Identification and characterization of a heparin-binding region on the L1 major capsid protein of recombinant human papillomavirus virus-like particles and its role in interactions of VLPs with cells

The L1 major capsid protein of human papillomavirus (HPV), a 55--56 kDa polypeptide, forms particulate structures with an average particle diameter of 50 to 60 nm when expressed in the yeast Saccharomyces cerevisiae . These structures resemble the native virion and assemble regardless of whether the minor capsid antigen, L2, is co-expressed. The virus-like particles (VLPs) retain important characteristics of intact virus including the ability to recognize conformationally---dependent monoclonal antibodies, generate neutralizing antisera, and bind to mammalian cells. Previous studies in animal and in vitro models have demonstrated that these VLPs are capable of eliciting protective responses against challenge with live virus, suggesting the feasibility of a prophylactic vaccine. However, the initial events of HPV infection, including cellular recognition and internalization, are still poorly understood.;The present study demonstrates for the first time that VLPs composed of the L1 protein of HPV types 11 and 16 interact with immobilized heparin as well as with glycosaminoglycans which resemble heparin on the surface of human keratinocytes and Chinese hamster ovary (CHO) cells. The interaction of VLPs with heparin was shown to exhibit a very high avidity and an affinity comparable to that of other viral heparin-binding proteins. Immobilized heparin affinity chromatography and surface plasmon resonance were used to show that this interaction can be specifically inhibited by free heparin and dextran sulfate and that the effectiveness of the inhibitor is related to its molecular weight and charge density. Related glycosaminoglycans such as chondroitin sulfate and chemically modified heparins were significantly less effective as inhibitors, suggesting a certain degree of specificity in the interaction; and fractionation of native heparin by anion exchange chromatography identified two major species with widely differing abilities to inhibit VLP binding.;Sequence comparison of nine human L1 types revealed a conserved region of the carboxy terminus containing clustered basic amino acids which bear resemblance to proposed heparin-binding motifs in unrelated proteins. Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocytes. Physical characterization of intact and truncated VLPs was used to determine that no structural changes had occured during enzymatic treatment, providing strong evidence that heparin-binding is localized to the C-terminus of L1. Alteration of anionic cell surface molecules was effected by treatment of keratinocytes with chlorate or substituted beta-D- xylosides, resulting in undersulfation or secretion of proteoglycan-associated glycosaminoglycan chains. These treatments led to reduced binding of VLPs, whereas treatment of cells with laminin, a substrate for the alpha6 integrin receptor, provided minimal inhibition. Heparan sulfate glycosaminoglycans on keratinocytes were enzymatically removed by treatment with heparinases, and this also resulted in a dramatic reduction of VLP binding. Similarly, binding of VLPs to a CHO cell mutant deficient in glycosaminoglycan synthesis was shown to be much less efficient than that observed for wild type cells.;The observations reported in this study establish for the first time that the carboxy terminal portion of HPV L1 binds to heparin, and that this region appears to be crucial for interaction with the cell surface. This offers a possible explanation for why HPV VLPS bind to a wide variety of cell types other than those for which they show a specific tropism, and suggests the possibility of a dual receptor mechanism involving a general, non-specific recognition by glycosaminoglycans followed by uptake mediated by a proteinaceous receptor.