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. 2011 Oct;85(19):10048-57.
doi: 10.1128/JVI.00643-11. Epub 2011 Jul 20.

Systematic analysis of the amino acid residues of human papillomavirus type 16 E7 conserved region 3 involved in dimerization and transformation

Biljana Todorovic  1 , Paola Massimi, Katherine Hung, Gary S Shaw, Lawrence Banks, Joe S Mymryk
Affiliations

Systematic analysis of the amino acid residues of human papillomavirus type 16 E7 conserved region 3 involved in dimerization and transformation

Biljana Todorovic et al. J Virol. 2011 Oct.

Abstract

The human papillomavirus (HPV) E7 oncoprotein exists as a dimer and acts by binding to many cellular factors, preventing or retargeting their function and thereby making the infected cell conducive for viral replication. Dimerization of E7 is attributed primarily to the C-terminal domain, referred to as conserved region 3 (CR3). CR3 is highly structured and is necessary for E7's transformation ability. It is also required for binding of numerous E7 cellular targets. To systematically analyze the molecular mechanisms by which HPV16 E7 CR3 contributes to carcinogenesis, we created a comprehensive panel of mutations in residues predicted to be exposed on the surface of CR3. We analyzed our novel collection of mutants, as well as mutants targeting predicted hydrophobic core residues of the dimer, for the ability to dimerize. The same set of mutants was also assessed functionally for transformation capability in a baby rat kidney cell assay in conjugation with activated ras. We show that some mutants of HPV16 E7 CR3 failed to dimerize yet were still able to transform baby rat kidney cells. Our results identify several novel E7 mutants that abrogate transformation and also indicate that E7 does not need to exist as a stable dimer in order to transform cells.

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Figures

Fig. 1.
Fig. 1.
(a) Multiple sequence alignment of CR3 regions from representative supergroup A HPVs. Perfectly conserved residues are highlighted in black, with less conserved residues shaded in progressively lighter shades of gray, corresponding to the degree of conservation. The numbering scheme corresponds to HPV16 E7 CR3, starting with residue 49. Residues that are involved in zinc coordination are indicated with asterisks at the bottom of the alignment. Highly conserved hydrophobic core residues which are predicted to stabilize the dimer are labeled with ovals. The panel of mutations created for the purposes of the described studies is labeled at the top and bottom of the alignment. (b and c) Ribbon diagrams of the HPV16 model showing the positions of substitutions introduced into the protein. The model was produced using the PDB coordinates of HPV45 (2F8B) and a sequence alignment between the two proteins. The figures show the dimeric HPV16 model (a) and a view across a portion of the dimerization interface (b). The surface-exposed side chains where substitutions were made are shown as sticks (pink), and the bound zinc atoms are indicated as spheres (cyan). The two protomers are shaded differently, and substitutions are shown on only one protomer for clarity.
Fig. 2.
Fig. 2.
E7 dimerization in vivo. (a) Controls for the yeast two-hybrid assay. L-40 yeast cells were cotransformed with a LexA DNA binding domain (bait) and a B42 activation domain (prey). The combination of vectors (bait/prey) used is indicated below each data bar, with “vector” representing an empty vector and “WT” indicating a vector expressing a wild-type HPV16 E7 fusion. Indicated constructs were expressed in L-40 yeast cells containing an integrated LexA-responsive β-galactosidase reporter. Cell extracts were prepared and assayed for β-galactosidase activity. (b) Dimerization of mutants with mutations targeting predicted surface-exposed residues as determined by the yeast two-hybrid assay. HPV16 E7 mutants were expressed as fusions to the LexA DNA binding domain and the B42 activation domain in L-40 yeast cells, and β-galactosidase activity was assessed. The upper horizontal line is set at 45% of wild-type activity, corresponding to a predicted 5-fold increase in Kd; the lower horizontal line corresponds to a predicted 10-fold increase in Kd and is set at 28% of wild-type activity. We considered any mutation with <45% of wild-type activity to be impaired for dimerization. The same criterion also applies to panel c. (c) Dimerization of mutants with mutations targeting predicted hydrophobic core residues and zinc-coordinating cysteines as assessed by the yeast two-hybrid assay. (d) Expression levels of LexA and B42 fusion proteins of dimerization-impaired E7 mutants. Yeast extracts cotransformed with either wild-type E7 or the E7 mutant as both LexA (bait) and B42 (prey) fusions were tested for protein expression by Western blotting using anti-LexA and anti-HA antibodies. An anti-G6PD blot served as a loading control.
Fig. 3.
Fig. 3.
Dimerization of E7 CR3 in vitro. Predicted surface-exposed mutants identified in the yeast two-hybrid assay as impaired for dimerization (>55% decrease in activity) were tested for the ability to form dimers in vitro. Both E7 CR3 fused to GST and that cleaved with TEV protease to obtain E7 CR3 alone were purified. A 300 nM concentration of GST-tagged wild-type or mutant E7 CR3 was mixed with 600 nM E7 CR3. The ability of the wild type or mutant to dimerize was assessed by determining the amount of recovered E7 CR3 in a GST pulldown assay. The proteins were resolved by SDS-PAGE, and the amount of E7 CR3 was determined by Western blotting. The bar graph shows band intensities for the representative Western blot, as determined by densitometry. (b) Coomassie blue-stained gels of purified GST-E7 CR3 (top) and TEV-cleaved E7 CR3 (bottom), used as a loading control for the in vitro dimerization assay.
Fig. 4.
Fig. 4.
Further characterization of a select panel of mutants. (a) E7 mutants with decreased transformation potential. The M84S and QKP96-98EEA mutants, as well as the CR2 del21-24 deletion mutant, which targets the LXCXE motif, were assessed for the ability to target pRb for degradation. Saos2 cells were transfected with a pRb expression plasmid and vectors expressing the indicated E7 mutant; pRb levels were determined by Western blotting at 48 h posttransfection. (b) The ability of E7 mutants to bind pRb was also assessed in a yeast two-hybrid assay. pRb was expressed as bait, with the indicated E7 mutant as prey. β-Galactosidase activity was measured and reported relative to that of wild-type E7. (c) The L67R mutant, with a mutation in the hydrophobic core of E7 which leads to decreased dimerization, and the QKP96-98EEA mutant, which dimerizes but fails to transform cells, were transfected into HEK293 cells together with a GFP expression plasmid. At 24 h posttransfection, cells were treated with cycloheximide for the indicated times and the levels of E7 determined by Western blotting. (d) Cellular localization of M84S and QKP96-98EEA mutants was assessed by immunofluorescence in U2OS cells.
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