PMC

Bacitracin does not affect exposure of the L2 RG-1 epitope. Cells were infected in medium with or without Bac for 0 h or 4 h before fixation and permeabilization for IF as described in Materials and Methods. L2 was stained with mouse monoclonal RG-1 and AF488 anti-mouse (green) with rabbit polyclonal K75 and AF555 anti-rabbit (red). Bar, 10 μm.

Bacitracin does not block endocytic trafficking or exposure of buried L1 epitopes. Cells were infected in medium with or without Bac for 4 h (A) or 8 h (B and C) prior to fixation and permeabilization for IF as described in Materials and Methods. (A) L1 was stained with mouse monoclonal H16.U4 and AF488 anti-mouse (green) with rabbit polyclonal K75 and AF555 anti-rabbit (red). (B) L1 was stained with mouse monoclonal L1-7 and AF488 anti-mouse (green) with rabbit polyclonal K75 and AF555 anti-rabbit (red). (C) L1 was stained with rabbit polyclonal K75 and AF488 anti-rabbit (green), and LAMP1 was stained with mouse anti-LAMP1 and AF555 anti-mouse (red). Nuclei of all samples were stained with DAPI (blue). Bars, 10 μm (A and B); 5 μm (C).

Bacitracin prevents vDNA localization at nuclear PML bodies. Cells were mock treated (A to E) or infected with HPV16 in medium alone (F to J) or in the presence of either Bac (K to O) or BafA (P to T) for 42 h prior to fixation and IF and as described in Materials and Methods. Nuclear PML bodies were stained with rabbit anti-PML and AF555 anti-rabbit (red). EdU-labeled vDNA was stained via click chemistry labeling with AF488-N₃ (green), as described in Materials and Methods. L1 was stained with the mouse monoclonal L1-7 and AF633 anti-mouse (purple pseudocolor). Nuclei were stained with DAPI. Bars, 5 μm.

Bacitracin blocks HPV16 infection via a cellular process. (A) Continuous infection of HaCaT cells in the presence of increasing concentrations of Bac. Infection was quantified after 48 h by luciferase assay. RLU, relative light units. (B) Virus was pretreated with 1 mM or 5 mM Bac in cell culture medium for 8 h at 37°C. Virus was then diluted into complete medium such that the final Bac concentration was well below the inhibitory concentrations, and infection was assayed by luciferase assay. Infections were performed in triplicate, with error bars representing standard errors of the means.

Cellular uptake of bacitracin. (A) Pulse-chase experiment. Cells were mock treated or pulsed with Bac for 1 h at 37°C before they were either fixed for IF or chased with normal medium for the indicated times before fixation and IF. Actin was stained with AF488-phalloidin (green), Bac was detected with sheep anti-Bac polyclonal antibody and AF555 donkey anti-sheep antibody (red), and nuclei were stained with DAPI (blue). (B) Colocalization of internalized Bac with cellular markers. HaCaT cells were grown in 5 mM Bac for 8 h prior to fixation and IF and as described in Materials and Methods. Bac was stained with sheep anti-Bac polyclonal and AF555 donkey anti-sheep (red) antibodies. Cells were counterstained with antibodies against the indicated markers (green), as described in Materials and Methods. Nuclei were stained with DAPI (blue). The inset shows colocalization of Bac signal with the indicated cellular markers, quantified by the Manders method, as described in Materials and Methods. (C) Colocalization of Bac with HPV16. Cells were infected in medium plus Bac for the indicated times prior to fixation for IF. HPV16 L1 was stained with rabbit anti-L1 K75 polyclonal and AF488 donkey anti-rabbit (green) antibodies. Bac was stained with sheep anti-Bac polyclonal and AF555 donkey anti-sheep (red) antibodies. Nuclei were stained with DAPI (blue). Bars, 10 μm (A); 5 μm (B and C).

siRNA knockdown of PDI family members. (A) HaCaT cells were transfected with siRNAs against PDI family members or a scrambled control (Scr) for 24 h prior to infection with HPV16, as described in Materials and Methods. Luciferase activity was measured 24 h postinfection. Results are from three independent experiments, each performed in triplicate, with error bars representing standard errors of the means; *, P < 0.001, paired two-tailed Student's t test. (B) Confirmation of specific siRNA knockdown by Western blotting of siRNA-transfected/infected-cell lysates. (C and D) HaCaT cells plated on coverslips were transfected with siRNAs prior to continuous infection with EdU-labeled HPV16 for 16 h. Cells were stained with rabbit anti-PDI in panel C and rabbit anti-ERp72 in panel D, both with AF555 anti-rabbit (red). EdU-labeled vDNA was stained with AF488-N₃ (green) as described above. (E and F) Localization of vDNA (green) and PML bodies (red) in Scr (E) or combined PDI/ERp72 (F) siRNA-transfected cells. Three representative images are shown for each condition. Arrows indicate colocalized signals; bars, 5 μm.

Bacitracin does not block γ-secretase but may inhibit a critical reductive function during infection. (A) Cellular γ-secretase activity assay. Cells were transfected with an irrelevant GFP plasmid (mock) or the γ-secretase reporter pAPP-C99-Myc expressing a Myc-tagged C99 fragment of the amyloid precursor protein for 6 h prior to replacement of growth medium with or without the indicated drugs. Cell lysates were harvested at 24 h for SDS-PAGE and Western blotting assays with the indicated antibodies. Cleavage of the C99 fragment by γ-secretase generates the smaller AICD fragment, marked with an arrow. The asterisk denotes a nonspecific cleavage product of C99, which has been reported by others in the literature using this same plasmid (). (B) Transient reduction can partially repress Bac inhibition. Cells were infected with virus with or without Bac for 8 h prior to removal of the virus and addition of fresh medium with or without Bac and supplemented with the indicated concentrations of β-ME. Cells were incubated with the reductant with or without Bac for 12 additional hours prior to switching back to medium with or without Bac without β-ME reductant and incubation for another 28 h (48-h total infection time) prior to measurement by luciferase assay. Infections were performed in triplicate, with error bars representing standard errors of the means.

Time-dependent effects of bacitracin. (A) Binding assay. Virus was bound to cells in medium with or without 5 mM Bac for 1 h at 4°C. Cells were then thoroughly washed and either trypsin or mock treated before harvesting of total protein by the addition of SDS-PAGE loading buffer. Cell-associated L1 was detected by Western blotting with an anti-L1 monoclonal antibody. The asterisk denotes a nonspecific band of cellular origin. (B) Quantification of the band intensities from three separate binding assays. (C and D) Virus uptake assay in the absence (C) or presence (D) of 5 mM Bac. Cells were mock infected (M, lanes 1 and 10) or prebound with virus and either processed immediately (0 h, lanes 2 and 11, the bound input virus) or trypsinized after various lengths of time at 37°C (lanes 3 to 9 and 12 to 18) before processing for nonreducing SDS-PAGE and Western blotting for L1. Molecular masses (kDa) are shown to the left, and positions of the L1 forms are denoted on the right. The asterisk again denotes the nonspecific band. (E) Bac-induced binding and entry occur through the infectious pathway. Infections were set up with or without Bac pretreatment and with or without Bac during virus binding as described in the text. Cells were washed and switched to 37°C, and infection was measured at 48 h by luciferase assay. (F) Time of addition/withdrawal experiments. Cells were bound with virus in medium with or without Bac and washed, and medium with or without Bac was replaced. After various times at 37°C, medium was changed to add Bac (time of addition, solid line) or to remove Bac (time of withdrawal, dashed line). The highest levels of infection for each experiment were normalized to 100%. All infections were performed in triplicate, with error bars representing standard errors of the means.