Interactions between papillomavirus L1 and L2 capsid proteins

Abstract

The human papillomavirus (HPV) capsid consists of 360 copies of the major capsid protein, L1, arranged as 72 pentamers on a T=7 icosahedral lattice, with substoichiometric amounts of the minor capsid protein, L2. In order to understand the arrangement of L2 within the HPV virion, we have defined and biochemically characterized a domain of L2 that interacts with L1 pentamers. We utilized an in vivo binding assay involving the coexpression of recombinant HPV type 11 (HPV11) L1 and HPV11 glutathione S-transferase (GST) L2 fusion proteins in Escherichia coli. In this system, L1 forms pentamers, GST=L2 associates with these pentamers, and L1+L2 complexes are subsequently isolated by using the GST tag on L2. The stoichiometry of L1:L2 in purified L1+L2 complexes was 5:1, indicating that a single molecule of L2 interacts with an L1 pentamer. Coexpression of HPV11 L1 with deletion mutants of HPV11 L2 defined an L1-binding domain contained within amino acids 396 to 439 near the carboxy terminus of L2. L2 proteins from eight different human and animal papillomavirus serotypes were tested for their ability to interact with HPV11 L1. This analysis targeted a hydrophobic region within the L1-binding domain of L2 as critical for L1 binding. Introduction of negative charges into this hydrophobic region by site-directed mutagenesis disrupted L1 binding. L1-L2 interactions were not significantly disrupted by treatment with high salt concentrations (2 M NaCl), weak detergents, and urea concentrations of up to 2 M, further indicating that L1 binding by this domain is mediated by strong hydrophobic interactions. L1+L2 protein complexes were able to form virus-like particles in vitro at pH 5.2 and also at pH 6.8, a pH that is nonpermissive for assembly of L1 protein alone. Thus, L1/L2 interactions are primarily hydrophobic, encompass a relatively short stretch of amino acids, and have significant effects upon in vitro assembly.

FIG. 1.

HPV11 GST=L2 specifically binds HPV11 L1 in coexpressing cells. Samples from HPV11 L1 coexpressed with GST alone (lanes 1 to 3) or with HPV11 GST=L2 (lanes 4 to 6) were immunoblotted with α-L1 antisera. Lanes 1 and 4 contain 5 μl of the starting material prior to glutathione-Sepharose chromatography, lanes 2 and 5 contain 100 μl of the last wash prior to elution, and lanes 3 and 6 contain 10 μg of the reduced glutathione eluate. Molecular size markers in are indicated at the left in kilodaltons.

FIG. 2.

L1/L2 stoichiometry in initial eluates and purified complexes. A total of 10 μg each of partially purified eluate (lane 1) or FPLC-purified L1+L2 complex (lane 2) from a coexpression of HPV11 L1 with HPV11 GST=L2(313-455) (Fig. 3) was separated by SDS-PAGE and stained with Coomassie blue, and then the ratio of L1 to L2, indicated below each lane, was determined by densitometry analysis. The positions of HPV11 L2 and HPV11 GST=L2(313-455) are indicated at the right, and molecular size markers are indicated at the left in kilodaltons.

FIG. 3.

HPV11 GST=L2 fusion proteins. White rectangles depict HPV11 L2 or deletion derivatives of HPV11 L2; gray rectangles depict GST fused to the amino terminus of L2 or L2 deletion derivatives. The L2 amino acids incorporated into each fusion protein are indicated beside each depiction. The ability of each fusion protein to form a complex with HPV11 L1 was determined by the ability to detect L1 in bacterial coexpression experiments (see the text for details and see Fig. 4 for examples).

FIG. 4.

Analysis of HPV11 L1 binding by using deletion derivatives of HPV11 L2. A total of 10 μg of reduced glutathione eluates was separated by SDS-PAGE and stained with Coomassie blue (A) or immunoblotted with α-L1 antisera (B). Lanes 1 through 8 in both panels correspond to coexpression of HPV11 L1 with HPV11 GST=L2, HPV11 GST=L2(1-156), HPV11 GST=L2(157-309), HPV11 GST=L2(313-455), HPV11 GST=L2(313-400), HPV11 GST=L2(346-455), HPV11 GST=L2(346-439), and HPV11 GST=L2(396-439). The asterisks in panel A represent the positions of the full-length fusion proteins; the position of HPV11 L1 and the bacterial chaperone proteins GroEL and DnaK are indicated at the right. Molecular size markers are indicated at the left of each panel in kilodaltons.

FIG. 5.

Alignment of the putative L1-binding domains of selected L2 proteins. Alignments were generated with the assistance of Vector NTI AlignX software (InforMax). Conserved residues are in blue. Boxed residues indicate the hydrophobic region between the conserved prolines at 413 and 419 of HPV11 L2; negatively charged residues within this region are highlighted in yellow. Asterisks indicate HPV11 L2 amino acids subjected to site-directed mutagenesis with the change indicated above each asterisk. The combined alanine- and leucine-to-glutamine changes at 417 and 418 of HPV11 L2, indicated in red, severely disrupted L1 binding. Underlined residues in the HPV5 sequence are those that are different from the National Center for Biotechnology Information (NCBI) reference sequence (Table 2). Amino acids included in the alignment are indicated in brackets at the right.

FIG. 6.

HPV11 L1 forms heterologous complexes with other papillomavirus L2s. A total of 10 μg of reduced glutathione eluates were separated by SDS-PAGE and stained with Coomassie blue (A) or immunoblotted with α-L1 antisera (B). Lanes 1 through 8 in both panels correspond to coexpression of HPV11 L1 with HPV11 GST=L2(313-455), HPV6b GST=L2(314-459), HPV16 GST=L2(321-473), HPV33 GST=L2(319-467), HPV1a GST=L2(347-507), HPV5 GST=L2(376-518), HPV12 GST=L2(360-518), COPV1 GST=L2(360-513), and BPV1 GST=L2(316-469), respectively. The asterisks in panel A represent the positions of full-length fusion proteins; the position of HPV11 L1 is indicated at the right. Molecular size markers are indicated at the left of each panel in kilodaltons.

FIG. 7.

Disruption of HPV11 L1 binding in site-directed mutants of HPV11 L2. A total of 10 μg of reduced glutathione eluates from coexpressions with wild-type GST=L2(313-455) (lane 1) and GST=L2(313-455) carrying the mutations AL417418EE, A417E, L418E, P413A, P416A, and P419A (lanes 2 through 7, respectively) were separated by SDS-PAGE and stained with Coomassie blue. The ratio of L1 to L2, indicated below each lane, was determined by densitometry analysis. Note the difference in the ratio of L1 to L2 in lane 1 compared to that in lane 2.

FIG. 8.

Stability of L1+L2 complexes. An immobilized complex of HPV11 L1 and HPV11 GST=L2(313-455) was treated with the following: lane 1, 1 M NaCl in buffer L (40 mM Tris [pH 8.0], 200 mM NaCl, 2 mM DTT, 1 mM EDTA); lane 2, 2 M NaCl in buffer L; lane 3, 1% NP-40 in buffer L; lane 4, 1% NP-40 plus 0.1% DOC in buffer L; lane 5, radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.2], 150 mM NaCl, 0.1% SDS, 1% NP-40, 1% DOC); lane 6, 0.5 M urea in buffer L; lane 7, 1 M urea in buffer L; lane 8, 1.5 M urea in buffer L; lane 9, 2 M urea in buffer L; lane 10, 2.5 M urea in buffer L; lane 11, 5 M urea in buffer L; lane 12, buffer L; and lane 13, SDS-PAGE loading buffer (63 mM Tris [pH 6.8], 10% glycerol, 2.3% SDS). A total of 50 μl of the supernatant from each treatment separated by SDS-PAGE and stained with Coomassie blue is shown in panel A. The positions of HPV11 L1 and HPV11 GST=L2(313-455) are indicated at the right. Molecular size markers are indicated at the left in kilodaltons. Panel B shows the relative amounts of L1 released from the complex by each treatment as determined by densitometry analysis of gels like that depicted in panel A. The bars correspond from left to right to the treatment indicated for lanes 1 to 13 in panel A. The values represent the average of three independent experiments.

FIG. 9.

An L1+L2 complex assembles at pH 6.8. HPV11 L1 alone or in complex with HPV11 L2(313-455) was dialyzed against 40 mM HEPES (pH 6.8) and 0.5 M NaCl in the presence or absence of 5 mM DTT. Representative fields are shown. The scale bars represent 100 nm.