Fig 2: Role of ZAP in restricting SARS-CoV-2 production. (A) SARS-CoV-2 RNA levels (top panel, n = 3 [biological replicates] ± SEM) and infectious titers (middle panel, n = 2 [biological replicates] ± standard deviations [SD]) in the supernatant of Calu-3 cells that were left untreated and/or uninfected or infected with SARS-CoV-2 and treated with the indicated amounts of IFNs. Cells were additionally transfected either with control siRNA (CTRL) or ZAP siRNA (ZAP) as indicated. Immunoblots of whole-cell lysates were stained with anti-ZAP, anti-KHNYN, anti-TRIM25, and anti-GAPDH (bottom panel). Relative expression levels of the long (L) or short (S) isoforms of ZAP are indicated below the blots. One representative blot of 2 biological replicates is shown. (B and C) SARS-CoV-2 RNA copy numbers (B) or infectious virus titers (C), quantified relative to the control in the supernatant of Calu-3 cells that were left untreated or treated with the indicated IFNs and transfected with control (C) or ZAP (ZAP) siRNA. Numbers above the samples indicate the average change in viral RNA copy numbers (vRNA) or infectious titer. P values: *, <0.05; **, <0.01; ***, <0.001; ****, <0.0001; unpaired Student's t test.

Fig 3: Cellular distribution of ZAP-S, ZAP-L and their CaaX motif mutants.(A) Confocal microscopy images of live HEK293T ZAP KO cells 24h after transfection with 250ng of GFP-tagged ZAP isoforms or ZAP-L with inactivated CaaX (C899S) and ZAP-S with added CaaX motif (+CVIS). Size bar 10µm. (B) Representative western blot and quantification of ZAP present in cell fractionation samples of parental HEK293Ts (mean of n = 5) or (C) ZAP KO cells following transfection of 60ng HA-ZAP constructs (mean of n = 3). Cytoplasmic (C), membrane (M) and insoluble (I) fractions are shown. Calnexin serves as a marker for membrane protein and G3BP and GAPDH are cytoplasmic protein controls.

Fig 4: ZAP cofactors KHNYN and TRIM25 bind most efficiently to wild type ZAP-L.Upper left panel: Representative western blot of GFP-ZAP isoforms and mutant proteins expressed in HEK293T ZAP KO cells. Upper right panel: GFP-ZAP, TRIM25 and KHNYN co-immunoprecipitated using GFP-binding magnetic beads. Input and pulldown samples were stained for GFP as well as endogenous KHNYN and TRIM25. Lower panels: quantification of KHNYN and TRIM25 co-immunoprecipitated with GFP-ZAP normalized to the relative GFP signal. Mean of n = 5 + SD. * p < 0.05 for the sample compared to ZAP-L. * p < 0.05 for the comparisons demarked by the lines.

Fig 5: Expression and antiretroviral effect of endogenous ZAP. (A) Representative Western blot of ZAP-L and -S expression levels in HeLa, THP-1, and primary CD4+ T cells and monocyte-derived macrophages (MDM) or HEK293T ZAP KO cells transfected with the indicated amounts of ZAP expression or empty control vector. Cells were stimulated with 500 U/ml IFN-a or 200 U/ml IFN-? (with or without IL-2) for 3 days or left untreated. (B) Quantification of ZAP-L and -S expression levels in primary CD4+ T cells and MDM from different blood donors. CD4+ T cells were treated with IFN as described for panel A or treated with IL-2 and anti-CD3/CD28 beads (Act.). *, P < 0.05; ***, P < 0.001. (C) Inhibitory effects of endogenous ZAP in HeLa cells transduced with HIV-1 strains in the presence or absence of IFN-a. Infectious virus yield from control HeLa cells expressing ZAP, measured 3 days postransduction by a TZM-bl reporter assay, was normalized to that obtained from HeLa ZAP KO cells (100%). Numbers in parentheses indicate the number of CpG dinucleotides in the ZAPsen region of each HIV-1 strain. Shown are the results obtained in three independent experiments, each performed in triplicate. Each dot represents the mean value from technical triplicates, with bars indicating means from three independent experiments (± SEM). (D) Correlation between the infectious virus yields in the presence of ZAP overexpression in HEK293T or endogenous ZAP expression in HeLa or THP-1 cells. (E) Correlation between the number of CpG dinucleotides in the ZAPsen region of the HIV-1 env gene and the infectious virus yield in the presence of ZAP in HeLa or THP-1 cells. Symbols in panels D and E represent the average value obtained for one IMC in at least three independent experiments, and infectious virus yields were normalized to those obtained in the absence of ZAP (100%).