Fig 2: LAPTM5 and LAPTM4A restrict HIV-1 infectivity.a–c HeLa cells were cotransfected with FLAG-tagged LAPTM5, 4A, 4B, or mock expression constructs along with Vpr-defective HIV-1 proviral vectors, as indicated. Two days after transfection, HIV-1 production was measured by p24 ELISA, and TZM-bl indicator cells were used to measure HIV-1 infectivity. Cells were lysed for western blotting to assess LAPTM5, 4A, 4B, gp120, and GAPDH expression. **P < 0.01 (two-tailed, unpaired Student’s t-test), data are plotted as mean ± SEM of three independent experiments. All western blot data are representative of three independent experiments; their full-size images are presented in the Source Data.

Fig 3: Vpr enhances HIV-1 infection in LAPTM5-expressing CD4+ T cells.a–c TZM-bl cells were transfected with FLAG-tagged LAPTM5 or mock expression constructs to express LAPTM5 at similar levels as in MDMs. The same number of TZM-bl cells or MDMs was lysed for western blotting to assess LAPTM5 and GAPDH expression (a). LAPTM5-positive or -negative-TZM-bl cells were infected with 100 ng of wild-type or Vpr-defective HIV-1 (b, c) for 3 days. At 1 dpi, cells were treated with raltegravir (2 μM) to block subsequent rounds of infection. HIV-1 capsids or Env-containing virions in culture were measured by p24 or gp120 ELISA, respectively. Data are plotted as mean ± SEM of three independent experiments. Cells were lysed for western blotting to assess gp120, Gag, LAPTM5 and GAPDH expression. d, e CD4+ T cells were stimulated and transduced with tetracycline-controlled LAPTM5 or mock expression lentiviral vectors to produce LAPTM5 at similar levels as in MDMs. The same number of CD4+ T cells or MDMs was lysed for western blotting to assess LAPTM5 and GAPDH expression (d). LAPTM5-positive or -negative-CD4+ T cells were infected with 100 ng of dual-trophic wild-type or Vpr-defective HIV-189.6 for 6 days (e). Viral production was measured by p24 ELISA at the indicated time points. f Working model of how Vpr counteracts LAPTM5 to promote HIV-1 infection. Data are plotted as mean ± SEM of three independent experiments. All western blot data are representative of three independent experiments; their full-size images are presented in the Source Data.

Fig 4: IPF fibroblasts do not modify the mitochondrial transmembrane potential or the ADP/ATP ratio but decrease the total levels of ATP. (A) Representative fluorescence microscopic image of control and IPF fibroblasts after staining with JC-1 (4 μm) in the presence or absence of oligomycin (1 μM) or FCCP (10 μM). The intensity of JC-1 reflects the level of mitochondrial transmembrane potential. (B) The bar graph shows the quantification of JC-1 signals in control and IPF fibroblasts in the presence or absence of oligomycin and FCCP using fluorescence microscopy. The results are expressed as the mean ± SEM. CTRL (n = 1) and IPF (n = 2) (C) Expression levels of ATP synthase in the lysate of control and IPF fibroblasts were determined via Western blot analysis. The expression levels of GAPDH were used as a loading control CTRL (n = 3) and IPF (n = 4) from three independent experiments. (D) ADP/ATP ratio was quantified by a luciferin/luciferase-based luminescence assay. The results are expressed as the mean ± SEM. The differences were analyzed using an unpaired t-test. CTRL (n = 4) and IPF (n = 4). (E) ATP production was quantified by luminescence assay in control and IPF fibroblasts. The results are expressed as the mean ± SEM. The differences were analyzed using an unpaired t-test. ** p < 0.01 vs. control. CTRL (n = 3) and IPF (n = 3).

Fig 5: LAPTM5 restricts HIV-1 infectivity.a HeLa cells were transfected with FLAG-tagged LAPTM5 or mock expression constructs. At 24 h after transfection, cells were infected with 100 ng of Vpr-defective HIV-1NL4-3.Luc.R-E- (VSV-G). At 2 dpi, cells were lysed to measure luciferase reporter activity, and western blotting was performed to assess exogenous protein expression. Data are plotted as mean ± SEM of three independent experiments. b HeLa cells were cotransfected with FLAG-tagged LAPTM5 or mock expression constructs along with a Vpr-defective pNL4-3.GFP.R−E− reporter vector combined with various expression constructs of HIV-1 ×4-, R5-trophic, or dual-trophic Env or VSV-G as indicated. Two days after transfection, TZM-bl indicator cells were used to measure HIV-1 infectivity. *P < 0.05, **P < 0.01; NS, not significant (two-tailed, unpaired Student’s t-test), data are plotted as mean ± SEM of three independent experiments. c, d HeLa cells were cotransfected with non-tagged LAPTM5 or mock expression constructs along with Vpr-defective HIV-1 proviral vectors (NL4-3 Vpr- or BaL Vpr-) and a monitoring expression plasmid of GFP in the presence or absence of a FLAG-tagged Vpr expression vector derived from NL4-3. Two days after transfection, TZM-bl indicator cells were used to measure HIV-1 infectivity (c). Cells were lysed for western blotting to assess LAPTM5, Vpr, GFP, and GAPDH expression (d). Data are plotted as mean ± SEM of three independent experiments. All western blot data are representative of three independent experiments; their full-size images are presented in the Source Data.