Construction of lentiviral vectors.

<p>A) The HIV-1 provirus NL4.3 and the HIV-1-vector V^Hgenomic are shown. Large parts of the <i>gag, pol</i> and <i>env</i> genes are deleted in V^Hgenomic (see white bars in the deleted regions marked by shaded areas). The remaining <i>gag</i> sequence contains parts of the encapsidation signal (Psi, Ψ) and the <i>env</i> fragments contain splicing regulatory elements as well as the RRE. Due to deletions (shaded squares) and frameshift mutations (black asterisks in <i>gag</i> and <i>rev</i>) no viral genes are expressed from V^Hgenomic. Both vectors are drawn to scale. B) Schematic representation of the lentiviral vectors V^Hgenomic, V^Henv and V^Hnef. The intron between SD1 and SA5 or the introns between SD1 and SA5 and between SD4 and SA7 were deleted from V^Hgenomic in V^Henv or V^Hnef, respectively. Unspliced and spliced transcripts with splice sites (5′ splice sites in green and 3′ splice sites in blue) and <i>cis</i>-acting splicing regulatory elements (in orange) are shown. Please note that the unspliced Msd1-sa5 RNA of V^Henv is identical in sequence to the singly-spliced SD1-SA5 RNA of V^Hgenomic. Furthermore, the unspliced Msd1-sa5+Msd4-sa7 RNA of V^Hnef is identical to the fully-spliced SD1-SA5+SD4-SA7 RNA of V^Hgenomic and the singly-spliced Msd1-sa5+SD4-SA7 RNA of V^Henv. Arrowheads represent RT-PCR primers. C) and D) After cotransfection of lentiviral vectors with <i>tat</i> and <i>rev</i> expression plasmids into HEK293T cells cytoplasmic RNA was isolated and analyzed by RT-PCR with primer pairs depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048688#pone-0048688-g001" target="_blank">figure 1B</a>. Agarose gel electrophoretic analyses of PCR products are shown. The amplification products were sequenced to verify splicing between the indicated splice sites.