Heparan sulfate-independent cell binding and infection with furin-precleaved papillomavirus capsids - PubMed (original) (raw)
Heparan sulfate-independent cell binding and infection with furin-precleaved papillomavirus capsids
Patricia M Day et al. J Virol. 2008 Dec.
Abstract
Papillomavirus infection normally involves virion binding to cell surface heparan sulfate proteoglycans (HSPGs). However, we found that human papillomavirus type 16 pseudovirions efficiently bound and infected cells lacking HSPGs if their L2 capsid protein was precleaved by furin, a cellular protease required for infection. The inability of pseudovirions to efficiently bind and infect cultured primary keratinocytes was also overcome by furin precleavage, suggesting that the defect involves altered HSPG modification. We conclude that the primary function of HSPG binding is to enable cell surface furin cleavage of L2 and that binding to a distinct cell surface receptor(s) is a subsequent step of papillomavirus infection.
Figures
FIG. 1.
(A) Furin cleaved L2 within the FPC pseudovirus. Pseudovirions were generated according to standard procedures or with the inclusion of furin (20 U; Alexis Biochemicals) in the lysis/maturation buffer. Both preparations were purified through an OptiPrep gradient. Western blot analysis detection using an anti-L2 monoclonal antibody (11) revealed cleaved L2 in the FPC preparation. We determined that approximately 35% of the L2 is cleaved by furin in this pseudovirion preparation. Quantification of the bands was performed on a Fuji LAS4000 imaging system. (B) Infectivity of the pseudovirus preparations on FD11 and FD11+ cells. The CHO-derived cell lines FD11, which is furin-deficient, and FD11+, which was transfected with a cDNA-expressing furin, were infected with untreated pseudovirus (white bars), the FPC virus (light gray bars), or untreated virus with exogenous furin (dark gray bars). The preparations were equilibrated by L1 content and titrated, threefold dilutions starting at 50 ng/ml. Infection was quantified by flow cytometric quantification of green fluorescent protein expression at 48 h. Exogenous furin was delivered in supernatant form (to 30% of the total volume) from the CHO-derived cell line, Δfur, which secretes a functional, truncated furin (6). Quantification of these supernatants reveals approximately 8 U/ml of active furin. The efficiency of in vitro cleavage of HPV16 L2 (not assembled into viral particles) was compared to cleavage with commercially calibrated furin (data not shown). (C) Infectivity of the pseudovirus on pgsa-745 cells. The same procedure was followed to evaluate the infectivity of the pgsa-745 cells. A titration of pseudovirus infectivity is shown with untreated pseudovirus (white bars), the FPC virus (light gray bars), or untreated virus with exogenous furin (dark gray bars). (D) Infectivity of the pseudovirus on keratinocytes. The first group of the three shows the infection of primary HFKs under the various conditions. The second and third groups show the infection of Ect1 E6/E7 and End1 E6/E7, respectively (the white bars represent infection with untreated pseudovirus, the FPC virus is shown in light gray, and the untreated virus with exogenous furin is shown in dark gray). All cells were cultivated in keratinocyte serum-free medium (Invitrogen) with added epidermal growth factor and bovine pituitary extract. The infection with the untreated virus was performed in the presence of the supernatant from FD11 cells to compare it to the addition of exogenous furin from the Δfur cells. This indicated that the addition of conditioned CHO medium did not affect the infectivity of the keratinocyte cells without the presence of furin.
FIG. 2.
Binding of pseudovirus preparations to HFK cells. Cells were seeded onto no. 01 thickness glass coverslips in a 24-well plate at a density of 1 × 105 cells/well and cultured overnight. To evaluate the capsid binding pattern, 50 ng of pseudovirus was added to the cells for 1 h. Unbound virus was removed by extensive washing, and the cells were fixed with 2% paraformaldehyde. Bound virus was detected with a rabbit polyclonal antiserum raised against HPV16 virus-like particles (24) and Alexa Fluor 488-conjugated donkey anti-rabbit immunoglobulin G (Molecular Probes). Alexa Fluor 594-conjugated phalloidin (Molecular Probes) was included in the secondary antibody stain at a dilution of 1/300. Untreated virus is shown in panels A and B. These two panels show the same cells in two different focal planes to emphasize the relative binding to the ECM (A) and the cell surface (B). FPC virus (C and D) was likewise imaged.
FIG. 3.
Cleavage of a furin-dependent ligand. Cells were seeded at 5 × 104 cells/well/ml of a 48-well plate and grown to confluence. Twenty-five micrograms of PA33 (17) was added for 1 h at 37°C, and furin cleavage was assessed by Western blot analysis using a polyclonal antiserum against PA (14). The lysates were loaded in the following order: lane 1, CHO-K1; lane 2, FD11+; lane 3, FD11; lane 4, pgsa-745; lane 5, HeLa; lane 6, HaCaT; lane 7, HFK; lane 8, Ect1 E6/E7; lane 9, End1 E6/E7. The ability of the cell line to be infected with untreated pseudovirus is indicated below the appropriate lane.
FIG. 4.
Binding of pseudovirus in the absence of HSPG. The binding of untreated pseudovirus to pgsa-745 cells is shown in panel A. FPC virus is shown in panel B. Panels C and D show binding to sodium chlorate-treated HaCaT cells. Cells were grown in 50 mM sodium chlorate for 2 days prior to being plated on coverslips. Untreated virus is shown in panel C. FPC virus is shown in panel D.
References
- Binley, J. M., R. W. Sanders, A. Master, C. S. Cayanan, C. L. Wiley, L. Schiffner, B. Travis, S. Kuhmann, D. R. Burton, S. L. Hu, W. C. Olson, and J. P. Moore. 2002. Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins. J. Virol. 762606-2616. - PMC - PubMed
- Bitoun, E., A. Micheloni, L. Lamant, C. Bonnart, A. Tartaglia-Polcini, C. Cobbold, T. Al Saati, F. Mariotti, J. Mazereeuw-Hautier, F. Boralevi, D. Hohl, J. Harper, C. Bodemer, M. D'Alessio, and A. Hovnanian. 2003. LEKTI proteolytic processing in human primary keratinocytes, tissue distribution and defective expression in Netherton syndrome. Hum. Mol. Genet. 122417-2430. - PubMed
- Buck, C. B., D. V. Pastrana, D. R. Lowy, and J. T. Schiller. 2005. Generation of HPV pseudovirions using transfection and their use in neutralization assays. Methods Mol. Med. 119445-462. - PubMed
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