Splicing regulatory elements (SREs) in the HIV-1 genome and mutational analysis of ESE
. (A) Top: The open reading frames (ORFs) are indicated by open boxes. The long terminal repeats (LTR) are located at both ends of the provirus. Center: All HIV-1 proteins are encoded in a single primary transcript. More than 40 different viral mRNAs are produced by alternative splicing allowing efficient translation of all ORFs within the infected cell. Intrinsic strength of the 5′ss (D1 to D4) and 3′ss (A1-A7) is indicated in parentheses (5′ss: HBond Score, http://www.uni-duesseldorf.de/rna; 3′ss: MaxEntScore, http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq_acc.html). Bottom: Positions of the SREs within the HIV-1 pre-mRNA: splicing enhancers (green) and silencers (red) are indicated. [ESE705-29 [14,40]; ESE-Vif ; ESEM ; ESE4932-62 ; ESE5005-32 ; guanosine (G) rich silencer G4 ; GI2-1 ; ESSV [43-45]; ESEvpr ; GI3-2 ; ESS2p ; ESE2 [15,47]; ESS2 [48-50]; guanosine-adenosine rich (GAR) ESE [13,37,51]; E42 fragment ; ISS ; ESE3 ; ESS3 [53-55] (adapted to [51,56]). Primers used in RT-PCR analyses are indicated by arrows (forward: E1, reverse: E4, E5, I4 and E7). (B) Top: HIV-1 exon 4 reference (pNL4-3) and mutant sequences used in this study. The ESEtat is indicated by a grey rectangle. Previously published SREs in this region are underlined. Bottom: HEXplorer score profiles for wild-type HIV-1 exon 4 reference (white) and mutant sequences (black). ESEtat is indicated by a grey rectangle. ESE2 is indicated by curly braces. Positions of mutated nucleotides are indicated by arrows.