FIG. 3.

Intracellular localization of ORF9 and ORF9 interactions with gE in vitro. (A) Cells infected with POKA were fixed, embedded, cryosectioned, and then incubated with a rabbit antibody to VZV ORF9 followed by incubation with protein A gold conjugates (panels a, b, and d). ORF9 (15-nm gold particles) is incorporated into VZV virions and present in the virion tegument (a, arrow). ORF9 was also detected in association with putative Golgi cisternae and Golgi-derived membranes in the cytoplasm (b, arrows) and with electron-dense materials in the nucleus (d, arrow). When cells were prepared by high-pressure freezing, ORF9 was also detected in association with putative Golgi cisternae and Golgi-derived membranes in the cytoplasm (c, arrows) and with electron-dense materials in the nucleus (e, arrows). ORF9 is absent from perinuclear VZV virions that have a primary envelope (e, arrowhead). Bars, 0.2 μm (a and e), 0.1 μm (b and c), and 0.5 μm (d). N, nucleus. (B) Coimmunoprecipitation of the gE protein with anti-ORF9 rabbit polyclonal antibody. Melanoma cells were infected with VZV and harvested in 1% NP-40 lysis buffer, and cells extracts were incubated with beads coated with ORF9 antibody and Western blotted with anti-gE mouse MAb 3B3. Lane 1, uninfected cell extract; lane 2, beads alone; lane 3, infected cell extract; lane 4, beads coated with ORF9 antibody. (C) Coimmunoprecipitation of the ORF9 protein with beads coated with anti-gE MAb 3B3. Infected cells extracts were incubated with anti-gE MAb 3B3, and the membrane was probed with anti-ORF9 antibody. Lane 1, uninfected cell extract; lane 2, beads alone; lane 3, beads coated with gE antibody; lane 4, infected cell extract. Molecular mass markers are indicated on the left.