823. Dissecting Overlapping Functions of Transcriptional Enhancer and Polyadenylation in the Lentiviral U3

Abstract

The transcriptional readthrough activity of the 3' long terminal repeat (LTR) of lentiviral vectors is critical to the safety profile of this vector system in future clinical applications. We have previously reported that the 3' polyadenylation (polyA) readthrough activity of the murine leukemia virus long terminal repeat (LTR) is much higher than that of the human immunodeficiency virus type 1 (HIV-1) LTR; however, deletion of the U3 or HIV-1 LTR to generate a self-inactivating (SIN) lentiviral vector results in a high transcriptional readthrough activity (J. Virol. 76: 7209-7219, 2002). This strongly suggests that other transcriptional termination signals (TS) besides the polyA signal (AAUAAA) in the R region are present in the U3 region. By serially inserting back different lengths of the U3 sequence, we demonstrated that the readthrough activity of the HIV-1 3' LTR was inversely correlated with the length of the U3, suggesting that the termination signals span across the entire U3 region. However, the transcriptional control region, co ntaining 124 nt of the U3 sequence, preserves |[sim]| 70-80% of the wild type TS function. Furthermore, deletion of the Sp1 and NF-kB binding sites abolished most of the TS function. Knocking down NF-kB expression by siRNA down-regulated the TS function. The introduction of foreign TS elements including human T cell leukemia virus type I (HTLV-I) polyA signal, human growth hormone gene intron, a tRNA tertiary structure, or a tetracycline responsive element (TRE) with a TRE-binding factor did not restore the TS function. Therefore, the lentiviral U3 contains an overlapping transcriptional enhancer and termination function which is difficult to be separated. Further improving the transcriptional termination but not the enhancer/promoter function of the 3' LTR will be critical to safety improvement of this vector system.