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. 2003 Mar;77(6):3531-41.
doi: 10.1128/jvi.77.6.3531-3541.2003.

Cell surface-binding motifs of L2 that facilitate papillomavirus infection

Rongcun Yang  1 , Patricia M Day, William H Yutzy 4th, Ken-Yu Lin, Chien-Fu Hung, Richard B S Roden
Affiliations

Cell surface-binding motifs of L2 that facilitate papillomavirus infection

Rongcun Yang et al. J Virol. 2003 Mar.

Abstract

Human papillomavirus type 16 (HPV16) is the primary etiologic agent of cervical carcinoma, whereas bovine papillomavirus type 1 (BPV1) causes benign fibropapillomas. However, the capsid proteins, L1 and L2, of these divergent papillomaviruses exhibit functional conservation. A peptide comprising residues 1 to 88 of BPV1 L2 binds to a variety of cell lines, but not to the monocyte-derived cell line D32, and blocks BPV1 infection of mouse C127 cells. Residues 13 to 31 of HPV16 L2 and BPV1 L2 residues 1 to 88 compete for binding to the cell surface, and their binding, unlike that of HPV16 L1/L2 virus-like particles, is unaffected by heparinase or trypsin pretreatment of HeLa cells. A fusion of HPV16 L2 peptide 13-31 and GFP binds (K(d), approximately 1 nM) to approximately 45,000 receptors per HeLa cell. Furthermore, mutation of L2 residues 18 and 19 or 21 and 22 significantly reduces both the ability of the HPV16 L2 13-31-GFP fusion protein to bind to SiHa cells and the infectivity of HPV16 pseudovirions. Antibody to BPV1 L2 peptides comprising residues 115 to 135 binds to intact BPV1 virions, but fails to neutralize at a 1:10 dilution. However, deletion of residues 91 to 129 from L2 abolishes the infectivity of BPV1, but not their binding to the cell surface. In summary, L2 residues 91 to 129 contain epitopes displayed on the virion surface and are required for infection, but not virion binding to the cell surface. Upon the binding of papillomavirus to the cell surface, residues 13 to 31 of L2 interact with a widely expressed, trypsin- and heparinase-resistant cell surface molecule and facilitate infection.

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Figures

FIG. 1.
FIG. 1.
A peptide comprising the first 88 amino acids of BPV1 L2 binds to HeLa cells and competes BPV1 infection. (A) Recombinant six-His-tagged HPV16 L2 (lane 1), BPV1 L2 full-length protein (lane 2), or fragments comprising residues 1 to 88 (lane 3), 45 to 173 (lane 4), 130 to 257 (lane 5), 216 to 340 (lane 6), 300 to 425 (lane 7), and 384 to 469 (lane 8) were affinity purified from E. coli, separated on an SDS-PAGE gel (15% polyacrylamide), and stained with Coomassie blue. (B) The six-His-tagged L2 polypeptides (5 μg in 0.5 ml of PBS) shown in panel A (heavy line) or buffer alone (fine line) was incubated with 2 × 106 HeLa cells for an hour on ice. After washing, bound L2 was detected in all samples by indirect immunofluorescence with a tag-specific monoclonal antibody and flow cytometric analysis. The bottom right panel shows the binding of full-length HPV16 L2 (dotted line), BPV1 L2 (heavy line), and buffer alone (fine line). Cont., control. (C) Monolayers of C127 cells, except for plate 2, were incubated with ∼200 FFU of purified BPV1, either alone (plate 1) or with 50 μg of BPV1 L2 1-88 (plate 3), 10 μg of BPV1 L2 1-88 (plate 4), 50 μg BPV1 L2 45-173 (plate 5), 10 μg of BPV1 L2 45-173 (plate 6), 50 μg of BPV1 full-length L2 (plate 7), 10 μg of BPV1 full-length L2 (plate 8), 50 μg of HPV16 full-length L2 (plate 9), or 10 μg of HPV16 full-length L2 (plate 10) in 1 ml of PBS for an hour at 37°C. The plates were washed, cultured for 3 weeks, and stained.
FIG. 2.
FIG. 2.
Trypsin-insensitive binding of BPV L2 residues 1 to 88 to a variety of cell lines. (A) Binding of the BPV L2 1-88 peptide at 4°C to the cell lines indicated was measured by indirect immunofluorescence and flow cytometric analysis. (B) Binding at 4°C of BPV L2 residues 1 to 88 to trypsin-treated (0.2%, 5 min at room temperature) (13) or untreated HeLa cells was measured by indirect immunofluorescence and fluorescence-activated cell sorter analysis. The heavy line indicates fluorescence in the presence of BPV L2 residues 1 to 88, and the lighter line corresponds to the fluorescence when the BPV1 L2 peptide was omitted. (C) Binding of HPV16 L1/L2 VLPs to HeLa cells to trypsin pretreated (0.2% for 5 min at room temperature) or control HeLa cells was measured by indirect immunofluorescence and flow cytometry.
FIG. 3.
FIG. 3.
HPV16 peptide 13-31 is highly conserved and binds to the cell surface. (A) ClustalW-formatted alignment of the L2 amino acid sequence of representative high-risk HPV genotypes and BPV1 between residues 13 and 31. (B) Purified GST-GFP fusion proteins including residues 299 to 333, 25 to 45, and 13 to 31 of HPV16 L2, either with or without PreScission protease (PreS.P) digestion, were separated on an SDS-PAGE gel (15% polyacrylamide) and stained with Coomassie blue (49). The positions of the PreScission protease and GST- and GFP-L2 fragments are indicated. (C) Flow cytometric analysis of SiHa cells in the presence of buffer (fine line) or GFP fusion proteins (heavy line). (C1) PreScission protease-digested GST-HPV16 L2 13-31-GFP fusion protein. (C3) Undigested GST-HPV16 L2 13-31-GFP fusion protein. (C2) PreScission protease-digested GST-GFP. (C4) GST-GFP. (D) Analysis by confocal fluorescence microscopy of the cell surface binding and uptake of GFP fusion protein containing L2 residues 13 to 31. SiHa cells were incubated with GFP-tagged HPV16 L2 residues 13 to 31 for an hour at 4°C (D1 and D3), and then the cells were washed and shifted to 37°C for 30 min (D2 and D4). The cells were viewed by phase-contrast light microscopy (D1 and D2) or confocal fluorescence microscopy (D3 and D4).
FIG. 4.
FIG. 4.
Scatchard analysis and competition studies of L2 binding to the cell surface. (A) Known concentrations of the HPV16 L2 13-31-GFP fusion protein were incubated for an hour at 4°C with 106 HeLa cells. The supernatants containing free peptide and cell-bound peptide were harvested separately, and their ratio was determined by Western blotting for Scatchard analysis, as described in reference . (B) Binding of GST-GFP either lacking or containing residues 13-31 of HPV16 L2 to HeLa cells after incubation for 20 min at 4°C, washing, and fixation was measured directly by flow cytometry and expressed as the mean fluorescent intensity of 104 cells. The binding to HeLa cells of the HPV16 L2 13-31-GFP fusion in the presence of BPV1 L2 1-88 peptide at the concentrations indicated or control BPV1 L2 peptides at 25 μg/ml was assessed.
FIG. 5.
FIG. 5.
Mutations, but not heparinase, eliminate binding of the HPV16 peptide 13-31 to cells. (A) The binding to SiHa cells of a 10-μg/ml sample of fusion protein comprising GST and GFP fused with either HPV16 L2 residues 13 to 31 (ASATQLYKTCKQAGTCPPD) or the L13L14V15V16 (LLVVQLYKTCKQAGTCPPD), A18A19 (ASATQAAKTCKQAGTCPPD), or V21V22 (ASATQLYKVVKQAGTCPPD) mutant peptides was assessed by flow cytometric analysis and plotted in panels A1 to A4, respectively. (B) The binding of a 10-μg/ml sample of fusion protein comprising GST and GFP alone (fine line) or fused with HPV16 L2 residues 13 to 31 (bold line) to SiHa cells pretreated with 2.5 U of heparinase I, II, and III per ml for 1 h at 37°C was analyzed by flow cytometry. (C) The binding of HPV16 L1/L2 VLPs to untreated (C1) or heparinase-treated (2.5 U each of forms I, II and III per ml for 1 h at 37°C) SiHa cells (C2) was detected with H16.V5 (heavy line) and FITC-conjugated anti-mouse IgG, or, as a negative control, secondary antibody alone (fine line).
FIG. 6.
FIG. 6.
Residues 91 to 129 of BPV1 L2 are not required for virion assembly or binding of virions to the cell surface, but are necessary for infectivity. (A) BPHE-1 cells were infected with recombinant SFV expressing BPV1 L1 alone or coinfected with SFV expressing BPV1 L1 and wild-type L2 or L2Δ91-129. Thirty hours postinfection, the cells were harvested and lysed by sonication. The presence of DNase I-resistant BPV1 genome that is immunoprecipitated with anti-VLP antiserum was detected by Southern blot analysis (upper panel). The lysates were also tested for the presence of infectious BPV1 virions by using the C127 focal transformation assay (see plates in lower panel). BPHE-1 cells were coinfected with BPV1 L1 and the wild type or L2Δ91-129 and harvested from the lysates 30 h postinfection by centrifugation through a 40% sucrose cushion and separated from empty VLP by further purification by rate zonal density centrifugation (10 to 40% sucrose, SW-41 rotor, 25,000 rpm, 4°C). Equivalent yields and incorporation of L2 and L2Δ91-129 into purified virions were demonstrated by Western blotting with rabbit BPV1 L1 antiserum (B) and rabbit anti-BPV1 L2 antiserum (C). Virion preparations of L1/L2 and L1/L2Δ91-129 were loaded in lanes 1 and 2, respectively (B and C). (D) Equal amounts of BPV1 L1/L2 and L1/L2Δ91-129 virions or buffer alone were incubated with C127 clone C cells at 4°C for 1 h, and surface-bound virions were detected by immunofluorescent staining with monoclonal antibody 5B6.
FIG. 7.
FIG. 7.
L2 residues 91 to 129 are partially exposed on the surface of BPV1 virions. (A) A microtiter plate coated with 300 ng of purified BPV1 virions per well was reacted for an hour with monoclonal antibody C6 to BPV1 L2 residues 116 to 123 (31), 5B6 to a conformationally dependent and neutralizing epitope on BPV1 L1 (41), or PAb122 to an irrelevant antigen. After washing, specific binding was detected with a peroxidase-linked anti-mouse IgG antibody. The plate was developed with ABTS, and the A405 was determined. (B and C) Microtiter plates were coated with a 300-ng/well sample of either BPV1 L2 45-173 peptide that had been purified by using its hexahistidine tag (B) or BPV1 virions from a bovine wart purified on cesium chloride gradients (C). The plates were reacted with preimmune (PI) or immune sera of rabbits immunized with KLH coupled to BPV1 L2 peptides 100-120 (K2 antiserum) and 115-135 (K3 antiserum). Upon washing, specific binding was detected with a peroxidase-linked anti-rabbit IgG antibody and ABTS. The A405 was determined.
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