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. 2020 May 4;94(10):e01923-19. doi: 10.1128/JVI.01923-19

FIG 1.

FIG 1

PIWIL4 inhibits HIV-1 replication through repressing the HIV-1 5′ LTR activity. (A) The relative expression of human PIWIs in resting and activated CD4+ T lymphocytes as well as in J-lat 10.6 cells was detected by quantitative RT-PCR (qRT-PCR), and results were normalized to the expression of GAPDH. (B) The expression of PIWIL4 in resting and activated CD4+ T lymphocytes from three different healthy donors was examined by immunoblotting. (C to E) Anti-CD3/CD28 antibody-stimulated primary CD4+ T lymphocytes from healthy donors were transfected with PIWIL4-specific siRNAs (si-PIWIL4) or nontarget control (si-NT). (C and D) The cells were collected at 48 h posttransfection, and PIWIL4 expression was determined by qRT-PCR and immunoblotting. (E) Cells were infected with replication-competent HIV-1/NL4-3. The levels of HIV-1 p24 in the supernatants were quantified by ELISA. dpi, days postinfection. (F to H) TZM-bl cells were transfected with si-PIWIL4 or si-NT. The efficiency of endogenous PIWIL4 knockdown by the indicated siRNAs was confirmed by qRT-PCR and immunoblotting (F and G), and cells were jointly transfected with HIV-1 Tat-expressing vector or treated with TNF-α (50 ng/ml for 24 h) (H). Cells were harvested and assayed for luciferase. The quantitation was normalized to the si-NT. (I) The wild-type or NF-κB-binding site-mutated HIV-1 5′ LTR cloned from pNL4-3 was inserted into a pGL3 basic vector. The endogenous PIWIL4 in HeLa cells was knocked down by specific siRNAs. After 12 h, these cells were further transfected with the wild type or mutated LTR promoter-driven luciferase reporter plasmid for an additional 36 h, harvested, and assayed for luciferase. The quantitation was normalized to the si-NT. (J) The 3′ LTR cloned from pNL4-3 was inserted into a psi-check-2 vector. The endogenous PIWIL4 in HeLa cells was knocked down by specific siRNAs. After 12 h, cells were further transfected with this newly constructed reporter plasmid for an additional 36 h. The firefly luciferase driven by the HSV-TK promoter was used to normalize transfection efficiency. Cells were harvested and luciferase expression was assayed. The quantitation was normalized to the si-NT. Results are representative of three independent experiments. Data are presented as means ± SEM. P values were calculated by Student's t test. **, P < 0.01; ***, P < 0.001.