FIG 7.

The majority of the L2 protein becomes exposed within the cytoplasm during infectious entry. (A) Cytoplasm-localized puncta were selected from the experiments shown in both Fig. 4 (PSV16, HPV16 pseudovirus) and 6 (PSV18, HPV18 pseudovirus) and quantified from infected HaCaT cells treated with either 0.5% Triton X-100 (TX) or 5 μg/ml digitonin (DIG). The ratio of L2 to EdU was determined by measuring the signal strength of each channel. (B) Histograms of signal strengths for all channels from select representative puncta from the experiments shown in both Fig. 4 and 6. Note the lack of L2-specific MAb K4 signal under selective permeabilization. (C) HeLa cells were infected with 18L2-R295/8A-harboring pseudovirions for 18 h. Cells were harvested, and lysates were prepared by mechanically disrupting the plasma membranes. Whole-cell extracts were treated with or without 1 μM bafilomycin A1 and/or 0.2% trypsin and/or Triton X-100 for 1 h. Western blotting was performed, and the L2 protein was detected using a cocktail of 18L2-specific MAbs. Detection of BiP protein served as an internal control. The protein levels were quantified by measuring the pixel intensity of each band using densitometry relative to their respective control lanes. Note that the L2 protein is sensitive to trypsin, regardless of the addition of 0.5% Triton X-100. ns, not significant.