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. 2024 Feb 6;27(3):109152. doi: 10.1016/j.isci.2024.109152

Figure 5.

Figure 5

PCID2 blocks HIV-1 RNA alternative splicing during latency in cell line models and cells obtained from people living with HIV

(A) Relative percentage of intron-containing unspliced (US) and completely spliced (CS) HIV-1 RNA in shControl and shPCID2 knockdown J-Lat 11.1. Bars and error lines represent mean and SEM respectively (n = 4). Statistical significance was determined by t test, ∗p < 0.05, ∗∗p < 0.01.

(B) Fold change in HIV-1 RNA splicing variants upon PCID2 knockdown relative to shControl. Values were normalized to GAPDH and UTR HIV-1 transcript. Bars represent mean and error lines represent SEM. Statistical significance was determined by t test; ∗p < 0.05, ∗∗p < 0.01.

(C) Percentage of sh Control and shPCID2 J-Lat cells expressing GFP and intracellular p24 as analyzed by flow cytometry. Bars represent mean of three independent infections and error lines represent SEM. Statistical significance was determined by t test; ∗p < 0.05, ∗∗∗p < 0.001.

(D) Western blot of PCID2-Flag in control or PCID2-Flag overexpressing J-Lat 11.1. Beta tubulin was used as loading control.

(E) HIV-1 unspliced (US) RNA decay dynamics in control and PCID2-Flag overexpressing J-Lat 11.1. Cells were treated with Actinomycin D (10 μg/mL) and RNA was collected at 0, 1, 2, 4, 6, 8 and 10 h after treatment. Gene expression of unspliced RNA was normalized to 18S RNA and values are calculated relative to time point 0 h. Symbols represent the mean of three independent experiments and error bars represent SEM. Statistical significance was determined by t test; ns = not significant.

(F) B2-microglobulin RNA decay dynamics in control and PCID2-Flag overexpressing J-Lat 11.1 as described in B. Gene expression of B2-microglobulin RNA was normalized to 18S RNA and values are calculated relative to time point 0 h. Symbols represent the mean of three independent experiments and error bars represent SEM.

(G) Timeline schematic of shRNA mediated PCID2 knockdown in CD4+ T cells obtained from people living with HIV-1.

(H) Gene expression analysis of PCID2 mRNA in shControl and shPCID2 knockdown CD4+ T cells from three PLWH donors 48 h after transfection. Bars and error lines represent mean and SEM respectively.

(I) Release in post-transcriptional blocks to latency calculated as a ratio of relative abundance in HIV-1 RNA splicing variants CS HIV-1 RNA and US HIV-1 RNA normalized to housekeeping in shControl and shPCID2 knockdown CD4+ T cells from three PLWH donors.