FIG 3.

Single-amino-acid substitutions in mMx1 that disrupt GTPase/GTP binding and nuclear localization abrogate antiviral activity against HSV-1. (a and b) LA-4 CTRL or cells with DOX-inducible overexpression of parental mMx1 or an mMx1(K49A), mMx1(T69A), or mMx1(R614E) mutant were seeded, cultured overnight, and then incubated in medium supplemented with or without 1 μg/mL DOX for 24 h. (a) After DOX induction, cells were detached, fixed, permeabilized, and stained for intracellular FLAG expression. Histograms show FLAG expression after incubation in the presence (open) or absence (shaded) of DOX. (b) After DOX induction, cells were incubated with (i) IAV strain HKx31 (MOI = 1) or (ii) HSV-1 KOS (MOI = 0.1) for 60 min and washed and cultured at 37°C. (c) 293T cells constitutively overexpressing CTRL or parental mMx1, an mMx1(K49A), mMx1(T69A), or mMx1(R614E) mutant, or mMx2 were stained for intracellular FLAG staining as described above. Representative histograms are shown. (d) 293T cells (5 × 10⁵) constitutively overexpressing CTRL or parental mMx1, an mMx1(K49A), mMx1(T69A), or mMx1(R614E) mutant, or mMx2 were infected with (i) IAV HKx31 (MOI = 1) or (ii) HSV-1 KOS (MOI = 0.1) for 60 min and washed and cultured at 37°C. (b and d) At 24 (IAV) or 48 (HSV-1) hpi, supernatants were removed and clarified. IAV supernatants were activated with 2 μg/mL TPCK-treated trypsin and then titrated on MDCK cells by ViroSpot assay. HSV-1 supernatants were titrated on Vero cells by plaque assay. Data show the mean (±SD) from triplicate samples and are representative of two independent experiments. The dashed line indicates the detection limit of plaque and VS assays. Statistical significance was determined by Student's t test. ***, P < 0.001. ns, not significant.