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

Abstract

The accessory protein Vpx from the red-capped mangabey or mandrill SIV (SIV_rcm/mnd-2) lineage has been reported to increase HIV-1 infection in resting CD4⁺ T cells without affecting SAMHD1, a known target of Vpx in HIV-1 infection. This indicates that Vpx, in addition to SAMHD1, circumvents other restriction factors for lentiviruses. To identify potential restriction factors, this study examined cellular proteins interacting with Vpx_rcm and found that keratin-72 (KRT72), an intermediate filament (IF) protein expressed in resting CD4⁺ T cells, is a host antiviral factor targeted by Vpx. Vpx_rcm/mnd-2 lineages could strongly promote KRT72 degradation, resulting in increased HIV-1 infection in resting CD4⁺ T cells. We discovered that KRT72 restricts HIV-1 replication by sequestering incoming HIV-1 capsids in cytoplasmic IFs. With KRT72, the capsid cores of HIV-1 become attached to IFs, and their trafficking toward the nucleus is inhibited. In contrast, without KRT72, HIV-1 capsids are transported to the nucleus, leading to high levels of integrated HIV-1 DNA. Thus, KRT72 is a Vpx-counteracted antiviral factor that binds the incoming capsids to cytoplasmic IFs, restricting HIV-1 infection in resting CD4⁺ T cells.

Fig. 1. KRT72 is expressed in resting CD4⁺ T cells and degraded by Vpx.

a Schematic representation of KRT72 interaction with VLP-VPx_rcm in resting CD4⁺ T cells. Resting CD4⁺ T cells were electroporated with a FLAG-tagged Vpx expression vector derived from SIV_rcm. Six hours after electroporation, cells were treated with VLP-Vpx_rcm for 8 h and treated with or without MG132 (0.75 µM) for 6 h. Cells were lysed for anti-FLAG IP to precipitate cellular Vpx_rcm-interacting proteins. b, c KRT72 is expressed in resting CD4⁺ T cells. Total RNA was extracted from primary monocytes, MDMs, MDDCs, PMA-treated or untreated THP-1, and CD4⁺ T cells stimulated with or without CD3/CD28 and IL-2 for 72 h in established cell lines (293T, HeLa, and Jurkat cells). KRT72 transcript levels were measured using qPCR and normalized to GAPDH protein levels (b). Western blotting assessed KRT72 and GAPDH protein levels (c). ***P < 0.001 (two-tailed, unpaired Student’s t-test), and P-value is 0.0004. Data are the mean ± standard error of the mean (SEM) of three independent experiments. d–f KRT72 is degraded by Vpx in a proteasome-dependent manner. Resting CD4⁺ T cells were electroporated (d) or not with FLAG-KRT72 (e, f) and treated with VLP particles with or without Vpx from SIV_mac239, SIV_mnd-2, SIV_rcm, or HIV-2_Rod with or without MG132. Western blotting assessed exogenous FLAG-KRT72, endogenous KRT72, SAMHD1, and GAPDH protein levels using specific antibodies (d, e). Total RNA was extracted for qPCR to measure KRT72 transcript levels normalized to GAPDH protein levels (f). n.s. not significant (two-tailed, unpaired Student’s t-test) and P-values are 0.53, 0.84, 0.47, respectively. Data are the mean ± SEM of three independent experiments. Western blotting data are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 2. KRT72 restricts HIV-1 infection in resting CD4⁺ T cells.

a–f KRT72 knockdown restores HIV-1 infection in resting CD4⁺ T cells. Resting CD4⁺ T cells were electroporated with KRT72 or control siRNA with or without a nontagged KRT72 expression vector. Twenty-four hours after electroporation, cells were spinoculated with HIV-1_{NL4-3.Luc.R-E-} (VSV-G) with or without EFV treatment (300 nM) and cultured with or without stimulation with CD3/CD28 plus IL-2. At 3 days postinfection (dpi), cells were lysed to measure luciferase activity (a), and genomic and circular viral DNA was extracted for qPCR to assess early RT (b), later RT (c), HIV-1 integration (d), and 2-LTR circular DNA (e). Data are the mean ± SEM of three independent donors. ***P < 0.001; **P < 0.01; *P < 0.05; n.s. (two-tailed, unpaired Student’s t-test). Aliquoted cells were also analyzed using flow cytometry to measure the surface levels of CD69, CD25, HLA-DR, and CellTrace violet (see Supplementary Fig. 3). Western blotting assessed KRT72, SAMHD1, and GAPDH protein levels using specific antibodies (f). Western blotting data are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 3. KRT72 restricts wild-type HIV-1 infection in resting CD4⁺ T cells.

a–e KRT72 suppresses HIV-1 late RT and 2-LTR circular DNA levels in resting CD4⁺ T cells. Resting CD4⁺ T cells were electroporated with KRT72 or control siRNA. Twenty-four hours after electroporation, cells were spinoculated with HIV-1_NL4-3 with or without EFV (300 nM). At 48 hpi, cells were analyzed by flow cytometry (a). FACS data are the mean ± SEM of three independent donors (b). Western blotting assessed KRT72, SAMHD1, and GAPDH protein levels using specific antibodies (c). At 24, 48, 72, and 96 hpi, genomic and circular viral DNA was extracted for qPCR to assess late RT (d) and 2-LTR circular DNA (e), respectively. Data are the mean ± standard deviation (SD) of three triplicates and are representative of three independent experiments. ***P < 0.001; **P < 0.01; *P < 0.05 (two-tailed, unpaired Student’s t-test), and P-values are 0.009, 0.003, 0.0044 respectively. f–j Resting CD4⁺ T cells were electroporated with siRNAs for KRT72 or control with or without an untagged KRT72 expression vector. Twenty-four hours after electroporation, cells were spinoculated with HIV-1_NL4-3 with or without EFV (300 nM). At 2 dpi, cells were analyzed by flow cytometry (f). FACS data are the mean ± SEM of three independent donors (g). Genomic and circular viral DNA was extracted for qPCR to evaluate later RT (h) and 2-LTR circular DNA (i), respectively. Data are the mean ± SEM of three independent donors. ***P < 0.001; **P < 0.01; *P < 0.05; n.s. (two-tailed, unpaired Student’s t-test), and P-values are 0.0385, 0.0003, 0.0003, 0.0002, 0.0092, 0.0082, 0.0003, respectively. Western blotting assessed KRT72, SAMHD1, and GAPDH protein levels using specific antibodies (j). Western blotting data are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 4. KRT72 restricts HIV-1 infection in resting CD4⁺ T cells and is counteracted by Vpx.

a, b Vpx promotes KRT72 degradation to restore HIV-1 infection in resting CD4⁺ T cells. Resting CD4⁺ T cells were pretreated with VLP with or without Vpx derived from SIV_mac239, SIV_rcm, and SIV_mnd-2 isolates. Cells were electroporated with siRNAs for KRT72, SAMHD1, or control. Twenty-four hours after electroporation, cells were spinoculated with HIV-1_{NL4-3.Luc.R-E-} (VSV-G) with or without EFV (300 nM). At 3 dpi, cells were lysed for Western blotting to assess KRT72, SAMHD1, and GAPDH protein levels using specific antibodies (a) or measure luciferase activity (b). c, d CD4⁺ T cells were electroporated with siRNAs targeting KRT72, SAMHD1, or control. Twenty-four hours after electroporation, cells were spinoculated with HIV-1*_{NL4-3-Luc.R-E-} (VSV-G) carrying or not carrying Vpx proteins derived from SIV_mac239 or SIV_rcm with or without EFV (300 nM). At 3 dpi, cells were lysed to measure luciferase activity (c) or determine KRT72, SAMHD1, and GAPDH protein levels using their respective antibodies (d). ***P < 0.001, **P < 0.01, *P < 0.05, n.s. (two-tailed, unpaired Student’s t-test). Data are the mean ± SEM of three or five independent experiments. e, f Vpx_mac293 mutants L25A, H39A, and W56A but not Q76A promote KRT72 degradation to enhance HIV-1 infection in resting CD4⁺ T cells. Resting CD4⁺ T cells were spinoculated with 100 ng HIV-1*_{NL4-3-Luc.R-E-} (VSV-G) carrying or not carrying WT or mutant FLAG-tagged (L25A, H39A, W56A, or Q76A) Vpx_mac293 with or without EFV treatment (300 nM). At 2 dpi, cells were lysed to measure luciferase activity (e) or for Western blotting to assess KRT72, SAMHD1, Vpx, and GAPDH protein levels using specific antibodies (f). **P < 0.01; n.s. (two-tailed, unpaired Student’s t-test). Data are the mean ± SEM of three independent experiments. Western blotting data are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 5. KRT72 inhibits HIV-1 infection in target cells.

a 293T cells were transfected with FLAG-tagged KRT72 or mock expression constructs at the indicated doses. At 24 hpi, 50 ng HIV-1_{NL4-3.Luc.R-E-} was used to infect cells (VSV-G). At 2 dpi, cells were lysed to measure the luciferase reporter activity, and total DNA was extracted for qPCR to measure early RT, late RT, and 2-LTR circular DNA. **P < 0.01; *P < 0.05; n.s. (two-tailed, unpaired Student’s t-test) and P-values are 0.0006, 0.65, 0.0008, and 0.0006, respectively. Data are the mean ± SEM of three independent experiments. b, c 293T cells were transfected with FLAG-tagged KRT72, MX2, or mock expression constructs. At 24 hpi, cells were infected with 1, 5, 10, 25, 50, or 100 ng HIV-1_{NL4-3.Luc.R-E-} (VSV-G) with or without EFV (300 nM). At 2 dpi, cells were lysed and the activity of the luciferase reporter was measured. Genomic and circular viral DNA were extracted for qPCR to assess HIV-1 late RT, HIV-1 integration, and 2-LTR circular DNA, respectively, and the results obtained from 1 ng HIV-1 infection with mock were set at a relative level of 1. Data are the mean ± SD of three triplicates and are representative of three independent experiments (b). Western blotting was conducted to assess exogenous protein expression using specific antibodies (c). d KRT72 inhibits HIV-1 infection in primary CD4⁺ T cells. Stimulated CD4⁺ T cells were electroporated with FLAG-tagged KRT72 or mock expression vectors at the indicated doses. Twelve hours after electroporation, cells were infected with 10 ng HIV-1_{NL4-3.Luc.R-E-} (VSV-G). At 2 dpi, cells were lysed to measure the luciferase reporter activity, and total DNA was extracted for qPCR to measure early RT, late RT, and 2-LTR circular DNA. **P < 0.01; *P < 0.05; n.s. (two-tailed, unpaired Student’s t-test), and P-values are 0.0002, 0,79, 0.0005, and 0.0019, respectively. Data are the mean ± SEM of three independent experiments. Western blotting data are representative of three independent experiments. Source data are provided as a Source Data file.

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

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-1_{NL4-3.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-1_NL4-3 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.