Identification of peptides that inhibit the DNA binding, trans-activator, and DNA replication functions of the human papillomavirus type 11 E2 protein

Abstract

Peptide antagonists of the human papillomavirus type 11 (HPV-11) E2-DNA association were identified using a filamentous bacteriophage random peptide library. Synthetic peptides antagonized the E2-DNA interaction, effectively blocked E2-mediated transcriptional activation of a reporter gene in cell culture, and inhibited E1-E2-mediated HPV-11 DNA replication in vitro. These peptides may prove to be useful tools for characterizing E2 function and for exploring the effectiveness of E2-inhibitor-based treatments for HPV-associated diseases.

FIG. 1.

Comparison of synthetic peptides by TRF. IC₅₀ results are reported in Table 1. One microgram of anti-E2 monoclonal antibody 93.2a12 (anti-E2 antibody generated at GlaxoSmithKline) was added to each well in a 96-well plate and incubated at 4°C overnight. Wells were blocked with 1% milk in Tris-buffered saline and then washed with TBSDM buffer. Peptide samples were serially diluted in TBSDM containing 10 nM FL-E2 and incubated at room temperature for 30 min. Annealed oligonucleotides were end labeled for 30 min at room temperature in the presence of 16 nM (each) dATP, dTTP, and dGTP plus 16 nM Eu-dCTP and 0.017 U of Klenow enzyme/μl. Eu-DNA (100 pM) was then added to each FL-E2 sample and incubated at room temperature for an additional 30 min. The Eu-DNA-FL-E2 reaction mixtures were transferred into the monoclonal antibody-coated wells and incubated at room temperature for 1 h. Each well was then washed, and 100 μl of TRF enhancement solution (EG & G Wallac, Gaithersburg, Md.) was added and incubated for 10 min at room temperature. Bound Eu-DNA fluorescence was then determined on a Victor 1420 multiple label counter (EG & G Wallac). Data from three independent experiments were analyzed with the equation f = V_max{1 − [x/(K + x)]}, where f is the signal from bound Eu-DNA, V_max is the maximum signal, x is the peptide concentration, and K is the IC₅₀ of synthetic peptides for inhibition of the FL-E2-Eu-DNA interaction.

FIG. 2.

Inhibition of E2-mediated reporter gene transcription by 6N40D peptide expressed in Vero cells. (A) Four tandem repeats of the E2-binding-site palindromes (nucleotides 28 to 66 within the HPV-11 origin of replication) were inserted upstream of the minimal simian virus 40 (SV40) promoter and sequence encoding a secreted form of human placental alkaline phosphatase to create the reporter plasmid pSEAP-2. (B) An HPV-11 E2 expression plasmid, pFastBac-E2, was generated by inserting the open reading frame sequence for full-length HPV-11 E2 into pFastBac-EF1αP. Expression from this vector is directed from the EF1α promoter. (C) A DNA cassette encoding the 6N40D E2 inhibitor peptide and a nuclear localization signal peptide (NLS) was inserted into the enhanced GFP fusion vector, pEGFP-N1 (Clontech), and then subcloned into the pFastBac-MAM-1 expression vector, yielding the baculovirus expression construct pFastBac-MAM/6N40D. pFastBac-MAM/6N40D derivative vectors were made which contained the NLS-GFP fusion with double Cys→Ser mutated 6N40D peptide sequence (pFastBac-MAM/6N40DCys→Ser) or the NLS-GFP fusion with a scrambled 6N40D peptide sequence (NH₂-SCGDEHMGLECGWVGF-CONH₂) (pFastBac-MAM/scrambled). CMV, cytomegalovirus. (D) To test the effects of 6N40D peptides on E2-mediated transcription control in vivo, Vero cells were transiently transfected with 0.125 μg of the reporter plasmid pSEAP-2 and/or 0.5 μg of the E2 transcription activator plasmid pFastBac-E2. The transfected cells were inoculated 24 h later with baculoviruses that expressed 6N40D, double Cys→Ser mutated 6N40D, or scrambled 6N40D-NLS-GFP fusion proteins, according to the bottom panel. Medium supernatant (50 μl) was assayed for SEAP activity 48 h after the transductions. Fold induction represents SEAP activity in Vero cells with mock-transfected cells as reference. At least three independent experiments were performed in triplicate, and the average values are shown. The error bars indicate the standard deviations.

FIG. 3.

Inhibition of HPV-11 in vitro DNA replication by peptides 6N40A and 6N40A dimer. (A) pUC18/7870-99, an in vitro DNA replication template that contains 182 bp (nucleotides 7870 to 0099) from the HPV-11 locus control region inserted into pUC18. (B) NdeI-digested replication products from cell-free replication assays using HPV-11 ORI-containing plasmid (pUC18/7870-99) and increasing concentrations of 6N40A, 6N40A dimer, and E2P. E1, E2, and peptide were added as noted. Lanes corresponding to initial and baseline intensities of counts (I₀ and I_base, respectively) are indicated. (C) Replication assays with increasing peptide concentrations were quantified by both PhosphorImager and filter-binding techniques, and the resulting data for both were fitted to two-parameter hyperbolic decay curves. 6N40A dimer inhibited replication (circles) with an IC₅₀ of 631 nM, while monomeric 6N40A inhibited replication (squares) less efficiently (IC₅₀ = 27.3 μM). Peptide E2P (triangles) did not inhibit in vitro DNA replication at the concentrations tested.