Extracellular Conformational Changes in the Capsid of Human Papillomaviruses Contribute to Asynchronous Uptake into Host Cells

Abstract

Human papillomavirus 16 (HPV16) is the leading cause of cervical cancer. For initial infection, HPV16 utilizes a novel endocytic pathway for host cell entry. Unique among viruses, uptake occurs asynchronously over a protracted period of time, with half-times between 9 and 12 h. To trigger endocytic uptake, the virus particles need to undergo a series of structural modifications after initial binding to heparan sulfate proteoglycans (HSPGs). These changes involve proteolytic cleavage of the major capsid protein L1 by kallikrein-8 (KLK8), exposure of the N terminus of the minor capsid protein L2 by cyclophilins, and cleavage of this N terminus by furin. Overall, the structural changes are thought to facilitate the engagement of an elusive secondary receptor for internalization. Here, we addressed whether structural changes are the rate-limiting steps during infectious internalization of HPV16 by using structurally primed HPV16 particles. Our findings indicate that the structural modifications mediated by cyclophilins and furin, which lead to exposure and cleavage, respectively, of the L2 N terminus contribute to the slow and asynchronous internalization kinetics, whereas conformational changes elicited by HSPG binding and KLK8 cleavage did not. However, these structural modifications accounted for only 30 to 50% of the delay in internalization. Therefore, we propose that limited internalization receptor availability for engagement of HPV16 causes slow and asynchronous internalization in addition to rate-limiting structural changes in the viral capsid.IMPORTANCE HPVs are the main cause of anogenital cancers. Their unique biology is linked to the differentiation program of skin or mucosa. Here, we analyzed another unique aspect of HPV infections using the prototype HPV16. After initial cell binding, HPVs display an unusually protracted residence time on the plasma membrane prior to asynchronous uptake. As viruses typically do not expose themselves to host immune sensing, we analyzed the underlying reasons for this unusual behavior. This study provides evidence that both extracellular structural modifications and possibly a limited availability of the internalization receptor contribute to the slow internalization process of the virus. These findings indicate that perhaps a unique niche for initial infection that could allow for rapid infection exists. In addition, our results may help to develop novel, preventive antiviral measures.

Keywords: furin; kinetic; papillomavirus; structural modification; virus entry.

FIG 1

FPC-HPV16 assemble correctly. (A) HPV16 and FPC-HPV16 particles were analyzed by electron microscopy after negative staining. Depicted are representative images of virions. Bars, 100 nm. (B) HPV16 and FPC-HPV16 particle composition by SDS-PAGE and Coomassie blue staining. Note the shift in the size of the furin-cleaved L2 protein (L2 cut). (C) Surface epitopes of HPV16-GFP (black) and FPC-HPV16-GFP (white) particles were targeted with neutralizing antibody H16.V5 (left) or H16.U4 (middle) or nonneutralizing control antibody CAMVIR-1 (right) at indicated dilutions. Presented are infection values (relative proportion of GFP-expressing cells) in HeLa cells relative to untreated controls in percentages ± standard deviations (SD). (D) Dependence on furin of HPV16 (black) or FPC-HPV16 (white) infection was tested in the presence of 10 μM furin inhibitor Dec-RVKR-CMK or, as a control, dimethyl sulfoxide (DMSO). Depicted are infection values relative to DMSO-treated controls in percentages ± SD. (E) To analyze structural processing, HPV16 or FPC-HPV16 and the L2-neutralizing RG-1 antibody were added to HeLa cells. Depicted are infection values relative to mouse serum-treated controls in percentages ± SD.

FIG 2

FPC-HPV16 and HPV16 enter host cells by the same entry pathway. HeLa cells were infected with HPV16 (black), FPC-HPV16 (white), or VSV (gray) in the presence of the indicated concentrations of gefitinib (A), EIPA (B), dynasore (C), cytochalasin D (D), jasplakinolide (E), NH₄Cl (F), cyclosporine (G), or aphidicolin (H). Depicted are infection values relative to solvent-treated controls in percentages ± SD.

FIG 3

Infectious internalization of FPC-HPV16 is faster and more synchronous than that of HPV16. (A) In an add-on experiment, HPV16 (dotted) or FPC-HPV16 (solid) was used to infect HeLa (left) or HaCaT (right) cells. At indicated times postinfection, extracellular virus was inactivated by a high-pH wash (pH 10.5). Depicted are the half-times of infectious internalization and the difference in the half-time between the two viruses in hours and percentages. (B) A seed-over experiment was performed as described for panel A. Infectious internalization values were normalized to the 48-h samples and are depicted in percentages ± standard errors of the means (SEM) for all experiments. Curves were fitted with the nonlinear regression function of GraphPad Prism v6. Statistical significance was tested by two-tailed unpaired Student's t test in GraphPad Prism v6; P values are indicated by asterisks: *, P < 0.05; **, P < 0.01. (C) pHrodo-HPV16 and pHrodo-FPC-HPV16 particles were added to cells, and the pHrodo signal was imaged at different time points p.i. and analyzed as relative intensity per cell.

FIG 4

KLK8 cleavage is not rate limiting for HPV16 internalization. (A) Kinetics of L1 cleavage by KLK8 was analyzed by incubation of HPV16 PsVs with 10 mg/ml heparin, binding to ECM, and incubation with conditioned medium or, as a control, DMEM. Samples were analyzed at the indicated times following conditioned medium incubation by SDS-PAGE and Western blotting using L1-specific CAMVIR-1 antibody. L1 cleavage bands (L1 A) were quantified by densitometry using Fiji. (B) Depicted is the increase of the cleaved L1 band (L1 A) normalized to the average maximal cleavage reached after 16 h of incubation with the batches of conditioned medium used in the experiments in percentages ± SD (n = 2). (C) Infection levels of HPV16 or FPC-HPV16 after 6 or 16 h of preincubation with conditioned medium (CM) relative to levels after preincubation with DMEM in percentages ± SD. (D) Schematic depiction of the experimental setup for KLK8 pretreatment. Virus particles are pretreated with heparin for structural activation (1) and then bound to HaCaT ECM and treated with conditioned medium or DMEM for 16 h (2). Seed-over infectious internalization (3) was performed as described before. Infection of HPV16 (left) or FPC-HPV16 (right) preincubated with growth medium (black) or conditioned medium (gray) for 16 h as described for panel A in a seed-over setup (compare with Fig. 3B). Depicted are infectious internalization values normalized to the 48-h samples in percentages ± SD.

FIG 5

CyPB activity modestly accelerates HPV16 internalization. (A) Amino acid sequences of the putative L2 CyPB binding motif (HPV16 versus HPV16 L2-GP-N). (B) The infectious internalization kinetics of HPV16 (black) and HPV16 L2-GP-N (dark gray) were compared in add-on experiments (as described for Fig. 3A). (C) Infectious internalization of HPV16 (black) and HPV16 L2-GP-N (dark gray) was analyzed in seed-over experiments (as described for Fig. 3B). (D) The infectious internalization kinetics of FPC-HPV16 (white) and FPC-HPV16 L2-GP-N (light gray) were compared in add-on experiments. (E) Infectious internalization of FPC-HPV16 HPV16 (white) and HPV16 L2-GP-N (light gray) were analyzed in seed-over experiments. Infectious internalization values are shown relative to 48-h infection samples in percentages ± SD for all experiments.

FIG 6

Infectious internalization kinetics of HPV16 are unaffected by cell cycle synchronization. (A) Schematic depiction of cell cycle synchronization by double thymidine block. (B) Synchronized and nonsynchronized cells were fixed with ethyl alcohol (EtOH) and analyzed for their cell cycles states at different times postrelease. Cell cycle phases were designated according to cellular DNA content by PI staining and flow-cytometric analysis. G₁, a single set of chromosomes (i.e., DNA content = 1); G₂/M, duplicated chromosomes (DNA content = 2); S, replicating chromosomes between the two states. Shown are the values from two independent experiments. (C) Infectious internalization of HPV16 (upper panels) and FPC-HPV16 (lower panels) with synchronized (black dotted line) and nonsynchronized (gray dotted line) cells was analyzed in add-on experiments. Cells were infected at 2 h, 6 h, or 14 h postrelease from thymidine block. Curves were fitted with the nonlinear regression function of GraphPad Prism v6. Relative infectious internalization values were normalized to 48-h infection samples and are depicted as percentages ± SEM.

FIG 7

FPC-HPV infection occurs independently of HSPGs, and particles show reduced binding affinity to heparin. (A) HPV16 (black) or FPC-HPV16 (white) infection of NaClO₃-treated HeLa or HaCaT cells in the seed-over setup (as described for Fig. 3B). Infectious values are shown in percentages ± SD relative to untreated cell samples for all experiments. (B) Heparin binding affinity of HPV16 (black) and FPC-HPV16 (gray) was analyzed using HiTrap heparin HP columns (GE) and subsequent elution by increasing salt concentrations (300 mM to 1,500 mM). Depicted is a representative Western blot for HPV16 L1 of the eluted fractions 1 to 11 of HPV16 (upper panel) or FPC-HPV16 (lower panel) during salt gradient elution. Representative chromatograms of the HPLC runs were aligned to the corresponding elution fractions (HPV16, black line; FPC-HPV16, gray line). Salt concentrations corresponding to the main elution peak from at least 3 independent experiments are summarized in the table.

FIG 8

The accessibility of the secondary receptor for virus interaction is limited. (A) HeLa or HaCaT cells were treated with 50 mM NaClO₃ for 2 days and subsequently infected with HPV16 (black) or FPC-HPV16 (white) in add-on experiments (as described for Fig. 3A). Relative infection values were normalized to 48-h infection samples of untreated cells in percentages ± SD. FACS, fluorescence-activated cell sorting. (B) AF488-labeled HPV16 (gray/green, upper panels) or FPC-HPV16 (gray/green, lower panels) particles were bound to untreated or NaClO₃-treated HeLa cells. Cells were fixed and stained for F-actin with Atto647N-phalloidin (red). Depicted are representative maximum intensity projections of confocal image stacks from at least three independent experiments. Virus signals were quantified relative to cell area for more than 150 cells per condition. (C) As described for panel B, using HaCaT cells.

FIG 9

(A) Indicated amounts of AF488-labeled HPV16 (gray/green, left panels) or FPC-HPV16 (gray/green, right panels) particles were bound to CHO wild-type (upper panels) or pgsA-745 (lower panels) cells for 1 h. Cells were fixed, stained for F-actin (red), and imaged as described for Fig. 8B. Shown are maximum intensity projections of representative cells from 1 of at least 3 independent experiments. (B) Add-on infection of pgsA cells with different amounts of HPV16 (black) and FPC-HPV16 (white) compared to parental CHO cells. Shown are average relative infection values in percentages ± SD from 3 independent experiments.