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

Abstract

A synthetic E7 gene of human papillomavirus (HPV) type 16 was generated that consists entirely of preferred human codons. Expression analysis of the synthetic E7 gene in human and animal cells showed levels of E7 protein 20- to 100-fold higher than those obtained with wild-type E7. Enhanced expression of E7 protein resulted from highly efficient translation, as well as increased stability of the E7 mRNA due to its codon optimization. Higher levels of E7 protein in cells transfected with synthetic E7 correlated with significant loss of cell viability in various human cell lines. In contrast, lower E7 protein expression driven by the wild-type gene resulted in a slight induction of cell proliferation. Furthermore, mice inoculated with plasmids expressing the synthetic E7 gene produced significantly higher levels of E7 antibodies than littermates injected with wild-type E7, suggesting that synthetic E7 may be useful for DNA immunization studies and the development of genetic vaccines against HPV-16. In view of these results, we hypothesize that HPVs may have retained a pattern of G + C content and codon usage distinct from that of their host cells in response to selective pressure. Thus, the nonhuman codon bias may have been conserved by HPVs to prevent compromising viability of the host cells by excessive viral early protein expression, as well as to evade the immune system.

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.

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 eE7Δ1 and E7Δ1, 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.

FIG. 3.

Expression of HPV-16 E7 and NPT mRNA and protein. HeLa cells growing on 6-cm plates (3 × 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 [α-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.

FIG. 4.

Immunofluorescence analysis of HPV-16 E7 expression in Vero cells transfected with plasmids expressing synthetic (eE7 and eE7Δ1) or wild-type (E7 and E7Δ1) 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 63× 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 (eE7Δ1) 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 (eE7Δ1), 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.

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 β-lactone (β-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.

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.

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.