Article Figures & Data
Figures
- FIG 1
CRISPR/Cas9-assisted generation of Jurkat T cells expressing HA epitope-tagged SERINC5 alleles. (A) Close-up of the CRISPR/Cas9-assisted homology-directed knock-in strategy. The guide RNA target sequence is underlined, with protospacer adjacent motif (PAM) in italics. HDR, homology-directed repair. (B) Putative topology of SERINC5 protein and indication of the HA epitope insertion (Protter) (31). (C) HA insertion at the interface of E290 and H291 of SERINC5 preserves the antiviral function of SERINC5 and its susceptibility to Nef-mediated counteraction. HEK293T cells were seeded into 12 wells and cotransfected with the indicated proviral DNAs and decreasing amounts of the indicated SERINC5-encoding plasmids (200 to 8 ng). At 2 days posttransfection, the infectivity of secreted virions was analyzed in a TZM-bl-based assay. Shown are the relative levels of infectivity, with 100% representing the infectivity of particles generated in vector-transfected controls. Values are arithmetic means ± SD from one representative experiment out of two independent experiments. (D) Illustration of the SERINC5 genomic locus with primer binding sites and location of the HA tag. (E) Genomic DNA from parental Jurkat cells and Jurkat SERINC5(iHA/iHA), SERINC5(iHA/KO), and SERINC5(KO/KO) clones was analyzed by PCR as indicated (also illustrated in panel D), and products were separated on a 7.5% acrylamide–TBE gel. Shown are data from one representative experiment out of two independent experiments.
- FIG 2
Characterization of expression of endogenous SERINC5 protein and SERINC5 mRNA. (A) Q-RT-PCR analysis of SERINC5 mRNA expression in parental Jurkat T cells and the indicated clones. SERINC5 mRNA expression in parental T cells was set to a value of 1. Error bars indicate SEM of data from three independent experiments. (B) Total RNA from parental T cells and the indicated clones was subjected to Northern blotting using SERINC5- and GAPDH-specific probes. Shown are data from one representative experiment out of two independent experiments. (C) Lysates from the indicated cell lines and clones were subjected to anti-HA and anti-MAPK immunoblotting. Shown are data from one representative experiment out of three independent experiments. (D) Lysates from the indicated cell lines and clones were loaded either directly (input [ø]) or following mock digestion (−) or PNGase digestion (+) in glycoprotein-denaturing buffer. (E) The indicated cell lines and clones were immunostained for HA surface expression and analyzed by flow cytometry. Shown are representative histograms from one experiment out of three independent experiments. (F) Quantification of the mean fluorescence intensity (MFI) of SERINC5(iHA) surface expression. Error bars indicate SEM from three experiments, including the one shown in panel D. (G) The indicated clones were stained with CTX-FITC, followed by anti-CTX antibody and anti-HA antibody staining. Cells were then PFA fixed and analyzed by confocal microscopy. Shown are data from one representative experiment out of three independent experiments. (H) The indicated clones were PFA fixed, permeabilized, and immunostained with anti-HA and anti-EEA-1 antibodies. Shown are data from one representative experiment out of three independent experiments.
- FIG 3
Type I interferon modulates cell surface expression of endogenous SERINC5 protein in the absence of modulation of mRNA and protein quantities. (A) Q-RT-PCR analysis of SERINC5 and IFIT1 mRNA expression in SERINC5(iHA/iHA) Jurkat clones 48 h after treatment with the indicated IFN-α subtypes (10 ng/ml) or mock treatment. SERINC5 and IFIT1 mRNA expression levels in mock-treated cells were set to a value of 1. Error bars indicate SEM of data from three independent experiments. (B) Parental cells and the indicated SERINC5(iHA/iHA) clones were treated with the indicated IFN-α subtypes (10 ng/ml) or mock treated. Lysates were then subjected to anti-HA, anti-ISG15, and anti-MAPK immunoblotting. Numbers indicate fold changes in levels of the indicated proteins. For each cell line, one representative immunoblot from two to three independent experiments is shown. (C) Parental cells and the SERINC5(iHA/iHA) clone P1E8 were treated for 18 h with ruxolitinib (10 μM) or mock treated, followed by treatment with the indicated with IFN-α subtypes (10 ng/ml) or mock treatment for an additional 48 h. Cells were then immunostained with anti-HA for surface SERINC5(iHA), PFA fixed, permeabilized, and immunostained for intracellular MXA/B. Numbers indicate mean fluorescence intensities for SERINC5(iHA) (green) and for MXA/B (red). Shown are representative dot plots from one experiment out of six independent experiments. (D) Quantification of relative cell surface SERINC5(iHA) protein expression in the indicated SERINC5(iHA/iHA) clones. Error bars indicate SEM of data from two to six independent experiments, including the one shown in panel C. Statistical analysis refers to each individual interferon subtype in the absence and presence of ruxolitinib. (E) The SERINC5(iHA/iHA) clone P1E8 was treated for 48 h with IFN-α2a (250 U/ml), labeled with CellTracker red CMTPX dye, and immunostained with anti-HA. Cells were then PFA fixed and analyzed by confocal microscopy. Mock-treated parental cells are shown as a specificity control.
- FIG 4
HIV-1 Nef-mediated enhancement of particle infectivity may occur in the absence of exclusion of endogenous SERINC5 from virions. (A) Parental Jurkat T cells and the indicated clones were infected with VSV-G-pseudotyped HIV-1 WT IRES-GFP and HIV-1 Δnef IRES-GFP. At 2 days postinfection, cells were analyzed for GFP expression by flow cytometry. Error bars indicate SEM from three independent experiments. (B) Infectivity of virions secreted from infected cells (shown in panel A) was analyzed in a luminometric TZM-bl infectivity assay. Shown are SEM from three independent experiments (corresponding to the data in panel A). RLU, relative luminescence units. (C) Parental cells, the indicated SERINC5(HA/HA) clones, and SERINC5(KO/KO) clone B1 were electroporated with a plasmid encoding the β-lactamase-Vpr chimeric fusion protein (pBlaM-Vpr) and proviral plasmids encoding WT HIV-1 or the HIV-1 Δnef mutant. Concentrated virus-containing supernatants were added, in the presence or absence of the HIV-1 fusion inhibitor T-20, to TZM-bl cells, and fusion was quantified as the change in the fluorescence emission of the cell-permeable CCF2 substrate upon cleavage by BlaM-Vpr by flow cytometry. (Left) Relative HIV-1 fusion, normalized to that of WT HIV-1, of clone B1; (right) corresponding relative HIV-1 infectivity. Shown are SEM from two to three independent experiments. (D) Concentrated supernatants from infected, uninfected, and pVSV-G-transfected cells were subjected to immunoblotting with the indicated antibodies. (E) Relative levels of HIV-1 p24-associated SERINC5(iHA) protein were quantified by Odyssey infrared-based imaging. Shown are SEM from three independent experiments.
- FIG 5
HIV-1 Nef modulates the subcellular localization and trafficking of endogenous SERINC5. (A) Parental Jurkat T cells and the indicated clones were infected with the indicated VSV-G-pseudotyped viruses. At 2 days postinfection, intact cells were immunostained for surface HA prior to PFA fixation and analyzed for SERINC5(iHA) surface expression by flow cytometry. In the dot plots, the SERINC5(iHA) surface levels are plotted against GFP. Numbers inside the gates indicate the MFI of the surface SERINC5(iHA) signal. Shown are representative dot plots from one experiment out of three independent experiments. (B) Relative level of SERINC5(iHA) surface expression. The MFI for cell surface-expressed SERINC5(iHA) was quantified on GFP-positive cells in the R3 gate relative to the MFI of GFP-negative cells in the R2 gate. Values obtained for cells infected with the HIV-1 Δnef mutant were set to 100%. Error bars indicate SEM from three experiments. (C) HIV-1 Nef enhances the rate of endocytosis of endogenous SERINC5(iHA). Shown are kinetics of the decrease of surface-exposed SERINC5 in SERINC5(iHA/iHA) clones infected with WT HIV-1 or the HIV-1 Δnef mutant. The values at time zero were set to 100%. Error bars indicate SEM from three experiments. (D) Parental cells and the indicated clones were infected with the indicated VSV-G-pseudotyped HIV-1 at a high MOI. The percentage of GFP-positive cells is indicated. At 2 days postinfection, cell lysates were subjected to immunoblotting with anti-HA, anti-HIV-1 p24CA, and anti-MAPK antibodies. Shown is one representative blot from one experiment out of three independent experiments. (E) Relative levels of cell-associated SERINC5 protein in infected cells were quantified by Odyssey infrared-based imaging. Shown is the quantification of the total HA signal, of the 51-kDa band only, and of the 35-kDa band only. Error bars indicate SEM from three independent experiments.
