Construction and characterisation of a recombinant fowlpox virus that expresses the human papilloma virus L1 protein

CaSki cells carrying multiple copies of integrated HPV-16 DNA, green monkey kidney (Vero) cells, and MRC-5 human lung fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated calf serum (Gibco/Invitrogen, Carlsbad, CA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (P/S). Specific-pathogen-free primary chick embryo fibroblasts (CEFs) were grown in DMEM with 5% heat-inactivated calf serum, 5% tryptose phosphate broth (TPB; Difco Laboratories, Detroit, MI, USA), and P/S.

The pQE30 expression plasmid (Qiagen, Valencia, CA, USA) engineered to contain the L1 gene of HPV-16 [30] (pQE30-L1-NLS-His) was kindly supplied by C. Giorgi (Istituto Superiore di Sanità, Rome, Italy). After its cloning into JM109 bacterial cells, this engineered plasmid was used for the production of the RGS/L1/His-tagged protein, according to the manufacturer instructions (Qiagen), with minor modifications. Briefly, JM109/pQE30-L1-NLS-His bacterial cells were lysed in phosphate lysis buffer (300 mM NaCl, 1% Triton X-100, pH 8, prepared in buffer A: 10 mM Tris, 100 mM Na₂HPO₄, 6 M guanidine-HCl, pH 8). After clarification for 30 min at 17,000 × g at 4°C, the supernatant containing the HPV-L1 preparation was supplemented with 1% Triton X-100/20 mM imidazole, pH 8, in buffer A. This was then incubated with Ni-NTA agarose resin (Qiagen) for 30 min at room temperature. After washing once with 1% Triton X-100 in buffer A, twice with buffer A, and multiple times with buffer C (100 mM Na₂HPO₄, 10 mM Tris, 8 M Urea, pH 6.3) up to a final optical density of 0.013, the protein was eluted into different fractions with 1 M imidazole, pH 8. After their analysis by 15% PAGE, the fractions enriched in the recombinant HPV-L1 were pooled. The HPV-L1 was dialysed overnight at 4°C using a slide-A-lyser cassette (10 kDa MW cut-off, Pierce, Rockford, IL, USA), and soaked in dialysis buffer (25 mM Tris-HCl, 100 mM NaCl). The protein was quantified, stored at -80°C and used as a control in the Western blotting.

The L1 gene of HPV-16 was amplified from the pUF3/L1 plasmid that contained the humanised HPV-L1 gene sequence (1,518 bp; accession number: AJ313179) [31] kindly supplied by M. Mueller (German Cancer Research Center, Heidelberg, Germany). This was inserted into the multicloning site (MCS) downstream of the early/late VVH6 promoter [32, 33] of the pFP-MCS-GFP plasmid. The enhanced GFP (green fluorescent protein) gene derived from pEGFP-N1 (BD Biosciences Clontech Laboratories, Inc., Mountain View, CA, USA) is therefore located downstream of the L1 gene, but in the reverse orientation and under the control of the synthetic SP promoter [34] (a gift from A. Siccardi, HSR, Milan, Italy). The L1 and GFP genes are both inside the two arms of the 3-β-hydroxysteroid dehydrogenase 5-delta 4 isomerase gene (DH) fowlpox gene, used for site-specific in-vitro recombination [35]. The DNA sequence encoding the complete L1 region of HPV-16 was amplified using the V178 (5'-GCC-GCG-CCC-GGG-AAG-CTT-ATG-AGC-CTG-TGG-CTG-CCC-AGC-GAG-3') and V179 (5'-GCC-GCG-GTC-GAC-AAG-CTT-TCA-CAG-CTT-CCT-CTT-CTT-CC-3') primers. The amplification was carried out starting from 250 ng DNA in a final volume of 50 μl, in a mixture containing 1 μM of each primer, 200 μM of each dNTP, 3 μM MgCl₂, 2.0 mM MgSO₄, and 0.025 U/μl Pwo DNA polymerase (Boehringer Mannheim, Indianapolis, IN, USA). The PCR conditions were: 95°C for 45 s, followed by 30 cycles at 95°C for 30 s, 70°C for 30 s, 72°C for 80 s, and 72°C for 7 min (PTC-200 thermocycler; MJ Research, Waltham, MA, USA). The HPV-L1 gene was first cloned into the pCR-BluntII-TOPO plasmid (Invitrogen, Carlsbad, CA, USA), which allows the insertion of blunt-ended sequences. The pCR-BluntII-TOPO/L1 was then cut out with HindIII (Fermentas, M-Medical, Milan, Italy) and ligated into the pFP recombinant vector, which had been previously linearised with HindIII for 1 h at 37°C. Ligation was carried out overnight at 16°C in 10 μl, using 1 U T4 DNA ligase (USB, Amersham-Pharmacia Biotech AB, Uppsala, Sweden) and 100 ng of the insert at an insert:vector molar ratio of 3:1. After transformation of supercompetent ECL bacterial cells, the colonies were screened by amplifying the HPV-L1 gene, and further analysed by digestion with the NruI/KpnI and HindIII/EcoRI restriction enzymes, to verify the correct orientation of the insert. The cloned recombinant plasmid was purified (Qiagen, Hilden, Germany) and sequenced (Genenco, MMedical, Milan, Italy) to exclude any possible mutation arising from PCR amplification. This cloned recombinant plasmid is henceforth referred to as pFP_L1 (9,726 bp).

The FP_L1 recombinant was obtained by in-vitro recombination in specific-pathogen-free primary CEFs, as described previously [35, 36], using the wild-type FP virus (FPwt) and pFP_L1. Briefly, the CEFs were infected with 0.5 PFU/cell FPwt in DMEM containing 2% foetal calf serum, and, after a 4-h incubation at 37°C, they were transfected by calcium phosphate precipitation using 125 μg pFP_L1 DNA in 1 ml of a mixture containing 125 μM CaCl₂ in 40 mM HEBS, pH 7, 300 mM NaCl, 1.4 mM Na₂HPO₄, 10 mM KCl, and 12 mM dextrose. Two days after the infection, the FP virus was released from the cells by three freeze-thaw cycles and used for further infection and selection of the recombinants. Recombinant plaques were identified by autoradiography after hybridisation with a [³²P]-labelled HPV-L1 probe, and then subjected to multiple cycles of plaque purification. One clone was selected for correct and highest expression of the HPV-L1 gene by Western blotting. The FP_L1 recombinant was amplified in CEFs and purified on a sucrose gradient, as described previously [33].

The expression of the HPV-L1 gene was first determined by RT-PCR, after infecting Vero cells with 5 PFU of FP_L1 recombinant. The experiments were performed in duplicate. RNA was extracted 1 day post-infection (p.i.) and every 3 days for 21 days, using Trizol LS (Gibco) to determine the level of HPV-L1 transcripts in non-permissive mammalian cells. The mRNAs from all of the samples were treated with 10 U RNase-free DNase I (Roche Diagnostics, Indianapolis, IN, USA) for 4 h at 37°C, to eliminate any cellular or viral DNA, then precipitated with 100% ethanol in the presence of 100 mM Na acetate. After washing in 75% ethanol and resuspension in diethylpyrocarbonate-treated water, RT-PCR was carried out using the Access RT-PCR System kit (Promega, Madison, WI, USA). Briefly, 50 ng RNA from each sample was used in a final volume of 20 μl, in the presence of 1 mM of each primer, 200 mM of each dNTP, 0.1 U/ml Tfl DNA polymerase, 0.1 U/ml AMV reverse transcriptase (AMV-RT), and 2.0 mM MgSO₄. Control samples without AMV-RT were also prepared. The presence of the HPV-L1 transcript was verified using the V191 (5'-CGA-CAC-CAG-CTT-CTA-CAA-C-3') and V190 (5'-TGT-TGA-ACA-GGT-GCC-TC-3') primers. Human β-actin was also amplified as an internal control, using the V84 (5'-CTG-ACT-ACC-TCA-TGA-AGA-TCC-T-3') and V85 (5'-GCT-GAT-CCA-CAT-CTG-CTG-GAA-3') primers, and a band of 518 bp was obtained. Mock-infected Vero cells were used as a negative control. The reverse transcriptase reaction was carried out at 48°C for 45 min, followed by 2 min at 94°C. PCR amplification was performed for 35 cycles at 94°C for 30 s, 60°C for 30 s, and 68°C for 1 min, followed by a final incubation at 68°C for 7 min. For human β-actin, the RNA (50 ng) amplification was carried out as described above, but with 1 mM MgSO₄. The thermal profile was at 48°C for 45 min followed by 94°C for 2 min and 40 cycles at 94°C for 30 s, 58°C for 30 s, 68°C for 1 min, and with a final extension of 68°C for 7 min. The PCR products were run on 1% agarose gels and the gel images were acquired using a Speedlight Platinum apparatus (Lightools Research, Encinitas, CA, USA). The RT-PCR products were quantified using the ImageJ software [37].

The cells were infected with 5 PFU/ml in DME methionine-, cystine-, and L-glutamine-free medium (DMEM met^-, cys^-, glut^-, MP Biomedicals Inc, DBA, Milan, Italy). After 2 h, 20 μCi/ml [³⁵S]-methionine and [³⁵S]-cysteine were added, using the same medium supplemented with 2% dialysed foetal bovine serum (Gibco). Sixteen hours p.i., the cells in 2 ml of medium were harvested by resuspending them in 1 ml lysis buffer (150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4, 0.2 mg/ml PMSF, 1% NP40, 0.01% sodium azide) and 0.6 TIU aprotinin (Sigma, St Louis, MO, USA) per Petri dish, with scraping into microcentrifuge tubes (Eppendorf, Milan, Italy). The lysate was clarified by centrifugation at 9,000 × g for 20 min at 4°C. Immunoprecipitation was performed either with 2 μl anti-HPV-L1 preadsorbed polyclonal mouse serum (C. Giorgi) or with a monoclonal antibody (CamVir-1; BD Biosciences, San Diego, CA, USA). Proteins were resolved using 12.5% SDS-PAGE, and fluorographed.

To determine whether HPV-L1 was expressed correctly by the FP_L1 recombinant, Vero cells were infected with 5 PFU/cell FP_L1 recombinant and examined by Western blotting. The pelleted cells were lysed in sample buffer (50 mM Tris, pH 6.8, 10% SDS, 1.5% dithiothreitol, 0.05% bromophenol blue), boiled for 5 min, and loaded onto polyacrylamide gels. After running through a 4% stacking gel for 1 h at 20 mA and through a 15% running gel for 1.5 h at 40 mA, the proteins were transferred onto 0.2 μm nitrocellulose membranes (Bio-Rad Laboratories, Milan, Italy) for 1 h at 100 V in cold transfer buffer (150 mM glycine, 20 mM Tris, 20% methanol) using a transblot apparatus (Bio-Rad). Soon after the protein transfer to the nitrocellulose, the positions of protein markers (Precision Plus Protein™ All blue standards, Bio-Rad) (5 μl/gel) were marked on the nitrocellulose. The nitrocellulose membranes were incubated for 30 min in 0.5% glutaraldehyde (Polysciences, Inc., Warrington, PA, USA), washed in H₂O, and incubated for 1 h with 5% skimmed milk (Merck, West Point, PA, USA) in Ca²⁺-free and Mg²⁺-free phosphate-buffered saline (PBS^-). After three rinses in wash buffer (20% Tween in PBS^-), the nitrocellulose membrane was incubated overnight at 4°C with a 1:5,000 dilution of the specific anti-HPV-L1 CamVir-1 antibody (BD Biosciences). The washing was followed by a 1-h incubation with a goat anti-mouse horseradish peroxidase antibody (1:10,000 dilution; Dako Cytomation), and 2-h washes, before the HPV-L1 was revealed using the ECL system (GE Healthcare, Buckinghamshire, UK). The densitometric analysis of the Western blotting bands of the L1 protein was performed using the ImageJ software [37].

The expression of HPV-L1 was also examined by immunofluorescence. Vero cells were seeded at a density of 5 ×10⁵/35-mm² dish on sterile glass coverslips, before infection with 5 PFU/cell FP_L1 recombinant at 37°C for 1 h. After a 6-h incubation at 37°C in DMEM supplemented with 2% foetal calf serum and P/S, the cells were washed twice with PBS^-, fixed with 2% paraformaldehyde (Polysciences) in PBS^- for 10 min at room temperature, followed by 100% cold acetone for 5 min at 4°C. The samples were incubated with either monoclonal CamVir-1 (BD Biosciences, 1:100) or mouse (C. Giorgi, 1:50) or rabbit (R20, from our laboratory, 1:500) polyclonal anti-HPV-L1 antibodies, followed by a 1:100 FITC antiserum (Cappel, MP Biomedicals, Inc., Aurora, OH, USA). The R20 antibodies were obtained by multiple inoculations of one rabbit with the FP_L1 recombinant. Before use, serum from R20 was immmunoadsorbed overnight with FPwt-infected Vero cells to remove antibodies raised against the vector. FPwt-infected Vero cells were used as a negative control, while CaSki and HeLa cells were used as positive controls. Samples were viewed under a Zeiss Axioskop fluorescence microscope.

Confluent Vero, MRC-5, and CEFs cells were infected with the FP_L1 recombinant and FPwt at 5 PFU/cell for 1 h at 37°C. On days 1 and 3 p.i., the cells were harvested for transmission electron microscopy (TEM). After centrifugation at 1,000 × g for 10 min at room temperature, the cells were fixed in 2.5% glutaraldehyde (Polysciences) in 0.1 M Na cacodylate buffer, pH 7.4, for 1 h at 4°C. They were then rinsed twice, and post-fixed in cacodylate-buffered 1% OsO₄ at 4°C for 1 h. The specimens were dehydrated through a series of graded ethanol solutions and propylene oxide, and embedded in Poly/Bed 812 resin mixture. Ultrathin sections were obtained using a Sorvall MT2B ultramicrotome equipped with a diamond knife, and they were stained with water-saturated uranyl acetate and 0.4% lead citrate in 0.1 M NaOH. VLPs from the commercial HPV Gardasil^® vaccine were negative-stained with water-saturated uranyl-acetate and used as a control. The specimens were viewed with a Philips CM10 electron microscope.

RNA was extracted from Vero cells infected with 2 PFU/cell FP_L1 recombinant, and harvested on days 1, 2, 7, 10, 14, 18 and 22 p.i.. The cells were rinsed twice with PBS^-, scraped from the Petri dishes with a rubber policeman, and centrifuged at 1,500 × g for 5 min. After lysis, the RNAs were extracted using the RNeasy mini kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer instructions. Reverse-transcriptase reactions were performed in a final volume of 50 μl, using 3 μg RNA and the High Capacity cDNA Archive kit (PE Applied Biosystems, Foster City, CA, USA). The reactions were performed at 25°C for 10 min, followed by 48°C for 30 min, and 95°C for 5 min. The cDNA (5 μl) was then added to each well of a MicroAmp Optical 96-well reaction plate (Applera, PE Applied Biosystems) in the presence of 2× Power SYBRA green master mix, and forward V392 (5'-GGA-GTA-CGA-CCT-GCA-GTT-CAT-CT-3') and reverse V393 (5'-CTG-CGT-TCA-TCG-TGT-GGA-TGT-3') primers to identify the HPV-L1 gene, in a final volume of 25 μl. The primers V385 (5'-AGC-AAA-GAC-CCC-AAC-GAG-AA-3') and V386 (5'-GGC-GGC-GGT-CAC-GAA-3') were used for the GFP gene, V380 (5'-TGG-AGA-TGA-ACT-CGG-ACC-TC-3') and V381 (5'-CGA-CCA-CCA-CCA-ACT-TCA-A-3') for the housekeeping RPS7 human gene. FPwt-infected Vero cells and Vero cells transfected for 24 h with the pUF3/HPV-L1 plasmid (kindly supplied by M. Mueller, German Cancer Research Centre, Heidelberg, Germany) or the pEGFP plasmid were used as negative and positive controls, respectively. All of the reactions were performed in an ABI PRISM 7700 apparatus (PE Applied Biosystems). The PCR conditions were 50°C for 2 min, 95°C for 15 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. A 20-min dissociation protocol was also applied. Two different analyses were performed and the copy numbers of the HPV-L1 and GFP transcripts were calculated using the comparative C_t method (also known as the 2^{-[delta] [delta]Ct} method), where [delta] C_{t, sample} = C_t,L1 - C_{t, RPS7}, [delta] C_{t, sample} is the C_t value for any sample normalised to the RPS7 endogenous housekeeping transcripts and [delta] [delta]C_t = [delta]C_{t, sample} - [delta]C_{t, wt}. In all of the samples, the 2^{-[delta] [delta]Ct} refers to an N-fold increase of HPV-L1/GFP copy number relative to the control.

The expression of the HPV-L1 transgene after infection by the FP_L1 recombinant was tested over time by RT-PCR. The mRNA isolated from infected Vero cells showed that the gene carried by the FP_L1 recombinant amplified as a band of 530 bp (Figure 1). In particular, HPV-L1 transgene expression was detectable from 3 days p.i., and extended up to day 21 p.i.. The expression levels were determined by densitometric analysis, and they showed that a very high level of expression was maintained up to day 9 p.i., which then constantly decreased. The similar levels of amplification of β-actin RNA (518 bp) over time confirmed the reliability of these differences in HPV-L1 expression. Mock-infected cells were always negative, as were cells infected with FPwt and those amplified in the absence of AMV-RT (data not shown).

To determine whether HPV-L1 transcripts were followed by the synthesis of the corresponding viral proteins, immunoprecipitation was performed on Vero, MRC-5, and CEFs cells (Figure 2A). HPV-L1 was detected only in MRC-5 cells (Figure 2A, lane 6), using either monoclonal or rabbit polyclonal antibodies. In the FP_L1-infected Vero cells, protein expression was not detectable (Figure 2A, lane 3) as well as in CEFs (data not shown), and in mock-infected (Figure 2A, lanes 1 and 4) and FPwt-infected (Figure 2A, lanes 2 and 5) cells. These data prompted us to confirm these results by Western blotting with the same cell lines (Figure 2B). The results showed expression of HPV-L1 not only in MRC-5 cells (Figure 2B, lane 5) but also in Vero cells infected by the FP_L1 recombinant (Figure 2B, lanes 2, 55-58 kDa). The densitometric analysis of the Western blotting bands of the L1 protein produced by the FP_L1 recombinant was compared to the band of a known amount of purified L1 protein used as a standard; this showed that 10⁷ Vero and MRC-5 cells produced 42 ng and 52 ng of protein, respectively. HPV-L1 produced by an engineered bacterial vector was used as a molecular-weight marker (Figure 2B, lane 6) and CaSki cells as a positive control (Figure 2B, lane 3). No specific bands were seen when Vero cells were infected with FPwt (Figure 2B, lanes 1 and 4) or in replication-permissive CEFs (data not shown). It is worth noting that although different monoclonal and polyclonal antibodies were used to determine the expression of HPV-L1, only the monoclonal BD antibody showed specificity and high avidity in all of the different tests.

To determine the localisation of HPV-L1, immunofluorescence (Figure 3) was performed on Vero, MRC-5, and CEFs cells infected with the FP_L1 recombinant. In all these different cell types, which were either non-permissive (Vero and MRC-5, Figure 3, 1b, 2b) or permissive (CEFs, Figure 3b) for replication, immunofluorescence was detected at the nuclear level using monoclonal or polyclonal antibodies. No fluorescence was seen in the respective different cell types infected with FPwt (Figure 3, 1a, 2a, 3a). As expected, CaSki and HeLa cells used as positive controls showed clear nuclear immunofluorescence (data not shown).

Structural capsid proteins can self-assemble into VLPs spontaneously. Non-permissive Vero (Figure 4A) and MRC-5 (Figure 4B) cells, as well as permissive CEFs (not shown), were infected with the FP_L1 recombinant, and VLP formation was examined at the ultrastructural level by electron microscopy. In both Vero and MRC-5 cells, FP_L1 recombinant virions were seen attached to the cell membrane (data not shown). Complete VLPs (T7, 72 pentamers) were seen in Vero cells at different times p.i., although in limited amounts and mainly enclosed in pseudo-vacuoles (Figure 4A). These pseudo-vacuoles seem to be the result of tangential sections of invaginations of the cell membrane, as shown by the unstructured background characteristics of the extracellular environment. In MRC-5 cells, only unassembled particles (T1, 12 pentamers) were seen in the nucleus (Figure 4B), whereas they could not be detected in FP_L1-infected CEFs (data not shown).

To determine whether the limited amount of VLPs can be ascribed to the interaction of the HPV-L1 and GFP genes driven by the VVH6 and SP promoters, respectively, a quantitative comparison between the HPV-L1 and GFP transcripts was performed over 3 weeks by real-time PCR at different days p.i. (Figure 5). The GFP mRNAs were expressed over time at a good level as well as the HPV-L1 transcripts, reaching their maximum expression level on day 7 p.i..