Gene transfer using human papillomavirus pseudovirions varies according to virus genotype and requires cell surface heparan sulfate

Oncogenic human papillomaviruses (HPVs) are difficult to study experimentally as they replicate at low levels in vivo. This has precluded the purification of high-risk HPV virions from in vivo lesions. Virus-like particles (VLPs) and pseudovirions from low-and high-risk HPV types can emulate various aspects of HPV virion attachment and infections. These studies suggest that HPV infection is mediated by ␣6-integrin and/or heparan-sulfonated receptors. However, whether VLPs and pseudovirions accurately reflect the infection process of HPV virions has not been verified. We generated infectious HPV31b virions from organotypic (raft) tissues and performed experimental infections in a variety of cells. Successful infection following viral attachment, internalization, and nuclear transport was assayed by detecting newly synthesized, spliced HPV transcripts using reverse transcription (RT)-PCR or RT-quantitative PCR. Most human epithelial cells were infected with HPV31b at a multiplicity of infection as low as 1 to 10 viral genome equivalents per cell. HPV31b infection was detected in other cell lines, including COS-7 monkey kidney cells, but higher viral multiplicities of infection were required. Heparin preparations of various molecular weights or heparinase I treatment of cells prevented HPV31b infection of COS-7 cells and C-33A human cervical cancer cells in reproducible and dose-dependent manners. However, these reagents were unable to block infection of human keratinocytes, including HaCaT and N/TERT-1 cells and low-passage human foreskin keratinocytes. These data suggest that HPV31b infection of human keratinocytes, the natural host cell for HPV infections in vivo, does not require a heparan-sulfonated receptor, whereas heparan sulfate is important for infection of some other cells.

Section: Vol 79 2005 Hpv31b Infections Do Not Require Heparan Sulfamentioning

confidence: 99%

Human Papillomavirus Type 31b Infection of Human Keratinocytes Does Not Require Heparan Sulfate

Patterson

Smith

Ozbun

2005

“…There is a growing body of evidence that heparan sulfate proteoglycans act as primary receptors for papillomaviruses and mediate viral attachment (5,10,12). Heparan sulfate proteoglycans interact with the carboxyl terminus of the HPV L1 protein (12).…”

mentioning

confidence: 99%

“…HPV-16 and -31 VLPs composed of L1 protein were produced in Sf21 insect cells by use of recombinant baculoviruses encoding the HPV-16 and -31 L1 open reading frames, respectively, and purified according to previously described procedures (4,5,7). VLPs composed of L1 and L2 proteins were also produced for HPV-31 and -58 (6).…”

mentioning

confidence: 99%

“…VLPs composed of L1 and L2 proteins were also produced for HPV-31 and -58 (6). For the generation of pseudovirions, 5 g of VLPs and 0.5 g of a linearized pCMVLuc plasmid (7.1 kbp) coding for luciferase under the cytomegalovirus promoter (Ozyme) were mixed and incubated for 30 min at room temperature before pseudoinfection of COS-7 cells seeded in 96-well plates according to a recently described procedure called direct interaction (4)(5)(6). Luciferase expression was measured by a luminescence assay (Luciferase reporter gene assay with constant light signal; Roche Molecular Biochemical, Meylan, France), and the results were expressed as counts per second (cps) per well.…”

mentioning

confidence: 99%

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Human Papillomavirus Types 16, 31, and 58 Use Different Endocytosis Pathways To Enter Cells

Bousarghin

Touzé

Sizaret³

et al. 2003

138

127

The early steps of the intracellular trafficking of human papillomavirus type 16 (HPV-16), -31, and -58 pseudovirions were studied by investigating the effects of drugs acting at defined points of endocytosis pathways on virus-like particle-mediated pseudoinfection by overexpression of a dominant-negative form of the Eps15 protein to inhibit clathrin-mediated endocytosis and by electron microscopy. The results obtained suggested the involvement of clathrin-mediated endocytosis in HPV-16 and HPV-58 entry and caveola-mediated endocytosis in HPV-31 entry.As a consequence of interactions with their receptors, viruses can follow a variety of pathways for entry into cells. Among nonenveloped DNA viruses, adenovirus (16), adenoassociated virus (2), and human polyomavirus JC (23) enter cells by means of clathrin-coated endocytic vesicles whereas simian virus 40 (SV40) and mouse polyomavirus are taken up into cells via caveolae (1,21,22,24).Papillomaviruses are small nonenveloped tumorigenic DNA viruses that infect the basal cells of the epithelium. Papillomavirions contain two proteins, L1 and L2, which encapsidate a closed, circular, double-stranded DNA of about 8 kbp. The viral capsid of 50 to 55 nm contains 72 pentamers of L1, and expression of the major capsid protein L1 results in the selfassembly of virus-like particles (VLPs) which have similar characteristics to the virions. Attempts to efficiently grow human papillomaviruses (HPVs) in cell culture have to date been unsuccessful, and consequently, surrogate systems using HPV pseudovirions have been developed to test for viral infectivity (4,13,25,26,29,30).It has previously been reported that in order to infect target cells, papillomaviruses bind to specific cell surface receptors (19,31), penetrate the plasma membrane by an unknown mechanism, and target their genomes to the cell nucleus (18,20,33). There is a growing body of evidence that heparan sulfate proteoglycans act as primary receptors for papillomaviruses and mediate viral attachment (5, 10, 12). Heparan sulfate proteoglycans interact with the carboxyl terminus of the HPV L1 protein (12). Although heparan sulfate proteoglycans are widely distributed on the surfaces of many cell types, they may not be sufficient to allow efficient HPV entry. Evander et al. (9) and McMillan et al. (17) have already shown that alpha-6 integrin is used by HPV type 6 (HPV-6) as a receptor for HPV entry into host cells.Electron microscopy examination has shown that bovine papillomavirus type 1 (BPV-1) virions are seen in the lipid layer-surrounded vesicles (33), suggesting that caveolae are involved in BPV-1 internalization and that they are associated with microtubules in the cytoplasm (15). In addition, uptake of HPV-33 by small and smooth endocytic vesicles (27, 31) suggests that this virus is internalized by caveolae. However, it was reported recently that this virus entry is not inhibited by nystatin, suggesting that it does not require a caveola-dependent pathway (27). The papillomaviruses then penetrate the nuclear me...

“…Using HPV33 L1L2 pseudovirions encapsidating a green fluorescence protein (GFP) marker plasmid, we identified cell surface heparan sulfate proteoglycans (HSPGs) as the primary attachment receptor required for efficient infection with HPV (18). Meanwhile, interaction with HSPGs has been shown to be essential for infection with various HPV types (11).…”

mentioning

confidence: 99%

Further Evidence that Papillomavirus Capsids Exist inTwo DistinctConformations

Selinka

Giroglou

Nowak

et al. 2003

117

115

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.Human papillomaviruses (HPVs) are highly species-specific epitheliotropic DNA viruses. Of the more than 100 different genotypes, HPV type 16 (HPV16), HPV18, HPV31, HPV33, HPV35, HPV45, and HPV58 are most closely associated with cervical epithelial neoplasias and members of the group of HPV imposing a "high risk" for malignant progression to invasive genital carcinomas (30). The nonenveloped papillomavirus is composed of 360 copies of the major capsid protein L1, organized in 72 capsomeres, and probably 12 copies of the minor capsid protein L2 (1, 43). The encapsidated genome is an 8,000-bp circularized double-stranded DNA associated with cellular histones.Despite their considerable clinical significance, the initial steps leading to infection with these viruses, as well as the mechanisms involved in virus entry into host cells, have not yet been completely elucidated due to the limited growth properties of HPV in cell cultures and the ubiquitous expression of HPV-binding proteins. The use of virus-like particles (VLPs) (20,25,36,46,49) has helped in the study of the initial interaction of papillomavirus particles with cell surfaces. It was established that VLPs of many HPV types compete for binding to the same highly conserved proteinaceous attachment receptor. In contrast to L1, L2 protein was not essential for binding, since L1 VLPs bound as efficiently as L1L2 VLPs (32,3...