Article Figures & Data
Figures
- FIG. 1.
HPV31 virions produced by a transfection-based method display normal morphology and infectivity properties. (A) Transfection-derived HPV31 virions visualized by TEM. (B) HaCaT cells were exposed to increasing doses of HPV31 (1, 10, and 100 vge/cell) as described in Materials and Methods. Monoclonal antibodies H31.A6 and H16.D9 were diluted in media and added to cells immediately following viral attachment. Total RNAs were subjected to RT-qPCR in triplicate to quantify spliced HPV31-E1⌃E4 transcripts indicative of infection (48 h p.i.). Error bars represent SEM. (C) AF594-labeled HPV31 (red) was bound to HKs at 10,000 vge/cell and visualized after 1 h at 4°C, after incubation at 37°C for 24 h (D), or after incubation at 37°C for 24 h in the presence of H31.A6 neutralizing antibody (E). Coverslips were mounted in the presence of DAPI to visualize nuclei. (F) HaCaT cells were inoculated with AF594-labeled HPV31 at the indicated doses; neutralizing antibody H31.A6 was used as in panels B and E. Infection was quantified as in panel B.
- FIG. 2.
Time course of HPV31 internalization. (A) Organotypic culture-derived or transfection-derived HPV31 virions were bound to HaCaT (black boxes), Ect1 (red triangles), or End1 (blue circles) cells for 1 h at 4°C at a dose of 50 vge/cell, and cells were washed and refed with regular growth media. At the indicated times postattachment, cells were treated with H31.A6 (dashed lines) or refed with new media (solid lines). The cells were harvested at 48 h p.i., and RNA was analyzed in triplicate by RT-qPCR for HPV31 E1⌃E4 levels; values were normalized to the average of the untreated controls at 0 h. Error bars represent SEM. Ab, antibody. (B) AF594-labeled HPV31 was bound to HaCaT cells on glass coverslips at 10,000 vge/cell for 1 h at 4°C. Cells were washed, refed with regular growth medium, and shifted to 37°C. At indicated times postattachment, cells were fixed and stained with DAPI to visualize nuclei. (C) AF594-labeled HPV16 was bound to HaCaT cells on glass coverslips at 10,000 vge/cell for 1 h at 4°C. Cells were washed, refed with regular growth medium, and shifted to 37°C. At indicated times postattachment, cells were fixed and stained with DAPI to visualize nuclei.
- FIG. 3.
Effects of dominant-negative endocytosis inhibitors on HPV31 entry and early transcription. (A to F) HaCaT cells transfected with GFP-tagged dominant-negative inhibitors of endocytosis and their corresponding controls. At 48 h posttransfection, AF594-HPV31 virions were bound to cells for 1 h at 4°C and cells were washed, refed with regular growth medium, and placed at 37°C. Cells were fixed and stained with DAPI 24 h after the 37°C shift to promote virus internalization. (A and B) Wild-type caveolin 1-GFP (Cav-1 control) and dominant-negative caveolin 1 (Cav-1 DN). (C and D) Wild-type Esp15 (Eps15 control) and Eps15 dominant-negative Eps15 (Eps15 DN). (E and F) Wild-type dynamin 2 (Dyn-2 control) and dominant negative dynamin 2 (Dyn-2 DN). Results are representative of three replicate transfection/infection experiments. (G) Quantification of HPV31 particle entry into transfected cells was performed by blinded evaluation of internalization state of AF594-HPV31 in a total of 100 transfected cells (expressing GFP). (H) Triplicate cultures of transfected HaCaT cells were infected 48 h posttransfection with HPV31 virions at 50 vge/cell. RNA isolated 48 h p.i. was quantified in triplicate for HPV31 E1⌃E4 transcript levels by RT-qPCR. Error bars represent SEM, and statistical significance was achieved with P < 0.05 (*) and P < 0.01 (**).
- FIG. 4.
Effects of biochemical endocytic inhibitors on HPV31 and HPV16 infection of HKs. HaCaT cells were pretreated with increasing concentrations of chlorpromazine (A [0, 10, and 20 μM]) or filipin (B [0, 0.8, and 1.6 μM]) for 1 h at 37°C. HPV31 (open boxes, dashed lines) or HPV16 (triangles, solid lines) virions at 50 vge/cell were bound for 1 h at 4°C to promote viral attachment. Cells were then washed, refed with medium/inhibitor, and placed at 37°C. RNA isolated at 48 h p.i. was quantified for HPV31 E1⌃E4 transcript levels by RT-qPCR. Infections were performed in duplicate, and RT-qPCR was performed in triplicate, with error bars representing SEM.
- FIG. 5.
HPV31 associates with detergent-resistant lipid rafts. (A) AF594-labeled ligands (CT-B, TF, HPV31, or HPV16) were bound to HaCaT cells for 1 h at 4°C and then washed and refed with regular growth medium before incubation at 37°C for 30 min. MβCD treatment for 30 min at 37°C was performed to disrupt cholesterol (right column). Cells were subjected to a control PBS wash (left column) or TX-100 wash (middle and right columns) on ice to remove detergent-soluble membranes. Cells were then fixed and stained with DAPI to visualize nuclei. (B) AF594-HPV31 was bound to cells for 1 h at 4°C and then washed, refed with regular growth medium, and placed at 37°C. At indicated times postattachment, cells were subjected to a control PBS wash (left column) or TX-100 wash (right column) before fixation. (C) HPV31 was bound to HaCaT cells for 1 h at 4°C, and cells were washed and refed with normal growth medium before incubation at 37°C for 30 min in the presence or absence of MβCD. Cells were then lysed, and the samples were subjected to centrifugation on a discontinuous density gradient. Fractions were collected from the top and from pelleted protein (P) and analyzed for HPV31 L1 protein by Western blotting.
- FIG. 6.
HPV31 and HPV16 fail to colocalize during HK entry. AF594-HPV16 (red) and AF488-HPV31 (green) virions (each at 10,000 vge/cell) were added to HaCaT cells for 1 h at 4°C, and cells were washed, refed with regular growth medium, and placed at 37°C. At the indicated time points, cells were fixed, DAPI stained, and visualized by confocal microscopy. Lower panels are higher magnifications of boxed areas in each upper panel.
