Fig. 6: KRT72 interacts with HIV-1 cores to inhibit their trafficking to the nucleus. | Nature Communications

Fig. 6: KRT72 interacts with HIV-1 cores to inhibit their trafficking to the nucleus.

From: Keratin-72 restricts HIV-1 infection in resting CD4+ T cells by sequestering capsids in intermediate filaments

Fig. 6

a–c KRT72 restricts HIV-1 cores trafficking toward the nucleus. 293T cells were transfected with mCherry-tagged KRT72 or mock expression constructs. At 24 hpi, cells were infected with HIV-1 labeled with INmNG with or without VSV-G. At 6 hpi (see Supplementary Fig. 12a, b) or 9 hpi, cells were analyzed for capsid colocalization with KRT72 and nuclei were stained with DAPI. Bar, 10 μm. Images (a) and quantification (b) of KRT72-mCherry signals associated with INmNG-labeled from 70 cells. Error bars, mean ± SEM. Micrograph data are representative of three independent experiments. Quantitative results of INmNG-labeled puncta (c) in the cytoplasm or nucleus per cell based on the analysis of 30 cells with or without KRT72. Data are the mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ****P < 0.0001; n.s. (two-tailed, unpaired Student’s t-test), and P-values in (c) are 0.96, 0.0023, and 0.0107, respectively. d, e KRT72 binds to HIV-1 cores during HIV-1 infection. 293T cells were transfected with a FLAG-tagged KRT72 expression construct. At 24 hpi, cells were infected with HIV-1NL4-3.Luc.R-E- (VSV-G); at 3 hpi, cells were washed thrice with PBS, lysed, and treated with or without benzonase for IP assays with an anti-FLAG antibody to precipitate KRT72 (d) or an anti-p24 antibody to precipitate incoming capsids (e). Western blotting was performed to detect FLAG-KRT72 and p24CA using specific antibodies. f, g KRT72 binds to HIV-1 cores in HIV-1-infected resting CD4+ T cells. Resting CD4+ T cells were spinoculated with HIV-1NL4-3 with raltegravir at 300 nM; at 12 h after spinoculation, cells were washed thrice with PBS, lysed, and treated with or without benzonase for IP assays with anti-KRT72 (f), anti-p24 (g), or IgG. Western blotting was performed to detect KRT72 and p24CA using specific antibodies. Western blotting data are representative of three independent experiments. Source data are provided as a Source Data file.

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