E6 proteins from diverse papillomaviruses self-associate both in vitro and in vivo

Abstract

Papillomavirus E6 oncoproteins bind and often provoke the degradation of many cellular proteins important for the control of cell proliferation and/or cell death. Structural studies on E6 proteins have long been hindered by the difficulties of obtaining highly concentrated samples of recombinant E6. Here, we show that recombinant E6 proteins from eight human papillomavirus strains and one bovine papillomavirus strain exist as oligomeric and multimeric species. These species were characterized using a variety of biochemical and biophysical techniques, including analytical gel filtration, activity assays, surface plasmon resonance, electron microscopy and Fourier transform infrared spectroscopy. The characterization of E6 oligomers is facilitated by the fusion to the maltose binding protein, which slows the formation of higher-order multimeric species. The proportion of each oligomeric form varies depending on the viral strain considered. Oligomers appear to consist of folded units, which, in the case of high-risk mucosal human papillomavirus E6, retain binding to the ubiquitin ligase E6-associated protein and the capacity to degrade the proapoptotic protein p53. In addition to the small-size oligomers, E6 proteins spontaneously assemble into large organized multimeric structures, a process that is accompanied by a significant increase in the beta-sheet secondary structure content. Finally, co-localisation experiments using E6 equipped with different tags further demonstrate the occurrence of E6 self-association in eukaryotic cells. The ensemble of these data suggests that self-association is a general property of E6 proteins that occurs both in vitro and in vivo and might therefore be functionally relevant.

Figure 1

Detection of soluble MBP-E6 oligomeric species. (A) Analytical gel filtration chromatography analysis. Affinity-purified MBP-E6 samples were cleared from soluble aggregates by ultracentrifugation. Concentrations were adjusted to approximately 2.5 μM and 100 μl volumes injected on a Superdex200 10/30 column. Elution peaks corresponding to monomer (MBP-E6₁, 60 kDa), dimer (MBP-E6₂, 120 kDa) and trimer (MBP-E6₃, 180 kDa) forms of MBP-E6 proteins are indicated. The shoulder at 15 ml corresponds to monomeric MBP arising from residual proteolytic activity in the protein preparations. Molecular size markers are reported on top of the figure. 1: V₀; 2: ferritin (440 kDa); 3: mouse Igg (150 kDa); 4: BSA (67 kDa); 5: ovalbumin (43 kDa); 6: RNase (13.7 kDa). The elution profiles correspond to wt E6 proteins from the following viral stains: HPV11 (magenta line), HPV16 (black line), HPV18 (blue line) and BPV1 (green line). (B) Molecular weight distribution of wt HPV18 MBP-E6 oligomers derived from sedimentation velocity experiments. C(M) indicates arbitrary units.

Figure 2

Gel filtration profiles of HPV11 MBP-E6 4C/4S preparations after separation from the MBP tag. Affinity-purified MBP-E6 preparations were cleared from soluble aggregates by ultracentrifugation and digested using TEV protease. Subsequently, 500 μl were injected on a Superdex75 10/30 column. (A) Peaks corresponding to monomeric (E6₁, 18 kDa), dimeric (E6₂, 36 kDa) and trimeric (E6₃, 54 kDa) E6 forms and MBP (MBP, 44 kDa) are indicated. Molecular size markers are reported on top of the figure. 1: V₀; 2: BSA (67 kDa); 3: ovalbumin (43 kDa); 4: Myoglobin (17.6 kDa); 5: RNase (13.7 kDa). Numbers 1–11 reported on the x-axis indicate fractions collected for SDS-PAGE analysis. (B) Lanes 1–11 correspond to fractions 1–11 indicated on the elution profile. Fractions were precipitated with 20% trichloroacetic acid. Pellets were re-suspended in 50 μl of loading buffer and applied to a 12% SDS-polyacrylamide gel. Bands corresponding to MBP, TEV and E6 are indicated.

Figure 3

Stability of wt HPV11 MBP-E6 oligomers upon dilution. (A) Affinity purified HPV11 MBP-E6 was cleared from soluble aggregates and applied onto a Superdex200 10/30 column. (B–C) Aliquots of fractions corresponding to the monomeric (B), dimeric (C) forms of MBP-E6 (corresponding to the indicated grey areas in (A)) were re-loaded individually onto the same gel filtration column at concentrations of 3.0 (continuous lines) and 0.4 (broken lines) μM. See also legend of Figure 1. (D) Dimer fractions of HPV11 MBP-E6 analyzed on a 12% SDS-PAGE in the presence (+βme) (Lane 1, L1) and in the absence (−βme) (L2) of the reducing agent β-mercaptoethanol. Samples were migrated on the same gel but were separated by two empty wells in order to avoid cross-contamination by β-mercaptoethanol. The migration of the molecular weight markers (in kDa) is reported on the left hand side.

Figure 4

Stability of wt HPV18 MBP-E6 oligomers upon dilution. (A) wt HPV18 MBP-E6 preparations were loaded on a Superdex200 10/30 column as described in Figure 3. Monomer (B) and dimer (C) fractions (indicated by the grey areas) were re-loaded a second time on the same column at concentrations of 3.0 (continuous lines) and 0.4 μM (broken lines). (MBP-E6)_n refer to larger oligomers eluting at the column’s void volume. See also legend of Figure 1. (D) Dimer fractions of HPV18 MBP-E6 analyzed on SDS-PAGE in the presence (+βme) (Lane 1, L1) and in the absence (−βme) (L2) of β-mercaptoethanol.

Figure 5

MBP-E6 oligomeric forms upon concentration. Affinity purified HPV11 (A) and HPV18 (B) MBP-fused wt E6 protein samples were ultracentrifugated to remove soluble aggregates. Aliquots of the supernatants were injected on a Superdex200 10/30 column (continous lines). The remaining samples were concentrated either 5-fold (A) or 2-fold (B) and injected on the same column (broken lines). See also legend of Figure 1.

Figure 6

Characterization of the activities of HPV18 wt E6 oligomers. Oligomeric MBP-E6 forms were fractionated on a Superdex 200 10/60 column. Protein concentrations were determined based on the calculated extinction coefficients of the monomeric forms. (A) Degradation of in vitro translated p53 by wt HPV18 MBP-E6 monomer (lanes (L): 1–4) and dimer (L: 5–8) species. p53 degradation reactions contained the following concentrations of HPV18 MBP-E6: 400 nM (L1 and L5); 200 nM (L2 and L6); 100 nM (L3 and L7); 50 nM (L4 and L8). The histogram indicates the residual p53 signal expressed as percentage relative to the intensity of the input control band (L9). (B) Binding of wt HPV18 MBP-E6 dimer to a peptide containing the LxxφLsh motif of E6AP as monitored by SPR. The figure illustrates the concentration dependent binding of MBP-E6 to approximately 600 RU of affinity-captured GST-E6AP peptide ligand. The MBP-E6 analyte concentrations for each sensorgram are indicated on the figure.

Figure 7

Spontaneous assembly of monomeric E6 and MBP-E6 proteins into ordered multimeric structures visualized by EM. (A) Partial field images of monomeric HPV16 E6 6C/6S taken at 0, 1/2, 3 and 7 days of incubation. Images of monomeric wt MBP-E6 proteins from (B) HPV11, (C) HPV16, (D) HPV18 and (D) BPV-1 viruses after one week incubation. Bar = 50 nm for images in (A) t=0, t=12 hours, t=3 days. Bar = 100 nm for images in (A) t=1 week and in (E). Bar=200 nm for image in (C). Bar = 500 nm for image in (B) and (D).

Figure 8

FTIR characterization of aggregates in samples of monomeric HPV16 E6 6C/6S. Superposition of IR spectra of E6 in buffer containing either 150 mM (A) or 400 mM NaCl (B) recorded at different time points of the incubation.

Figure 9

Detection of wt HPV18 E6 self-association in vivo using nucleo-cytoplasmic delocalization techniques. (A) Images of untransfected H1299 cells. (B–D) Localisation of Flag-E6, NLS-Myc-E6 and NES-Myc-E6 in H1299 cells transiently transfected: with: 0.7 μg of Flag-E6 and 2.3 μg of empty pXj vector (B); 2.3 μg of NLS-Myc-E6 and 0.7 μg of Flag-E6 (C) and 2.3 μg of NES-Myc-E6 and 0.7 μg of Flag-E6 (D).

Figure 10

Distribution of the ratios of nuclear/cytoplasmic Flag-E6 expression in the populations of H1299 transfected cells transfected with Flag-E6 alone (black line), NLS-Myc-E6 and Flag-E6 (green line) and NES-Myc-E6 and Flag-E6 (magenta line). Positions of the mean values for the three distribution curves are indicated by dashed lines. See also legend of Figure 9.