Fig. 3.

Effect of mutagenesis of the gE aa27-90 region on gE cellular localization and expression and VZV entry into human T cells in vitro. Melanoma cells were infected with rOka (A, D, and G), rOka-ΔP27-G90 (B, E, and H), or rOka-ΔY51-G90 (C and F), and fixed at 24 h (A–C) and 72 h (D–H) after infection. Cells were labeled with MAb anti-gE (Chemicon) (red), and nuclei were stained with Hoechst 33342 (blue). Insets are enlargements of the images in the panels. (Magnfication: A–F, ×400; G and H, ×630.) (I) Lysates from melanoma cells infected with rOka or gE mutant viruses were collected at 24 and 72 h after infection (p.i.) and analyzed by Western blot using MAb anti-gE 3B3 (Top), or rabbit anti-IE63 (Middle). MAb anti-αtubulin was used for normalization (Bottom). Lane 1, uninfected cells; lane 2, rOka; lane 3, rOka-ΔP27-G90; lane 4, rOka-ΔY51-G90. (J) Primary human tonsil T cells were infected by coculturing with HELF-infected monolayers for 26 h, stained with anti-CD3 antibody and anti-VZV immune serum, and analyzed by flow cytometry. Error bars indicate SD. The titer of the infected monolayers is indicated in the table.