Packaging cell lines are important tools for transferring genes into eukaryotic cells. Human immunodeficiency virus type 1 (HIV-1)-based packaging cell lines are difficult to obtain, in part owing to the problem that some HIV-1 proteins are cytotoxic in a variety of cells. To overcome this, we have developed an HIV-1-based packaging cell line which has an inducible expression system. The tetracycline-inducible expression system was utilized to control the expression of the Rev regulatory protein, which in turn controls the expression of the late proteins including Gag, Pol, and Env. Western blotting (immunoblotting) demonstrated that the expression of p24 gag and gp120 env from the packaging cells peaked on days 6 and 7 postinduction. Reverse transcriptase activity could be detected by day 4 after induction and also peaked on days 6 and 7. Defective vector virus could be propagated, yielding titers as high as 7 ؋ 10 3 CFU/ml, while replication-competent virus was not detectable at any time. Thus, the cell line should enable the transfer of specific genes into CD4 ؉ cells and should be a useful tool for studying the biology of HIV-1. We have also established an inducible HIV-1 Env-expressing cell line which could be used to propagate HIV-1 vectors that require only Env in trans. The env-minus vector virus titer produced from the Env-expressing cells reached 2 ؋ 10 4 CFU/ml. The inducible HIV-1 Env-expressing cell line should be a useful tool for the study of HIV-1 Env as well.
SummaryAntisense oligonucleotides complementary to the 5' end of the mRNA encoding the Ly-6A protein were used to block the expression of that protein . Using this approach we could inhibit the expression of Ly-6A by 60-80% in antigen-primed lymph node (LN) T cells as well as in the D10 T cell clone. Inhibition of Ly-6 expression resulted in the inability to restimulate in vitro, antigen-primed T cells . It also blocked the activation of normal spleen cells by Con A, monoclonal antibody (mAb) to CD3, and mAb to Ly-6. In contrast, stimulation of normal spleen cells with the pharmacological agents PMA + ionomycin were unaffected by the inhibition of Ly-6 expression . Similar results were obtained with the D10 T cell clone; stimulation with Con A + interleukin 1 (IL-1), antigen-presenting cells (APC), or the clonotypic antibody + IL-1 was greatly reduced in the presence of antisense oligonucleotides to Ly-6. Stimulation with PMA + ionomycin was again unaffected. We also studied the effect ofantisense oligonucleotides on stimulation of preactivated D10 cells . Preactivation of D10 cells with Con A + Ilrl renders them receptive to secondary stimulation by other lymphokines . In this case, antisense oligonucleotides to Ly-6 had no effect on secondary activation with ID2, IIr4 + IITl, or PMA + ionomycin . We conclude from these studies that Ly-6 expression is required for T cell receptor (TCR)-mediated T cell activation.
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