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. 2019 Sep 24;10(5):e02016-19. doi: 10.1128/mBio.02016-19

FIG 2.

FIG 2

An HIV-enhanced lincRNA transcript (HEAL), linc02574-201, regulates HIV-1 replication. (A and B) Efficient silencing of HEAL in MT4 cells inhibits HIV-1 replication. MT4 cells expressing control vector or one of three shRNAs targeting HEAL were infected with HIV-1. HEAL (A) and GP120 mRNA (B) were quantified by RT-qPCR at 2 days postinfection. Signals were normalized to GAPDH mRNA levels. n = 3, mean ± SD; *, P < 0.05. (C) Antisense oligonucleotides targeting HEAL inhibit HIV-1 replication. H9 cells were infected with lentiviruses encoding a nontargeting control (NTC) oligonucleotide or a HEAL-specific antisense oligonucleotide (HEAL ASO) for 2 days and then infected with HIV-1. The cells were reseeded for 3 days and then reinfected with NTC or ASO lentiviruses. Four days after the second ASO treatment, GP120 mRNA levels were analyzed by RT-qPCR. Signals were normalized to GAPDH mRNA levels. n = 3, mean ± SD; *, P < 0.05; **, P < 0.01. (D) sgRNA targeting HEAL inhibits HIV-1 replication. MT4 cells were transduced with lentiviruses containing a nontargeting control sgRNA (sgNC) or a HEAL-specific sgRNA (sgHEAL) for 7 days and then infected with LAI at an MOI of 0.025 or 0.5. GP120 mRNA levels were measured by RT-qPCR 2 days after infection. n = 3, mean ± SD; **, P < 0.01; ***, P < 0.001. (E) Knockdown of HEAL inhibits HIVBaL replication. Microglial cells expressing control vector or shHEAL-1 were infected with HIVBaL. HEAL and GP120 mRNAs were quantified by RT-qPCR at 9 days postinfection. Results are the mean ± SD from three independent experiments. Signals were normalized to GAPDH mRNA levels. *, P < 0.05; **, P < 0.01. (F) Knockdown of HEAL inhibits HIV89.6 replication in primary PBMCs. Activated primary PBMCs were transduced with control or shHEAL-1 lentivirus for 7 days. After activating for the second time for 3 days, cells were infected with HIV89.6 at an MOI of 0.01. HEAL and GP120 mRNA were quantified by RT-qPCR at 3 days postinfection. Signals were normalized to GAPDH mRNA levels. n = 3, mean ± SD; **, P < 0.01; ***, P < 0.001. (G) HEAL knockdown did not affect IFI6 expression. IFI6 mRNA expression was quantified in MT4 cells expressing control vector or shRNA targeting HEAL. ns, not significant. (H) Overexpression of HEAL enhances HIV-1 replication. HEAL was overexpressed in MT4 cells using a pCDH lentiviral vector, and the cells were infected with HIV-1 2 days later. HEAL and GP120 mRNA levels were measured by RT-qPCR 2 days after infection. Signals were normalized to GAPDH mRNA levels. n = 3, mean ± SD; **, P < 0.01; ***, P < 0.001.