7; see below) or with glyceraldehyde-3-phosphatedehydrogenase (GAPDH) probes labeled by primer extension.

Studies on protein stability. Cells growing on six-well plates were transfected with pIRES-Neo2-wtE7or pIRES-Neo2-eE7 by using Effectene. After 4 h, the transfectionmedium was aspirated and replaced with 2 ml of fresh growthmedium. The medium was aspirated 24 h later, the cells werewashed once with PBS, and fresh medium containing either 40µg of cycloheximide (Sigma) per ml, 5 mM clasto-lactacystinß-lactone (Calbiochem), or both was added. At varioustimes, the cells were washed with cold PBS, scraped from theplates, washed again with cold PBS, and lysed with SDS loadingbuffer. A control dish of untreated, nontransfected cells wasprocessed similarly.

Cell viability assay. A colorimetric assay for quantification of cellular proliferation,viability, and cytotoxicity was used. Cells were plated at adensity of 10⁵/well in a 96-well plate with 200 µl ofmedium per well. At 24 h after transfection, 10 µl ofthe tetrazolium salt MTT [3(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazoliumbromide; Roche] (5 mg/ml in PBS) per well was added, and theplate was incubated for another 2 h. Then the plate was centrifugedat 250 x g for 5 min, and the supernatant was discarded. Theprecipitated MTT was dissolved with 100 µl of acidic isopropanol(0.1 N HCl) per well until crystals were completely dissolved(as monitored under the microscope). Measurement of absorbancewas at 570 nm. Negative control wells containing medium butnot cells and positive controls consisting of nontransfectedcells were run in parallel.

Nucleotide sequence accession number. The GenBank accession number for the synthetic E7 gene is AF373107.

RESULTS

Top

Results

Construction of a synthetic HPV-16 E7 gene consisting entirely of preferred human codons. HPVs have a codon usage that differs substantially from thatof most human genes. A detailed analysis of the codon compositionof the E7 gene showed that 13 amino acids are encoded by codonsrarely used by highly expressed human genes, as occurs withother early (transforming) and late (capsid) HPV-16 genes (Fig.1A). This can be explained by the particular base compositionof HPV genomes. As shown in Fig. 1B, A and T are preferred inthe third position of a majority of their codons (65 to 74%).In contrast, only 42% of codons in human genes contain A orT at the third position (31), and the number is even lower amonghighly expressed genes (23%). This is in good correlation withthe low G+C content of these viruses (35 to 43%), which is markedlylower than the content in human genes (53 to 57%) (Fig. 1B).There are only minor differences between the main oncogenic(HPV-16 and -18) and nononcogenic HPVs (HPV-6 and -11). However,HPV-16 has the highest rate of A or T as the third base andlowest G+C content among genital HPVs (35%). Such biased useof the genetic code by HPV-16 could account, at least in part,for the small amount of E7 protein detected in transfected cellseven when the E7 gene is expressed under the control of strongpromoters.

To investigate whether differences in codon usage between theviral and the human genomes could account for the poor expressionof E7, we generated a mutant synthetic E7 gene containing exclusivelycodons that are found preferentially in highly expressed humangenes (17) (Fig. 1A). The resulting gene contained 63% G+C (20%more than wild-type E7) and no A or T in the third positionof its codons (Fig. 1C). The synthetic E7 gene was named eE7(for "enhanced E7"; see below and Fig. 2 to 4) and was constructedby PCR based on the sequence of the prototype virus (37) (GenBankaccession number K02718) by using oligonucleotides with overlappingsequences as described in Materials and Methods. As a control,a wild-type gene which was amplified from a plasmid containingthe prototype E7 gene of HPV-16 was used. In addition, a 5'deletion mutant lacking the first 35 amino acids of E7 was constructedby PCR using the full-length synthetic eE7 and wild-type E7genes as templates, which were named eE7 ${Delta}$ 1 and E7 ${Delta}$ 1, respectively.Upstream sequences flanking the coding regions of the geneswere converted to a Kozak consensus translation initiation site(27). In addition, to facilitate detection of the protein, asequence encoding the Flag epitope (DYKDDDDK) was fused to the3' ends of the genes. The wild-type and mutant chimeric geneswere inserted separately in a mammalian expression vector (pIRESneo2)under the control of the immediate early promoter of the humancytomegalovirus (CMV) and expressed as a bicistronic mRNA, withthe second part of the transcript being an NPT-II gene.

View larger version (37K):
[in this window]
[in a new window]
FIG. 2. Expression of synthetic and wild-type HPV-16 E7 genes in transient-transfection assays demonstrated by Western blot analysis. HeLa cells growing on six-well plates (10⁵ cells/well) were transfected with 2 µg of plasmid pIRES-neo2 (Clontech) (pIn2) or plasmids expressing synthetic eE7 or wild-type E7 genes or their truncated derivatives eE7 ${Delta}$ 1 and E7 ${Delta}$ 1, respectively. The efficiencies of transfection, monitored by cotransfecting 0.5 µg of a plasmid expressing a GFP gene (pQBI-25-fPA) with each of the above plasmids and counting fluorescent cells, were identical in all cases. The cells were harvested 24 h after transfection and lysed, and half of the lysate was subjected to SDS-PAGE. After blotting, complete and truncated E7 proteins were detected with anti-Flag antibodies, and the filter was rehybridized with antiactin antibodies to show that equal amounts of protein had been loaded. A representative blot is shown.

View larger version (33K):
[in this window]
[in a new window]
FIG. 4. Immunofluorescence analysis of HPV-16 E7 expression in Vero cells transfected with plasmids expressing synthetic (eE7 and eE7 ${Delta}$ 1) or wild-type (E7 and E7 ${Delta}$ 1) E7 genes. Cells were fixed 48 h after transfection and incubated with specific antibody as indicated, followed by Cy2-conjugated goat anti-mouse antibody. (A) The synthetic eE7 gene expressed higher levels of protein than the wild type. E7 protein was detected with anti-Flag antibody. Immunofluorescence was analyzed with a Leica TCS SP confocal microscope using a 63x objective and exactly the same laser and photomultiplier intensities, with the pinhole set at 1 Airy disk unit. Single sections corresponding to the plane of the highest intensity of fluorescence are shown. (B) Confocal sections of cells transfected with plasmids expressing full- length (eE7) and truncated (eE7 ${Delta}$ 1) synthetic genes. Anti-Flag antibody shows the intracellular distribution of E7 (left); nuclei were counterstained with propidium iodide (middle), which labels particularly strongly the nucleoli, as seen in the merged picture (right). Both forms of E7 protein are scattered throughout the nuclear matrix and excluded from the nucleoli. (C) Confocal section of a cell transfected with a plasmid expressing a truncated E7 mutant lacking the first 35 amino acids (eE7 ${Delta}$ 1), without the Flag tag. The protein localized to the nucleus, as detected with a monoclonal anti-E7 antibody followed by a Cy2-conjugated goat anti-mouse antibody.

Expression of synthetic and wild-type E7 in mammalian cells. To ascertain the efficacy of codon replacement in enhancingsynthesis of HPV-16 E7 protein, we compared the expression ofsynthetic and wild-type E7 genes after transient transfectioninto human, mouse, and monkey cell lines. In all cell typestested, the level of E7 protein was significantly higher fromthe synthetic gene. This was determined, in transient-transfectionexperiments, by Western blot hybridization (Fig. 2) and by proteinlabeling and immunoprecipitation assays (data not shown). Transfectionefficiencies were monitored by cotransfecting the E7 plasmidswith a plasmid expressing a GFP gene. As illustrated in Fig.2 for HeLa cells, the synthetic eE7 gene expressed markedlyhigher levels of E7 protein than the wild-type E7 gene. Theexpression of E7 protein by the eE7 gene in different transfectionexperiments was estimated to be 20- to 100-fold higher thanthat by the wild-type E7 gene, depending on the experimentalconditions. Similar differences in amounts of protein were observedin transfections with the deletion mutants eE7 ${Delta}$ 1 and E7 ${Delta}$ 1 (Fig.2), indicating that subfragments of eE7 yield enhanced proteinexpression with the same efficiency as the full-length gene.

The full-length E7 protein was detected in Western blots asa doublet, the upper band of which represents a protein of about19 kDa (Fig. 2), as in previous studies using anti-E7 antibodies(15). This doublet was also observed by immunoprecipitation(data not shown). It has been speculated that the less-abundant,higher-mobility peptide could represent a protein that coprecipitateswith E7 or shares an epitope with it (15). However, this seemsunlikely in our experiments, since we used anti-Flag antibodiesfor detection of E7. Rather, it may be a product resulting fromintracellular processing of E7.

In a different series of experiments, we investigated whetherhigher protein expression by the eE7 gene was due to enhancedtranslation of its message. To this end, RNA and protein levelsof E7 and NPT were compared after transient transfections ofHeLa cells (Fig. 3). As described above, wild-type E7 as wellas synthetic eE7 coding sequences were expressed with the pIneo2plasmid from a CMV promoter as bicistronic mRNAs joined to theNPT coding sequence through an internal ribosome, entry site.Results of a representative experiment are shown in Fig. 3.As expected, the amount of E7 protein expressed by plasmid pIneo2-eE7-IRES-NPTwas markedly higher (over 50-fold in this particular experiment)than that by pIneo2-E7-IRES-NPT. In contrast, the amount ofNPT protein expressed from the eE7-IRES-NPT message was onlyslightly higher (less than 1.5-fold) (Fig. 3A). The amountsof bicistronic mRNA transcribed from each plasmid were comparedin Northern blots of total and cytoplasmic RNA isolated fromtransfected cells of the same experiment by hybridization witha radioactive NPT probe (Fig. 3B). The eE7-IRES-NPT mRNA wasmore abundant than the E7-IRES-NPT mRNA (about two- to three-fold,as determined with the NIH Image program). Nearly the same differenceswere observed for total and cytoplasmic RNA isolates.

View larger version (60K):
[in this window]
[in a new window]
FIG. 3. Expression of HPV-16 E7 and NPT mRNA and protein. HeLa cells growing on 6-cm plates (3 x 10⁵ cells/plate) were transfected with the GFP-expressing plasmid pQBI-25-fPA (-) alone or cotransfected with this plasmid and a plasmid carrying either synthetic (pIn2-eE7) or wild-type (pIn2-E7) E7. The latter plasmids are transcribed into bicistronic mRNAs harboring sequences for expression of the E7 and NPT proteins. After 24 h, cells were harvested and lysates were processed for total or cytoplasmic RNA purification and for protein extraction. (A) Western blot analysis. Note the remarkably small amount of E7 protein expressed in cells transfected with pIn2-E7 compared with that in cells transfected with pIn2-eE7. In contrast, the difference in the amount of NPT expressed by the two plasmids is much lower (about 1.5 times higher by pIn2-eE7, as estimated densitometrically). Hybridization with GFP antibodies showed equal efficiency of transfection. (B) Northern blot analysis showing levels of eE7-IRES-NTP and E7-IRES-NTP bicistronic RNA in transiently transfected cells. Total RNA was loaded at 10 µg per lane. Detection was with [ ${alpha}$ -³²P]dCTP-labeled probes. A labeled NPT probe was used to detect the eE7-IRES-NPT and E7-IRES-NPT messages (arrowhead). The blot was stripped and rehybridized with a radioactive GFP probe to show nearly equal transfection efficiencies (middle). Staining of the gel with ethidium bromide after electrophoresis is shown in the lower panel. The positions of the 28S (4.7-kb) and 18S (1.9-kb) rRNAs are indicated.

These data suggested that mRNA containing synthetic eE7 wastranslated more efficiently into E7 protein (but not NPT protein)than the message bearing wild-type E7. The differences seenin the amounts of the respective mRNAs were too low to explainthe higher levels of E7 protein expressed from eE7 and may reflecta higher stability of the eE7 mRNA resulting from its secondarystructure or its longer binding to polysomes, thus remaininginaccessible to RNase.

Intracellular localization of E7. HPV-16 E7 is a phosphoprotein associated with the nuclear matrix(15) that has been also found in the cytoplasm (39). The highexpression of E7 protein from the eE7 gene allowed us to assessits intracellular localization in transiently transfected cells.As shown in Fig. 4, E7 localized mainly to the nucleus, althoughit could also be detected with a pattern of intensely fluorescentdots in the cytoplasm surrounding the nucleus. Within the nucleus,the protein appeared to be uniformly distributed through thenuclear matrix. However, a localization of E7 in the nucleoliwas not observed. Rather, E7 appeared to be completely excludedfrom nucleolar structures, as shown in confocal sections ofspecimens in which DNA and RNA were stained with propidium iodideto label the nucleoli (red fluorescence in Fig. 4B). The samelocalization pattern was observed in HeLa, SiHa, and other humancell lines. Like the full-length E7, the E7 ${Delta}$ 1 deletion mutantlocalized mainly in the nuclei of transfected cells and wasalso excluded from the nucleoli (Fig. 4B, lower panel). Thepossibility that E7 was excluded from the nucleoli due to theFlag tag was discarded by expressing E7 without tag and detectingthe protein with anti-E7 antibodies. As shown in Fig. 4C, E7was again excluded from the nucleoli. These data are in contrastwith a previous report describing nucleolar localization ofE7 (53) and in agreement with previous work showing associationof E7 with the nonchromatin nuclear structure (15).

Half-life of the E7 protein in eE7-transfected cells. Next, we examined whether the enhanced steady-state levels ofE7 protein in cells transfected with eE7 might be due, at leastin part, to delayed degradation of the protein. To this end,the stability of the protein was investigated in cells in theabsence and presence of the protein synthesis inhibitor cycloheximideand the proteasome inhibitor lactacystin. As the levels of E7protein expressed by the eE7 gene were sufficiently high fordetection in Western blots, a nonradioactive protocol was chosenfor analysis of protein expression after SDS-PAGE. Cotransfectionwith plasmid pQBI-25-fPA expressing GFP was used to confirmequality of transfection efficiency by counting green fluorescentcells. Actin was used as an internal control for the amountof protein loaded per lane. Inhibition of both protein synthesisand degradation caused a reduction in levels of the E7 proteinto about 50% in 1 h (Fig. 5, compare lane 3 with lanes 2 and4). During the same time, no change in the actin content ofthe cells was observed, as could be expected from its longerhalf-life (nearly 6 h) (6). These results are in agreement withprevious work showing that E7 is degraded via ubiquitin-mediatedproteolysis, with an estimated half-life of about 30 to 40 min(35, 39).

View larger version (49K):
[in this window]
[in a new window]
FIG. 5. The half-life of E7 was not affected by its enhanced expression. HeLa cells were transiently cotransfected with plasmids pIneo2-eE7 and pQBI-25-fPA (expressing GFP). At 24 h after transfection (Tf), protein synthesis was inhibited with cycloheximide (Chx), and protein degradation was blocked with clasto-lactacystin ß-lactone (ß-Lact), a cell-permeative proteasome inhibitor, as indicated. Then the cells were lysed, subjected to SDS-PAGE, and transferred to polyvinylidene difluoride membranes. Anti-Flag antibody was used to detect E7 protein, and antiactin was used to show equality of protein loading. Equal transfection efficiency was monitored by counting green fluorescent cells. A representative blot is shown.

Effect of high expression of E7 protein on cell viability. In the course of transient-transfection experiments with thesynthetic eE7 gene, we observed that increasing numbers of deadcells detached from the plates after the second day of transfection,a phenomenon that was not seen in transfections with the wild-typeE7 gene. This effect was observed in HeLa cells as well as C-33cells, another cervical carcinoma cell line that does not carryany known HPV DNA. Furthermore, our attempts to obtain a stablecell line with the plneo2-eE7 plasmid failed. In these experiments,over 30 clones resistant to neomycin were found not to containE7 (data not shown), suggesting selection against cells expressingthis protein. Silencing of E7 expression in those clones mightbe due to point mutations, since no deletions were noted infragments amplified by reverse transcriptase PCR. These resultsprompted us to use the MTT assay to investigate the possibilitythat sustained elevated levels of E7 protein may cause cellsto die (18, 29). MTT is a tetrazolium salt that is cleaved toformazan by the succinate reductase system, which is part ofthe respiratory chain of mitochondria and is active only inviable cells. Accordingly, the amount of formazan formed, whichcan be measured in a colorimetric assay, is in direct correlationwith the number of living cells in a culture.

HeLa cells were transiently cotransfected with plasmids expressingsynthetic HPV-16 eE7 (pIneo2-eE7), wild-type E7 (pIneo2-E7),or plasmid pIneo2 as a control and with a plasmid expressingGFP (pQBI25-fPA) as an indicator of the efficiency of transfection.This ranged from 20 to 25%, as determined by counting the numberof green fluorescent cells at 24 h and dividing it by the numberof seeded cells. The cells were processed for the MTT viabilityassay 24, 48, and 72 h after transfection. As shown in Fig.6, comparable numbers of viable cells were observed with thethree plasmids 24 h after transfection. However, pIneo2-eE7-transfectedcells underwent decreases in viability of 15 and 26% after 48and 72 h, respectively, compared to mock (pIneo2)-transfectedcells. In contrast, pIneo2-E7-transfected cells showed survivalrates of 12 and 15% over that of the control after 24 and 48h, respectively. Considering the transfection efficiency rates,it can be concluded that most cells carrying pIneo2-eE7 werenot viable after 72 h. Furthermore, cells transfected with pIneo2-E7seemed viable and proliferating at about a 37% higher rate thannontransfected cells, as the 12% increase in the total numberof viable cells should be attributed to 25% transfected cells.

View larger version (14K):
[in this window]
[in a new window]
FIG. 6. Effect of HPV-16 E7 expression on viability of HeLa cells. HeLa cells were seeded in 96-well microplates (5,000 cells per well in 200 µl of medium). One day later, cells were cotransfected with plasmid pIneo2, pIneo2-eE7, or pIneo2-E7 and with plasmid pQBI25-fPA expressing GFP, which served to monitor efficiency of transfection. At the indicated time points after transfection, cell viability was measured by the MTT method (see the text). The efficiency of transfection (number of GFP-expressing cells divided by number of seeded cells), estimated at 24 h, ranged from 20 to 25%, with minimal variation from well to well. Results are averages of three independent transfection experiments. Note the lower survival rates of cells transfected with pIneo2-eE7.

DNA immunization of mice with plasmid expressing E7. The ability of synthetic and wild-type E7 genes to induce effectiveanti-E7 humoral immunity in mice was tested after injectionof the corresponding plasmids. Three groups of naïve micewere injected three times subcutaneously at 2-week intervalswith either PBS or plasmid DNA expressing eE7 or wild-type E7.The titers of antibody elicited against E7 were markedly higherin mice injected with plasmid expressing eE7 than in those inoculatedwith the wild-type E7 gene, in which the antibody was almostundetectable (Fig. 7). Titers ranged from 100 to 400. Theserelatively low titers are likely due to the small amounts ofplasmid injected with the method used and the fact that theplasmid solutions were injected subcutaneously. However, thisapproach was preferred to methods using larger amounts of plasmidsand more invasive injections (e.g., intraperitoneal), becauseit resembles better the standard immunization method used inclinical practice.

View larger version (18K):
[in this window]
[in a new window]
FIG. 7. Plasmid expressing eE7 induces anti-E7 specific antibodies in mice. Animals were inoculated three times subcutaneously either with no plasmid (PBS) or with 4 µg of plasmid expressing wild-type E7 or synthetic eE7 genes. Six weeks after the first inoculation, mice were bled, and the sera were analyzed for E7-specific antibodies by ELISA. Antibody titers are expressed as the reciprocal of the lowest serum dilution that gave an absorbance of 0.1 unit above the background. Each symbol represents an antibody titer for an individual mouse.

DISCUSSION

Top

Discussion

Compared to human coding sequences, HPV genomes have at leastthree distinct features, as can be deduced from Fig. 1A and B.First, the overall G+C content of HPVs is significantly lowerthan that of human genes. Second, HPV genes contain A or T inthe third position of two-thirds of their codons, while only40% (average) and 23% (highly expressed) of human codons haveA or T in the third position (17, 31). Third, HPV genes havea codon usage that differs substantially from that of humangenes. For instance, all codons used preferentially by HPV-16fall among those less frequently used by highly expressed humangenes (Fig. 1A), with slight differences between early and lategenes (e.g., codons for amino acids A, E, and P). Interestingly,HPV-16 is the genital HPV type with the lowest G+C content andhighest proportion of A+T at the third position of its codons.Nevertheless, although they are markedly different from humangenes, the HPV types compared in Fig. 1B have only minor differencesbetween them, in spite of their very different biological andclinical significance (types 16 and 18 are oncogenic and types6 and 11 are nononcogenic).

A likely consequence of having codon biases divergent from thatof human genes is that the tRNA species required for translationof HPV mRNAs may be underrepresented in the host cell and henceviral protein expression severely limited. This would explainwhy high levels of viral mRNA are accompanied by low levelsof viral protein in HPV-bearing tumors and cell lines. Furthermore,HPV-16 variants carrying mutations that optimize codon usageare not rare in cervical cancers (4, 13).

It was the aim of this study to determine to what extent suchstriking differences between HPV and human codon usage couldinfluence expression of HPV-16 genes. The HPV-16 E7 gene waschosen for this purpose because it is a major HPV oncogene,has a short sequence, and is known to express very small amountsof the protein (15) (Fig. 2). A synthetic E7 gene (eE7) whosecodon usage was conveniently optimized to contain exclusivelycodons used preferentially by highly expressed human genes wasgenerated and characterized (Fig. 1A). The resulting gene (Fig.1C) has a G+C content above that of human genes and does notcontain A or T in the third position of its codons, in contrastto human genes, which have A or T in that position in 23% oftheir codons. As expected, the eE7 gene expressed larger amountsof E7 protein than the wild-type E7 gene. The difference inexpression was estimated, in transiently transfected cells,as 1 to 2 orders of magnitude higher (Fig. 2 to 4). Interestingly,enhancement of protein expression was observed in human, monkey,and mouse cells.

The use of a bicistronic expression system proved useful fordetermining the different translation rates of synthetic andwild-type E7 having NPT as a common internal control. Thus,translation of synthetic eE7 from bicistronic eE7-IRES-NPT mRNAproduced about 50-fold more protein than translation of thewild-type gene contained in the E7-IRES-NPT message (Fig. 3A).In contrast, the amount of NPT protein was only 1.5 times higher,indicating that translation of eE7 but not NPT was significantlyenhanced from the eE7-IRES-NPT mRNA. Additional evidence insupport of enhanced translation of eE7 came from quantitativeanalysis of Northern blots using NPT as probe, which shouldhybridize equally with both bicistronic mRNA species, showinga two- to threefold larger RNA amount for eE7-IRES-NPT thanE7-IRES-NPT (Fig. 3B). This difference was too small to accountfor the 50-fold increase in E7 protein expression seen in Fig.3A. However, this result was somewhat surprising, as the transcriptionrates of these plasmids were expected to be the same, sincethey carry the same CMV promoter. Therefore, we concluded thatthe eE7-IRES-NPT mRNA should have a longer half-life than itsE7-IRES-NPT counterpart, likely because it remains bound topolyribosomes longer, and hence inaccessible to degradation,and/or because the eE7 sequence, with a G+C content exceedingby 20% that of wild-type E7, confers stability on the bicistronicmRNA.

Another parameter that could contribute to the increase in thesteady-state levels of E7 protein could be an extended half-liferesulting from its higher expression levels. However, the half-lifeof E7 in cells transfected with pIneo2-eE7 was estimated tobe close to that described previously for cells expressing thewild-type E7 gene (35) (Fig. 5). Therefore, we concluded thatenhanced translation of the eE7 message should account for thehigh levels of E7 protein that observed.

The results of three series of experiments carried out in thisstudy illustrated the utility of our synthetic eE7 gene. Inthe first series, the eE7 gene was used to assess the intracellularlocalization of the protein (Fig. 4). The main functional propertyof E7 is to deregulate control of cell cycle progression, therebyinducing cells to enter S phase. This is achieved through interactionof E7 with nuclear proteins such as retinoblastoma protein,which suggests its nuclear localization. However, E7 has alsobeen shown to interact with M2-pyruvate kinase (57), suggestingthat it also has cytoplasmic functions. Indeed, E7 has beenlocalized to the cytoplasm (39) but has also been reported tobe in the nucleus (15) and to have a nuclear localization signal(12). We found that E7 localized mainly in the nucleus witha pattern suggestive of its association with the nuclear matrix(Fig. 4), in agreement with previous reports (15). However,in a previous study the E7 protein was found primarily boundto nucleoli (53). This discrepancy may be due to artifactualbinding of the antibody used in that study to nucleoli. Thisview is supported by previous studies by Greenfield et al. (15)and Kanda et al. (25), who observed that epitopes in two immunodominantregions of E7 could not be detected with monoclonal antibodiesunless chromatin was removed or a polyclonal antibody was usedinstead, suggesting that the epitopes were masked by cellularproteins. Furthermore, in a recent study, Guccione et al. (16)described a pattern of expression for a hemagglutinin-taggedE7 that matches the one described here.

The nuclear localization of the E7 ${Delta}$ 1 deletion mutant was somehowunexpected, since it was reported previously that a peptideconsisting of amino acids 16 to 41 of E7 localized to the nucleus(12) and residues 1 to 35 are absent in the E7 ${Delta}$ 1 mutant. However,E7 does not contain any consensus nuclear localization signal,and the mechanisms of its import into the nucleus are stillunknown.

Second, the eE7 gene was used to determine the effect of elevatedlevels of E7 protein on cell viability. Transient-transfectionexperiments showed that the high levels of E7 protein expressedby the eE7 gene eventually caused the death of all transfectedcells after 72 h of transfection. In contrast, lower levelsof protein expressed by wild-type E7 seemed not to cause damagebut rather stimulated cell proliferation (Fig. 6). These observationssuggest that, above a threshold, the ability of the E7 proteinto interfere with essential cellular functions exceeds its capacityto stimulate cell proliferation. It has been shown that expressionof HPV-16 E7 induces apoptosis in human fibroblasts and keratinocytes(1, 20, 44, 51) and in the lens of transgenic mice (22, 33).However, E7 has also been found to inhibit apoptosis inducedby TNF- ${alpha}$ in human fibroblasts (46), and E7-binding peptide aptamersinduced apoptosis in cervical cancer cells (32). According toour results, differences in the level of E7 protein expressedin the various systems used for those studies may provide anexplanation for such discrepancies. Nevertheless, in view ofthe transforming properties of E7 and the relevance of the cellularproteins with which it interacts, the elucidation of mechanismsby which E7 induces cell death is of considerable interest.To this end, the synthetic eE7 gene described here could bevery useful if the appropriate promoters are used for its expression.

In a third use of the eE7 gene in this study, it proved effectivefor DNA immunization studies in mice. The results of the analysisof the humoral response in animals inoculated with the plasmidsdescribed here showed that the eE7 gene had a higher immunogeniccapability than wild-type E7 (Fig. 7). The higher antibody responsein mice injected with plasmids carrying the synthetic eE7 revealedthe importance of ensuring high levels of expression of thetarget protein. Further work should determine the ability ofeE7 to trigger cytotoxic immune responses to E7. Since mucheffort is being applied to the development of vaccines againstoncogenic HPVs, the eE7 synthetic gene described here demonstratesthat codon optimization may be an essential strategy.

Finally, taken together, our data provide support for the hypothesisthat selective pressure has forced HPVs to evolve a codon usagesubstantially different from that of human genes in order tominimize deleterious effects to the host due to excessive expressionof early viral genes, thereby evading the immune system. Whileit may seem contradictory that human viruses have conservedcodon biases distinct from that of their host cells, this maybe the consequence of selective pressure to minimize the pleiotropiceffects of the viral proteins and presentation of viral antigenthat may result in more effective immune response. In orderto prove this hypothesis, these studies need to be extendedto other HPV synthetic genes generated with the same criteriaas the eE7 gene.

ACKNOWLEDGMENTS

We thank A. Aguilar, F. Rösl, and U. Soto for sharing reagentsand stimulating discussions and H. Delius, W. Nicklas, J. Valcárcel,and F. Gebauer for advice and valuable suggestions. We are gratefulto K. Müller for excellent technical assistance and W.Weinig for the synthesis and purification of oligonucleotides.

FOOTNOTES

* Corresponding author. Mailing address: Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany. Phone: 49 6221 424917. Fax: 49 6221 424902. E-mail: cid{at}dkfz.de.

REFERENCES

Top