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J. Biol. Chem., Vol. 282, Issue 38, 27913-27922, September 21, 2007

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Surface-exposed Amino Acid Residues of HPV16 L1 Protein Mediating Interaction with Cell Surface Heparan Sulfate^*

Maren Knappe, Sabrina Bodevin, Hans-Christoph Selinka, Dorothe Spillmann, Rolf E. Streeck, Xiaojiang S. Chen^¶, Ulf Lindahl, and Martin Sapp^||1

From the Institute for Medical Microbiology, University of Mainz, D-55101 Mainz, Germany, Department of Medical Biochemistry and Microbiology, The Biomedical Center, Uppsala University, SE-751 23 Uppsala, Sweden, ^¶Department of Molecular and Computational Biology, University of Southern California, Los Angeles, California 90089, and ^||Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, and Feist Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana, 71130

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.

Received for publication, June 21, 2007

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB490/E2 (to M. S. and R. E. S.), by the National Center for Research Resources, a component of the National Institutes of Health (Grant P20-RR018724, entitled "Center for Molecular and Tumor Virology"), and by Swedish Research Council Grant 15023, the Swedish Cancer Society, Swedish Foundation for Strategic Research Grant A303:156e, and Polysackaridforskning AB (Uppsala, Sweden). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932. Tel.: 318-675-5760; Fax: 318-675-5764; E-mail: msapp1{at}lsuhsc.edu.

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