Figure 1

Schematic design of WPRE elements and vectors. (a) The post-transcriptional regulatory element (PRE) from Woodchuck Hepatitis Virus B (WHV) is drawn as a gray bar; nucleotide numbering is according to Genbank accession no. J02442. Five different PRE fragments have been used in the current study. Based on secondary structure predictions, three different PRE fragments were tested: aPRE (450 nt.), bPRE (600 nt.) and cPRE (900 nt.).⁴ The PRE fragment used in lentiviral vectors (nt. 1093–1684) was termed LPRE. The partially overlapping open reading frame (ORF) for the WHV X protein is drawn as a thick black line. The X protein promoter is depicted as a box (P). All four ATGs (marked by asterisks in the original sequence), which are followed by an ORF encoding more than 25 amino acids were mutated and this construct was named bPRE4^*. The oPRE is derived from bPRE4^* and is completely devoid of the X protein promoter. (b) Gammaretroviral and lentiviral vectors used in the present study are shown in their configuration after integration. Gammaretroviral vectors are based on murine leukemia virus,⁴ lentiviral vectors on human immunodeficiency virus.^{3, 25} Gammaretroviral LTR-driven vectors (MP110, MP71), gammaretroviral SIN vectors (Sin11.CMV) and lentiviral SIN vectors (LentiCMV) are depicted. MP stands for myeloproliferative sarcoma virus.²⁸ The leader region of MP110 is devoid of any splice signals in order to evaluate effects of the PRE independently of splicing. In contrast, MP71 contains a splice-competent intron and a destabilized GFP version (d2GFP). The gammaretroviral and lentiviral self-inactivating vector (Sin11.CMV and LentiCMV, respectively) lack the entire enhancer–promoter of the U3 region¹⁵ and use the internal CMV promoter for expression in the target cells. In addition, the lentiviral SIN vector contains a Rev-responsive element (RRE) and a central polypurine tract (cPPT; not shown) in the leader region. The different PRE variants are located in the 3′ UTR. Primer binding site (θ), splice signals (SD, SA) and packaging signal (Ψ) are marked. The probes used for the Northern blots are shown as black lines. Details on the cloning procedures are available on request (schambach.axel@mh-hannover.de).