Figure 2

Sam68ΔC maintains translational repression of viral mRNAs when perinuclear bundles are dissolved. To analyze the requirement of the cytoskeleton for the formation of Sam68ΔC-induced perinuclear bundles, the effect of disrupting microtubules or microfilaments on viral RNA subcellular distribution was examined. As a comparison, HeLa cells were co-transfected with Rev, the HIV-1 env expression plasmid pgTat and pcDNA (a) or Sam68ΔC (b) and untreated prior to fixation (none). In parallel, Hela cells transfected with pgTat, Rev and Sam68ΔC were treated 48 h post-transfection with nocodazole (Noc.) (c), colcemid (Col.) (d) to disrupt microtubules or cytochalasin D (cyto. D) (e) or latrunculin B (Lat. B) (f) to depolymerise microfilaments for 2 hours prior to fixation. Locations of the unspliced pgTat (env) RNA and Sam68ΔC were determined by in situ hybridization and immunofluorescence respectively. Nuclei were stained with DAPI (g) To determine the impact of altered HIV-1 RNA subcellular distribution on its translation, HeLa cells were co-transfected with Rev, Gag-RRE and pcDNA or Sam68ΔC. 48 hours post-transfection, cells were treated with the indicated drug for 2 hours to depolymerise either the microtubules or microfilaments. Then ³⁵S-methionine was added to the media and incubated for 4 hours prior to harvest. Cell lysates were prepared and incubated with anti-Gag antibody. Immunoprecipitates were run out on 10% SDS-PAGE and exposed to a phosphor screen. The position of the Gag p55 band is indicated.