FIG. 6.

Blocking of cORF-mediated nuclear export by LMB, a CRM1 inhibitor. (A) Cotransfection of reporter and effector plasmids into HLtat cells without (−) or with (+) 4 nM LMB. Panels 1 and 2, pHIVgagRRE and pREV; panels 3 and 4, pHIVgagU3RIII and pcORF. Staining was achieved with a mouse monoclonal α-HIV p24 antibody (all panels) and with a rabbit α-cORF antibody (panels 3 and 4). (B) Determination of the levels of HIV Gag precursor in lysates of cells that were or were not treated with LMB, using the HIV p24 capture assay. HLtat cells were transfected with the vector combinations listed in the table below. Each variant was analyzed without (−) and with (+) 5 nM LMB. The cutoff value was determined by the extinction of the lysis buffer. (C) Detection of the HIV Gag precursor by Western blot analysis. The p55 Gag precursor was present only in the absence of LMB in Rev-RRE combinations (lane 1), cORF-RcRE (lane 9), and (very faintly) Rev-RcRE (lane 7). LMB completely blocked HIV Gag expression at a 5 nM concentration. The positions of molecular mass markers are indicated on the left.