Journal: Molecular Therapy. Nucleic Acids

Article Title: microRNA-422a promotes HIV replication and innate immune evasion by targeting MECP2

doi: 10.1016/j.omtn.2026.102844

Figure Lengend Snippet: HIV infection induces miR-422a in CD4+ T cells via expression of Nef (A) Primary CD4+ T cells were stimulated with anti-CD3/CD28 beads for 3 days, then infected with HIV-1 NL4-3 . The spreading infection was measured by evaluating supernatant p24 concentrations by ELISA at each time point. (B and C) The primary (B) and mature miR-422a expression levels (C) were measured by RT-qPCR after infection with HIV-1 in primary CD4+ T cells, as described in (A). (D) Stimulated primary CD4+ T cells were infected with an HIV-1 single-round reporter virus (VSVG-pseudotyped HIV NL4-3 ΔEnv EGFP reporter virus) for 3 days. Cells were then sorted based on EGFP expression using the Sony MA900 multi-application cell sorter and harvested for RNA isolation. miR-422a expression was measured by RT-qPCR. (E) Stimulated primary CD4+ T cells were infected with HIV-1 single-round reporter virus (VSVG-pseudotyped HIV NL4-3 ΔEnv ΔNef GFP reporter virus) for 3 days. Cells were then sorted based on GFP expression for RNA isolation. miR-422a expression was measured by RT-qPCR. (F) Nef was nucleofected into stimulated primary CD4+ T cells for 48 h. Cells were then collected to evaluate miR-422a expression by RT-qPCR. G. Nef was nucleofected into stimulated primary CD4+ T cells for 48 h. Cells were then stained with anti-CD25 and anti-69 antibodies to detect CD25 and CD69 expression by flow cytometry. Each dot represents data from one donor. Data are representative of the results of three independent experiments ( n = 3 biologically independent samples, mean ± SEM). Statistical significance was analyzed by unpaired or paired student t tests. p ≤ 0.05 [∗], p ≤ 0.01 [∗∗], p ≤ 0.001 [∗∗∗], p ≤ 0.0001 [∗∗∗∗].

Article Snippet: Specifically, VSVG-pseudotyped HIV NL4-3 ΔEnv EGFP reporter virus stocks were produced by co-transfecting the plasmid encoding HIV NL4-3 ΔEnv EGFP (BEI, #ARP-11100) and the plasmid encoding VSVG (Addgene, #8454) at a ratio of 3:1 into HEK-293T cells at 50%–60% confluency in culture flasks.

Techniques: Infection, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Virus, Isolation, Staining, Flow Cytometry