Figure 4: HIV-1 HA/His₆-Vpr as a valid alternative.

(a) Strategy used to construct HA/His6-Vpr starting from sequence verified gBlocks^®. This was cloned in the pNL4-3 HIV HSA plasmid using the previously described cloning strategy (Supplement 1s). Numbers indicate amino acid position of Vpr (black), and number of amino acids added by tagging (grey). (b) In silico mRNA folding predictions of NL4-3 HIV-1 HA/His6-Vpr genomic region p5041–9017, using Genebee RNA secondary structure prediction algorithm. The circle specifies the region where predicted folding is altered, compared to the WT NL4-3 HIV-1 predicted folding, numbers indicate the position in the NL4-3 genome, starting from the 5′ LTR sequence. (c) In silico predictions of full HIV-1 HA/His6-Vpr genome mRNA folding structure, using PPfold3.1 bioinformatics tool. The rectangle zoom-out and arrows specify the region where predicted folding is altered. (d) In silico mRNA folding predictions of NL4-3 HIV-1 WT, HIV-1 HA/FLAG-Vpr and HIV-1 HA/His6-Vpr genomic region p5000–6500 (or extended with tags length), using Genebee. Location of 5′ and 3′ ss is indicated as green dots, based on⁷. The position of the inserted tags within the constructs is highlighted with a blue line. (e) Western blot for HA and His₆ expression of pNL4-3 WT HIV-1 or pNL4-3 HIV-1 HA/His₆-Vpr plasmid transfected 293T cells, 48h post-transfection. 10 μg of protein was loaded and β-actin was stained as loading control. (f) Splicing of mRNA, isolated from 293T cells, 48 h post-transfection with pNL4-3 WT HIV-1 or pNL4-3 HIV-1 HA/His₆-Vpr plasmids, as described in Fig. 3a. Graph shows expression of unspliced (US), singly spliced (SS) and multiple spliced (MS) mRNA relative to WT levels, normalized for reference gene expression and corrected for total HIV-1 RNA. Error bars indicate the standard error of the mean (SEM).Wilcoxon signed-rank test, N = 6, p = 0.2188 (p2 + 4), 0.1563 (p5 + 6), 0.03125 (p3 + 8), 0.0625 (p2 + 8), 0.03125 (p3 + 9), 0.09375 (p7 + 9).