Binding and internalization of human papillomavirus type 33 virus-like particles by eukaryotic cells

Abstract

Infection of cells by human papillomaviruses (HPVs) associated with malignant genital lesions has not been studied because of the lack of an in vitro system and the unavailability of virions. We have now used virus-like particles (VLPs) of HPV type 33 to analyze the initial events in the interaction of the HPV capsid with cell lines. Binding of VLPs to HeLa cells was observed in biochemical assays and by immunofluorescence. VLP binding was inhibited by antisera raised against VLPs but not by monoclonal antibodies recognizing either L1 or L2 epitopes accessible on VLPs. Under saturating conditions, approximately 2 ؋ 10 4 VLPs were bound per cell, with a dissociation constant of about 100 pM. VLPs composed of L1 alone bound as well as VLPs composed of both capsid proteins, indicating that L2 is not required for initial binding. VLPs dissociated into capsomers did not bind, demonstrating that intercapsomer contacts are required. Neither capsomers nor simian virus 40 virions competed with VLP binding. Uptake of VLPs by small and smooth endocytic vesicles was demonstrated by immunoelectron microscopy. Cellular binding of VLPs was sensitive to trypsin but not to sialidase, N-glycosidase, or octyl-␤-D-glycopyranoside treatment, suggesting that a cell surface protein is involved in the VLP binding. Cell lines originating from a variety of tissues and organisms as distantly related as insects and humans bound VLPs with similar efficiency and specificity. Therefore, the putative receptor mediating VLP attachment should be highly conserved and cannot be responsible for the species and tissue specificity of HPVs.

In vitro generation and type-specific neutralization of a human papillomavirus type 16 virion pseudotype

“…Results from a number of laboratories have suggested that despite their strict host range, papillomaviruses bind to a wide variety of cell types derived from diverse species (24,29,41). The ability of HPV16{BPV1} virions to induce focal transformation of C127 cells implies that C127 cells express the cell surface receptor for HPV16 virions and are competent to perform the subsequent steps of internalization and uncoating that are required for initiating viral infection.…”

An Update on Non-clathrin-coated Endocytosis

“…Uptake of VLPs occurs via small, smooth endocytic vesicles with a 70-80 nm diameter and not by clathrin-coated vesicles. 72 It remains to be investigated whether these vesicles represent caveolae or micropinosomes, although uptake in CV-1 cells is observed to be inhibited by cytochalasin B, suggesting that caveolae may be involved in this process.…”

Highly efficient transport of carboxyfluorescein diacetate succinimidyl ester into COS7 cells using human papillomavirus‐like particles

“…Upon synthesis in eukaryotic cells using various expression systems, L1 and L2 proteins spontaneously self‐assemble into recombinant virus‐like particles (VLPs) [5–8]. Comparative studies of native virions and recombinant VLPs produced in Escherichia coli [9], yeast [8] and insect cells [5–7] showed that they are similar with regard to the morphological structure and cell binding [10,11]. Therefore VLPs represent a useful model system to study different aspects of the HPV infection cycle, in particular their cell binding and uptake.…”