HPV-16 E5 variants: Leu⁴⁴Val⁶⁵ (AJ244882); Thr³⁷Leu⁴⁴Val⁶⁵ (AJ44863); Thr⁶⁴(AJ244840) 24; Tyr⁸Leu⁴⁴Val⁶⁵, (AJ24481); the HPV-16 reference E5 sequence 2526 and HPV-6b E5 were amplified and cloned into pcDNA3.1Myc-His. For each HPV-16 E5 variant, a control construct, containing a TAA 'stop' codon (at codon position three) was also cloned into pcDNA3.1Myc-His.

All constructs and 'stop' controls had the correct DNA sequences after cloning (data not shown). T7 'run-off' transcripts were prepared and translated in cell-free wheat-germ expression assays spiked with ³⁵S-labelled cysteine (all have 4 cysteines). Autoradiographs of polyacrylamide gel elecprophoresis (PAGE) gels revealed equivalent levels of in vitro translation for all HPV-16 variants, but no evidence of protein products from the equivalent 'stop' controls (Figure 3A), confirming the fidelity of TAA ('stop') codons. However, different HPV-16 E5 variants were transcribed at different levels in stably-transfected (G418-selected) NIH-3T3 cell-lines, with Tyr⁸Leu⁴⁴Val⁶⁵ being expressed at higher level than either Thr³⁷Leu⁴⁴Val⁶⁵, or Leu⁴⁴Val⁶⁵, whilst the Thr⁶⁴ variant and reference sequence were transcribed at low level (Figure 3B). To assist data interpretation, cell-lines for all 'stop' constructs were pooled prior to subsequent experiments.

HPV-16 E5 co-operates with EGF to stimulate ³H-thymidine incorporation into DNA of human keratinocytes and murine fibroblasts 10. Here we selected to analyse E5-induced mitosis by cell-cycle profiles as this may provide more detailed information than levels of ³H-thymidine incorporation alone (e.g. Figure 4A). To confirm the validity of this approach we determined whether there was a synergistic relationship between the reference sequence of HPV-16 E5 and EGF. Such a relationship between the EGF and HPV-16 E5 reference sequence was demonstrable (e.g. Figure 4B), in agreement with a previous study using a ³H-thymidine readout 10.

Compared to the 'stop' control, cells transfected with HPV-6b E5 or the HPV-16 E5 Thr⁶⁴ variant did not induce changes in G₀G₁-phase cell percentages (versus 'stop', both p > 0.05: Figure 5). All other HPV-16 E5 variants caused reductions of G₀G₁-phase (all p < 0.05), this effect being greatest for Leu⁴⁴Val⁶⁵ and the reference isolate (respectively, -16.2% & -15.5% c.f. 'stop'). Modest reductions in G₀G₁-phase were observed for the Tyr⁸Leu⁴⁴Val⁶⁵ (-6.1%) and Thr³⁷Leu⁴⁴Val⁶⁵ (-7.4%) variants. The Thr⁶⁴ variant had a similar cell-cycle profile to the 'stop'.

Cells containing HPV-6b E5 or the HPV-16 E5 Thr⁶⁴ variant resembled the 'stop' in their S-phase profiles (both p > 0.05). Increases in S-phase were high for the reference isolate (+ 17.6% c.f. the 'stop') and for the Leu⁴⁴Val⁶⁵ variant (+ 12.2%), but lower for Tyr⁸Leu⁴⁴Val⁶⁵ (+9.4%) and for Thr³⁷Leu⁴⁴Val⁶⁵ (+ 6.3%: all p < 0.05). There were also increases in G₂M-phase percentages for cells containing Leu⁴⁴Val⁶⁵ (+ 5.8%: p < 0.05) or Thr³⁷Leu⁴⁴Val⁶⁵ (+ 2.8%: p > 0.05). Other E5 variants induced insignificant increases of G₂M-phase percentages (Thr⁶⁴ +1.8%; HPV-6b E5, + 1.6%; reference isolate, + 0.6%; and, Tyr⁸Leu⁴⁴Val⁶⁵, + 0.2%: all p > 0.05).

Static analyses of cell-cycle profiles can be difficult to interpret as the percentage values are inter-dependent variables. Thus, different cell populations could have identical cell-cycle profiles, but vastly dissimilar growth rates 2728. This problem was addressed by determining cell-growth at 24, 48 and 72 h in media containing EGF and minimal (2% v/v) serum supplement. Cells transfected with Leu⁴⁴Val⁶⁵ and Thr³⁷Leu⁴⁴Val⁶⁵ variants exhibited most growth (Figure 6: both p < 0.02 versus the 'stop' at 72 h), whilst the HPV-16 E5 reference isolate induced a smaller increase in cell number (p < 0.05 versus the 'stop' at 72 h). Cells containing the Tyr⁸Leu⁴⁴Val⁶⁵ and Thr⁶⁴ variants, or HPV-6b E5 grew slowly (all, p > 0.05 versus the 'stop' at 72 h). Alternate expression of cell-growth as cell-doubling times gave equivalent results (e.g. for: Leu⁴⁴Val⁶⁵ mean doubling time = 56.6 h; Thr³⁷Leu⁴⁴Val⁶⁵ = 49 h; and, Tyr⁸ Leu⁴⁴Val⁶⁵= 118 h).

HPV-16 E5 can repress p21 transcription 29. We thus determined levels of p21 and cyclin B1 proteins in cells containing the HPV-16 reference E5 sequence as well as a variant with a high-growth rate (Leu⁴⁴Val⁶⁵) and one with a low-growth rate (Tyr⁸Leu⁴⁴Val⁶⁵). Cells containing Tyr⁸Leu⁴⁴Val⁶⁵ maintained high levels of p21 protein throughout the time course, whereas p21 was much lower in cells containing the reference isolate and, near undetectable for those containing Leu⁴⁴Val⁶⁵ (Figure 7). There was also a delay before cyclin B1 could be detected in cells containing Tyr⁸Leu⁴⁴Val⁶⁵ as compared to those stably transfected with the other two E5 proteins. These effects were not artefactual as levels of protein loaded were similar as indicated by the β-actin controls.

HPV-16 E5 variants isolated from clinical samples: Ted (Leu⁴⁴Val⁶⁵, EMBL accession number AJ244882), 9785 (Thr³⁷Leu⁴⁴Val⁶⁵, AJ44863) and Twp3 (Thr⁶⁴, AJ244840) 24 were studied. Permission for the collection of clinical specimens was provided by the Research Ethics Committee of St Thomas' Hospital. In addition, E5 DNA from: HPV-16 containing CaSki cells (Tyr⁸Leu⁴⁴Val⁶⁵, AJ24481); the reference isolate of HPV-16 2526; and, from the low cancer-risk virus HPV-6b were investigated.

HPV-16 E5 genes were amplified in polymerase chain reactions (PCR) using rTth™ DNA polymerase in two separate reactions, so that E5 open reading frames (ORF) between nucleotides (nt) 3866 and 4077 (encoding E5 amino acids 6–76) were obtained. The first PCR utilised an upstream primer (³⁸³⁶GGA

GCTAGC

TAA

GGATCC

A cell-free wheat-germ expression assay (Promega Ltd.) was used to determine protein translation of T7 mRNA transcripts with individual reactions supplemented with 5 μl of ³⁵S-labelled cysteine (1 Ci/l: Amersham International Ltd.). Radiolabelled cysteine was selected since all HPV-16 E5 variants contain 4 cysteine residues. Proteins were subjected to PAGE (below) and radioactivity detected by autoradiography.

NIH-3T3 cells (ATCC Ltd.) were maintained in Dulbecco's minimum essential medium supplemented with 40 mM L-glutamine, 2 × 10⁶ U/l benzyl-penicillin, 2 g/l streptomycin sulphate (DMEM) and 10% (v/v) fetal calf serum (DMEM/FCS), in a humidified atmosphere of 5% (v/v) carbon dioxide in air. Cells were transfected with E5 containing plasmids using Lipofectin™ and grown in DMEM/FCS containing 1 g/l of G418 for 3 weeks (a dose cytotoxic for non-transfected NIH-3T3 cells within one week). Individual colonies of G418-selected cells were isolated and expanded in DMEM/FCS containing G418 to produce cell-lines for each variant. Individual cell-lines for each 'stop' construct were pooled. Variant cell-lines and the 'stop pool' were tested intermittently for mycoplasma infections: all were negative. Cell lines were tested for E5 mRNA expression using an adaption of the method described by Biswas et al. 5. Briefly reverse transcriptase (RT) reactions were primed using the HPV-16 E5 downstream primer (⁴¹¹⁰TACAGGATCCTTATGTA ATTAAAAAGCGTGCAT⁴⁰⁷⁸) with Moloney murine leukemia virus reverse transcriptase then samples were subjected to a nested PCR using internal primers located within the E5 ORF 5.

NIH-3T3 cells transfected with HPV-16 variants, HPV-16 'stop', or HPV-6b were seeded at 0.1 × 10⁶ cells in 5 ml of DMEM/FCS (containing no G418) into 25 cm³ flasks. Cells were serum-starved for 24 h in DMEM, media was then replaced with fresh DMEM with (or without) recombinant human EGF (20 μg/l: Boehringer-Mannheim Ltd.) and cells incubated for a further 24 h. Bromo-deoxyuridine (BrdUrd: 10 μM in DMEM) were added and cells washed twice by centrifugation through 10 ml of Dulbecco's phosphate buffered saline (PBS) for 5 min at 200 g at room temperature (rt) and then fixed with 2 ml of ice-cold aqueuous 70% (v/v) ethanol. Fixative was removed and cells incubated in 1 ml of 0.1 M HCl containing 1 g/l pepsin for 12 min at 37°C. Reactions were halted by centrifugation (as above) through, and re-suspension in, PBS. Murine monoclonal anti-BrdUrd IgG₁ heavy and kappa light chains (Becton-Dickenson Ltd: 250 μl per litre of PBS which contained 0.5% [v/v] Tween-20™ and 1% [v/v] FCS) was added for 1 h at rt, cells were washed with PBS and then incubated for 30 min at rt in the dark with fluorosceine-isothiocyanate (FITC) labelled Fab₂ fragments of rabbit anti-mouse immunoglobulin (Dako Ltd.) at rt. After a further wash in PBS cells were re-suspended in, and stained with, 1 ml of propidium iodide/RNAse solution (50 g propidium iodide and 200 g RNAse/l PBS) for 15 min at rt before analysis on a Becton-Dickenson flow cytometer (FACSCalibur™). Data from 10,000 events (i.e. stained cells) were analysed (for a minimum of four times) for each reading after separation of single intact nuclei from debris and cell-clumps by gating on an FL2 area/width plot. Cells were then characterised on the basis of detection of green (FITC) and red (propidium iodide) fluorescence.

Cells were grown in 2% (v/v) in DMEM containing 2% (v/v) fetal calf serum, supplemented with EGF (20 μg/l), after seeding at a density of 0.1 × 10⁶ cells in 20 cm³ Petri dishes. Cells were fed daily with fresh media containing EGF and cultures harvested at 24, 48 and 72 h by detachment from Petri dishes by exposure to Versene (5 ml for 2 min at 37°C). One millilitre of DMEM/FCS was added to inactivate the trypsin then cells were pelleted by centrifugation (200 g for 15 min at rt). Cell pellets were re-suspended in DMEM/FCS and viable cell numbers determined immediately by trypan-blue exclusion staining 38.

NIH-3T3 cells (0.1 × 10⁶ cells in 5 ml of DMEM/FCS containing no G418) into 20 cm³ Petri dishes and left overnight. Cells were serum-starved for 24 h in DMEM, grown in DMEM supplemented with EGF (as above), then harvested over an 18 h period. Cells were lysed by suspension in RIPA buffer and cellular debris removed by centrifugation at 10,000 g for 30 sec at rt. Protein concentrations were determined using a commercial assay (BioRad Ltd.) and adjusted to enable 5 μg of total protein to be added to each well of a PAGE gel. Cellular proteins were subjected to PAGE under reducing conditions through a 4–12% Tris/glycine gradient gel (Novex Ltd.) and blotted onto Hybond-P™ membranes (Amersham International Ltd.). Each blot was next cut into strips containing the proteins of interest: cyclin B1 (60 kDa), β-actin (42 kDa) and p21 (21 kDa). Strips were blocked overnight at rt using 5% (w/v) dried milk powder (Marvel™, Premier Brands UK Ltd.) in Tris-buffered saline/Tween™ (20 mM Tris [hydroxymethyl]-amino methane; 0.2 M sodium chloride and 0.1% [v/v] Tween-20™, pH 7.5: TBS-T).

Blot strips were washed three times with TBS-T then incubated for 1 h at rt with rabbit anti-β-actin (1/1000 dilution), mouse anti-p21 (2.5 mg/l) or, mouse anti-cyclin B1 (2 mg/l: Pharminogen Ltd.) antibodies. After two washes in TBS-T strips were immersed in the appropriate horse-radish peroxidase-labelled secondary antibody (sheep anti-mouse immunoglobulin or donkey anti-rabbit immunoglobulin: Amersham International Ltd. each at a dilution of 1/250) for 1 h at rt. Bound antibody was detected using an ECL-plus™ chemiluminescence kit and ECL Hyperfilm™ (Amersham International Ltd.).

Students' t-tests were used to assist the interpretation of data.