Purified Py, and antibodies against VP1 and large T antigen were provided by Tom Benjamin (Harvard Medical School). The CFP-Rab5a, dominant negative CFP-Rab5a (S34N), constitutively active CFP-Rab5a (Q79L), YFP-Rab7, dominant negative YFP-Rab7 (N125I) and LAMP1-YFP constructs were generous gifts from Joel Swanson (University of Michigan). The CFP-Heme Oxygenase-2 construct was from Melissa Rolls (Penn State). A monoclonal antibody against GD1a was purchased from Millipore, purified GD1a and GM1 from Matreya, monoclonal Myc antibody, Quantum dots 655, Texas Red-X, and Alexa Fluor 594 from Invitrogen, and proteinase K, trypsin, and NH4Cl from Sigma.
Preparation of Texas Red or Alexa Fluor 594 labeled Py
Purified Py was labeled with Texas Red–X succinimidyl ester (1 mM) or Alexa Fluor 594 succinimidyl ester (1 mM) following the manufacturer's protocol (Invitrogen). The labeled Py was separated from excess labeling reagent using a Micro Bio-Spin 30 Column (Bio-Rad Lab).
Preparation of Quantum dot coated with a GD1a, Myc, or TfR antibody
Quantum dot 655 (goat F (ab') 2 anti-mouse IgG conjugate) (1 µM) was incubated with a monoclonal antibody against GD1a (0.1, 1, or 10 mg/ml), Myc (10 mg/ml), or TfR (10 mg/ml) in 30 µl PBS at 4°C for 16 hrs with mixing. Protein A agarose beads were added to the sample to precipitate the excess GD1a, Myc, or TfR antibodies. GD1a-, Myc-, or TfR-coated Quantum dots were present in the resulting supernatant.
NIH 3T3 cells were transfected using Effectene (Qiagen) with constructs encoding wild-type and mutant CFP-Rab5, or wild-type and mutant YFP-Rab7. 24 hrs post-transfection, cells were incubated with Py (multiplicities of infections were approximately 100 PFU/cell or 1×104 particles/cell), washed after 24 hrs, and incubated for an additional 24 hrs. Cells were then fixed and subjected to immunofluorescence (IF) with an antibody against the virus-encoded large T antigen. Phase and IF images were collected with a Nikon TE2000-E microscope using the Plan Fluor Ph2 40×/Na 0.75 objective. Only those cells expressing the transfected protein were analyzed. Where indicated, GD1a (180 µM) or GM1 (180 µM) were incubated for 24 hrs prior to infection. For characterizing the effect of bafilomycin A1 and NH4Cl on Py infection, cells were treated with bafilomycin A1 (0.2 µM) or NH4Cl (50 mM) 2 hrs pre-infection, simultaneously with infection, or 3 hrs post-infection. Cells were then infected with crude Py for 3 hrs and the unbounded virus was removed by washing. The cells were incubated at 37°C for additional 48 hrs, fixed and subjected to T antigen expression analysis.
Time-lapse live fluorescence microscopy and image analysis
NIH 3T3 cells were transfected using Effectene (Qiagen) with the indicated constructs for 1 to 2 days, and where indicated, GD1a was added 24 hrs pre-infection. Cells were incubated with labeled Py (or Q-dot) at 4°C for 0.5 hr and the unbounded virus (or Q-dot) was removed by washing. The cells were incubated at 37°C for the indicated time, and observed with a Nikon TE2000-E microscope equipped with 100× objective. Images were acquired at 5 s or 10 s intervals.
For co-localization of Py (or Q-dot) and endolysosomal markers (CFP or YFP), different color images were taken sequentially with Nikon filter cubes for Texas Red (96313), CFP (96341) and YFP (96345). For co-localization of Py with ER (CFP-HO2), the ECFP/DsRed filter set (51018, Chroma) was used to simultaneously image the two colors. The dual-color image was split to two channels by Dual-View image splitter (Optical Insight) and projected to the two halves of a CCD camera (CoolSnap EZ2, Photometrics). To correct the imaging mis-alignment between different channels, Py or Q-dot images were registered to the other channels by bilinear transformation. To define the boundaries of the ER clearly, the ER images were subjected to filtering with the Fast Fourier Transform Bandpass Filter embedded in Image J (NIH). The filtering settings were set to 15 pixels with large structures and up to 3 pixels with small structures, and a tolerance of direction of 5%. Co-localization was defined as overlapping of the objects of interest in the two channels for at least 30 s in a movie.
Cells were fixed with formaldehyde (3%), permeabilized with Triton X-100 (0.2%), and incubated with either an antibody against Py large T antigen or VP1. Cells were then washed, and incubated with a fluorescently tagged secondary antibody (rhodamine labeled donkey anti-rat (for large T) or anti-rabbit (for VP1).
Cell surface binding and entry
Control or GD1a-supplemented cells were incubated at 4°C, infected with Py, and either continued to be incubated at 4°C for 1 hr or incubated at 37°C for 1 hr to allow entry. Cells were harvested and treated with proteinase K (30 µg/ml) where indicated. Proteinase K was heat-inactivated, and the lysate was subjected to SDS-PAGE followed by immunoblotting with a VP1 antibody.
Low pH-induced Py conformational change
Py was initially incubated in phosphate buffered saline (PBS) at pH 7.5, 6.0, or 5.0 for 60 min at 37°C. Virus incubated at pH 6.0 or 5.0 were then neutralized to pH 7.5 by addition of PBS (pH 10.0). The virus was subsequently incubated with a low concentration of proteinase K (2.5 ng/ml) or a high concentration of trypsin (1 mg/ml) for 30 min at 4°C, and subjected to SDS-PAGE followed by immunoblotting with a VP1 antibody.
ER-dependent conformational change
Py incubated at pH 7.5, or pretreated at pH 5 and neutralized to pH 7.5, was analyzed as described in 
Sucrose flotation of Q-dot
Sucrose flotation analysis is described in 
, except that Q-dot coated with a GD1a antibody was used instead of Py, and a monoclonal secondary antibody fused to HRP was used during immunoblotting.