Analysis of the infectious entry pathway of human papillomavirus type 33 pseudovirions.

Human papillomavirus type 33 (HPV-33) pseudovirus infection is a slow process dependent on the initial interaction with cell-surface heparan sulfate (T. Giroglou, L. Florin, F. Schafer, R. E. Streeck, and M. Sapp, 2001a, J. Virol. 75, 1565-1570). We have now further dissected the initial steps of pseudovirus uptake using removal of cell-surface proteoglycans and selective inhibition of entry pathways. Treatment of cells with heparinase I, but not with phosphoinositol-specific phospholipase C (PIPLC), prevented binding of papillomavirus-like particles and infection with HPV-33 pseudovirions, indicating that GPI-linked proteoglycans (glypicans) are not required for productive infection. The slow entry of pseudovirions was inhibited by cytochalasin D and nocodazole in a concentration-dependent manner, suggesting actin polymerization and intact microtubuli be required. Inhibitors of the caveolae-mediated uptake did not significantly affect pseudoinfection. Interestingly, pseudoinfection was blocked by selective inhibitors of endosomal acidification up to 12 h postinfection. Together, our results suggest that binding of HPV pseudovirions to heparan sulfate proteoglycans, most likely syndecans, is followed by delayed internalization via the endosomal pathway.

Further evidence that papillomavirus capsids exist in two distinct conformations.

Cell surface heparan sulfate proteoglycans (HSPGs) serve as primary attachment receptors for human papillomaviruses (HPVs). To demonstrate that a biologically functional HPV-receptor interaction is restricted to a specific subset of HSPGs, we first explored the role of HSPG glucosaminoglycan side chain modifications. We demonstrate that HSPG O sulfation is essential for HPV binding and infection, whereas de-N-sulfated heparin interfered with VLP binding but not with HPV pseudoinfection. This points to differences in VLP-HSPG and pseudovirion-HSPG interactions. Interestingly, internalization kinetics of VLPs and pseudovirions, as measured by fluorescence-activated cell sorting analysis, also differ significantly with approximate half times of 3.5 and 7.5 h, respectively. These data suggest that differences in HSPG binding significantly influence postbinding events. We also present evidence that pseudovirions undergo a conformational change after cell attachment. A monoclonal antibody (H33.J3), which displays negligible effectiveness in preattachment neutralization assays, efficiently neutralizes cell-bound virions. However, no difference in H33.J3 binding to pseudovirions and VLPs was observed in enzyme-linked immunosorbent assay and virus capture assays. In contrast to antibody H33.B6, which displays equal efficiencies in pre- and postattachment neutralization assays, H33.J3 does not block VLP binding to heparin, demonstrating that it interferes with steps subsequent to virus binding. Our data strongly suggest that H33.J3 recognizes a conformation-dependent epitope in capsid protein L1, which undergoes a structural change after cell attachment.