Figure 4
From: Tailored enrichment strategy detects low abundant small noncoding RNAs in HIV-1 infected cells
Functional analysis of HIV-1 sncRNAs. (A) CD4+ T-lymphocytes of three healthy donors were infected with the indicated five different HIV-1 primary isolates and screened for the presence of HIV-1 sncRNAs in 3 contigs (contigs 2, 43, and 58) identified in HIV-1JR-FL infected cultures. Cell cultures from three HIV-1 negative donors were probed for each virus, and the cultures which scored positive for the respective HIV-1 sncRNAs are depicted in red squares. (B) Primary macrophages and CD8+ T-cell depleted PBMCs of two healthy donors were infected with HIV-1JR-FL and HIV-1 sncRNA contig 2 (green) and contig 58 (orange) which were quantified by qPCR 14 (macrophages) and 6 (CD8+ T-cell depleted PBMC) days post infection. Amplification was unsuccessful in non-infected cells (data not depicted). As controls, the cellular miRNAs hsa-miR-21 (white and light grey for non-infected cells) and hsa-miR-223 (black and dark grey for non-infected cells) were quantified. < d.l., below detection limit of 1 RNA copy/103 cells. (C) Predicted secondary structures of sncRNALTR6, hybrids of sense and antisense orientated clones from contig 58, namely sncRNAenv183, sncRNAenv184 and sncRNAenv185, and the positive control siRNA-M184 pol [24] (Additional file 3: Table S3) are depicted. (D) Inhibition of HIV-1 replication in primary macrophages by HIV-1 sncRNAs. Macrophages were infected with HIV-1JR-FL seven days before transfection with sncRNAs. On day 0 cells were transfected with 50 nM of the indicated sncRNA (sncRNALTR6 (green), sncRNAenv183&185 (orange), sncRNAenv184&185 (red)) and viral replication was monitored by p24 ELISA on days 0, 5, 9, and 13 post transfection. Mock transfected (black), scrambled sncRNA (blue) and siRNA nonsense (grey) were used as negative control, and siRNA-M184 pol [20] (open circles) as positive control. (E) Western blot of primary macrophage lysates probed with anti-MxA antibody or anti-β-actin antibody as control. Primary macrophages were transfected with siRNA nonsense, sncRNAenv183&185, and sncRNAenv184&185 or treated with interferon-αA/D (positive control). The negative control comprised of untreated and untransfected macrophages.