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Abstract
The L1 major capsid protein of human papillomavirus (HPV) type 11, a 55-kDa polypeptide, forms particulate structures resembling native virus with an average particle diameter of 50-60 nm when expressed in the yeast Saccharomyces cerevisiae. We show in this report that these virus-like particles (VLPs) interact with heparin and with cell-surface glycosaminoglycans (GAGs) resembling heparin on keratinocytes and Chinese hamster ovary cells. The binding of VLPs to heparin is shown to exhibit an affinity comparable to that of other identified heparin-binding proteins. Immobilized heparin 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. Sequence comparison of nine human L1 types revealed a conserved region of the carboxyl terminus containing clustered basic amino acids that bear resemblance to proposed heparin-binding motifs in unrelated proteins. Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocyte (HaCaT) cells. Removal of heparan sulfate GAGs on keratinocytes by treatment with heparinase or heparitinase resulted in an 80-90% reduction of VLP binding, whereas treatment of cells with laminin, a substrate for alpha6 integrin receptors, provided minimal inhibition. Cells treated with chlorate or substituted beta-D-xylosides, resulting in undersulfation or secretion of GAG chains, also showed a reduced affinity for VLPs. Similarly, binding of VLPs to a Chinese hamster ovary cell mutant deficient in GAG synthesis was shown to be only 10% that observed for wild type cells. This report establishes for the first time that the carboxyl-terminal portion of HPV L1 interacts with heparin, and that this region appears to be crucial for interaction with the cell surface.
Highlights
Papillomaviruses are non-enveloped, double-stranded DNA viruses containing a circular genome of approximately 8,000 base pairs
We show in this report that these virus-like particles (VLPs) interact with heparin and with cell-surface glycosaminoglycans (GAGs) resembling heparin on keratinocytes and Chinese hamster ovary cells
Papillomaviruses can bind to a wide variety of cell types [20, 21], their productive tropism is generally limited to epithelial keratinocytes and fibroblasts [20]
Summary
HaCaT cells were maintained as monolayer cultures at 37 °C, 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Inc., Rockville, MD) supplemented with 10% fetal calf serum (FCS; Life Technologies, Inc.) and 1% penicillin/streptomycin (Life Technologies, Inc.). For sulfate-free medium, the DMEM formulation was the same except that magnesium sulfate was omitted and dialyzed FCS (Sigma) was used. CHO-K1 and pgsA-745 cell lines were obtained from American Type Culture Collection (ATCC; Rockville, MD) and were grown as monolayer cultures at 37 °C, with a 5% CO2 atmosphere in F-12K medium (ATCC) supplemented with 10% FCS and 1% penicillin/streptomycin. Cells were routinely subcultured every 14 –21 days
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