Abstract
Efficient infection of cells by human papillomaviruses (HPVs) and pseudovirions requires primary interaction with cell surface proteoglycans with apparent preference for species carrying heparan sulfate (HS) side chains. To identify residues contributing to virus/cell interaction, we performed point mutational analysis of the HPV16 major capsid protein, L1, targeting surface-exposed amino acid residues. Replacement of lysine residues 278, 356, or 361 for alanine reduced cell binding and infectivity of pseudovirions. Various combinations of these amino acid exchanges further decreased cell attachment and infectivity with residual infectivity of less than 5% for the triple mutant, suggesting that these lysine residues cooperate in HS binding. Single, double, or triple exchanges for arginine did not impair infectivity, demonstrating that interaction is dependent on charge distribution rather than sequence-specific. The lysine residues are located within a pocket on the capsomere surface, which was previously proposed as the putative receptor binding site. Fab fragments of binding-neutralizing antibody H16.56E that recognize an epitope directly adjacent to lysine residues strongly reduced HS-mediated cell binding, further corroborating our findings. In contrast, mutation of basic surface residues located in the cleft between capsomeres outside this pocket did not significantly reduce interaction with HS or resulted in assembly-deficient proteins. Computer-simulated heparin docking suggested that all three lysine residues can form hydrogen bonds with 2-O-, 6-O-, and N-sulfate groups of a single HS molecule with a minimal saccharide domain length of eight monomer units. This prediction was experimentally confirmed in binding experiments using capsid protein, heparin molecules of defined length, and sulfate group modifications.
Highlights
Cell surface heparan sulfate proteoglycans (HSPGs), mainly syndecans and glypicans, are composed of a protein core with covalently attached HS chains
L2 protein does not seem to contribute to the initial binding to HSPG as virus-like particles (VLPs) and pseudovirions composed of L1 protein alone can bind to cell surface-exposed proteoglycan as well as to heparin, a highly sulfated form of HS secreted by mast cells
Similar results were obtained with HPV type 16 (HPV16) L1L2 pseudovirions. These results indicate that 2-O-sulfate groups, glucosamine N-sulfate, and in particular glucosamine 6-O-sulfate groups of the polysaccharide all contribute to the interaction with human papillomaviruses (HPVs)
Summary
Cell surface heparan sulfate proteoglycans (HSPGs), mainly syndecans and glypicans, are composed of a protein core with covalently attached HS chains. Double, or triple lysine residues 278, 356, and 361 for arginine did not reduce infectivity of pseudovirions, suggesting that overall charge distribution rather than sequence specificity determines binding to GAGs (Fig. 3B).
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