p30 specifically recruits PA28γ to trap viral tax/rex RNA into the nucleus. (A) Simultaneous in situ hybridization detection of the tax/rex viral mRNA, immunolocalization of endogenous PA28γ and GFP-p30 show nuclear accumulation of tax/rex mRNA in the presence of GFP-p30. (B) To quantify the level of colocalization between tax/rex RNA detected by RNA FISH and PA28gamma, we measured their signal intensity profiles in cells with and without p30. The signal intensity profiles indicate that there is an increased level of PA28gamma at the sites of tax/rex RNA localization in the presence of the p30 protein in comparison to the cells without p30. (C) RNA immunoprecipitation shows specific recruitment of PA28γ onto the tax/rex mRNA in the presence of p30. HTLV-1 molecular clone pBST was transfected along with p30HA and/or FLAG-PA28γ into FT-27 PA28γ knockdown cells. Lysates were immunoprecipitated with anti-FLAG antibody and RNA extracted TRIzol and analyzed by RT-PCR for the presence of tax/rex or p21rex mRNA. (D) Western blot was performed on cell lysates to confirm p30HA expression and similar expression of transfected FLAG-PA28γ. Similar amounts of PA28γ were immunoprecipitated using the FLAG antibody as shown by Western blotting of immunoprecipitates.