Supporting Figures

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Supporting Figure 5
Supporting Figure 6

Fig. 5. Localization of de novo synthesized L2 proteins. HeLa cells were transfected with L2-CHA (A), L2-NHA (B), R9S-CHA (C), or R9S-NHA (D) and processed for immunofluorescent staining 24 h after transfection as previously described. The hemagglutinin epitope was detected with an anti-hemagglutinin antibody.

Fig. 6. Localization of L2 and the viral pseudogenome in cells treated with furin inhibitor. For the Lamp-1 colocalization we used C127 cells, a murine line commonly used to evaluate BPV1 infection, because detection of the Lamp-1 protein in these cells was most compatible with our staining protocol, although we have observed a similar colocalization in HeLa cells (data not shown). C127 cells were allowed to internalize BPV-CHA pseudovirions assembled in the presence of 20 mM 5-bromodeoxyuridine (BrdUrd). After 24 h, cells were fixed and processed. (A–C) The same field processed for codetection of the hemagglutinin-derived (HA) epitope and Lamp-1. Mouse anti-HA staining is shown in the green channel (A) and rat anti-Lamp-1 (clone 1D4B) is shown in the red channel (B). The merge is shown in C. (D–F) The same cells, with the green channel (D) representing the mouse anti-BrdUrd staining and the red channel (E) showing the rat anti-Lamp-1. The merge is shown in F. In these cells, as described for other murine fibroblasts, Lamp-1 is detectable in a ring pattern at the perimeters of late endosomes and lysosomes (1). Localization of the L2-CHA and pseudogenome within the boundaries defined by Lamp-1 staining is evident, indicating entry of the uncoated virions into the late endosomal/lysosomal compartments.

1. Chen, J. W., Murphy, T. L., Willingham, M. C., Pastan, I. & August, J. T. (1985) J. Cell Biol. 101, 85–95.