Figure 8. Effect of Nup and NTR depletion on HIV-1 infection and antiviral activity of MX2.

(A) Infectivity of HIV-1 GFP reporter virus in dividing (top) and non-dividing (bottom) HeLa cells stably transduced with doxycycline-inducible MX2 in the presence (open circles) or absence (filled circles) of doxycycline 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A). Middle, summary of localization of MX2 from immunoflouresence images in Figure 6. Aberrant localization following siRNA transfection in ≥~80% of cells is indicated by an ‘x’ and normal localization is indicated by a dot. Titers are mean ±sem, n = 3 technical replicates, representative of four independent experiments. (B) Infectivity of HIV-1 GFP reporter virus in dividing (top) and non-dividing (bottom) HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open triangles) or absence (filled triangles) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A). Middle, summary of localization of MX2 from immunoflouresence images in Figure 6. Aberrant MX2 localization following siRNA transfection in ≥~80% of cells is indicated by an ‘x’ and normal localization is indicated by a dot. Titers are mean ±sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210). (C) Schematic representation of the nuclear pore complex and target genes included in siRNA library color coded by subcomplex, as in Figure 4A.

Figure 8—figure supplement 1. Effect of Nup and NTR depletion on HIV-1 infection in HOS cells.

(A) Infectivity of HIV-1 GFP reporter virus in dividing (top) and non-dividing (bottom) HOS cells 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A). Titers are mean ±sem, n = 3 technical replicates, representative of three independent experiments.

Figure 8—figure supplement 2. The amino-terminal domain of MX2 fused to a heterologous protein exhibits antiviral activity but with altered Nup requirements.

(A) Deconvolution microscopic images (single optical sections) of immunoflourescently stained myc-tagged ARFAPTIN2 or MX2(N91)-ARFAPTIN2 fusion proteins (green), NUP98 (red), and DAPI-stained DNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. (B–C) Western blot analysis of ARFAPTIN2 fusion protein expression (α-myc and α-LAMIN B1) in B) HeLa or C) HT1080 cells in the presence or absence of doxycycline. (D) Infection of HeLa cells stably transduced with doxycycline-inducible ARFAPTIN2 myc-tagged fusion proteins with various GFP reporter viruses in the presence (white bars) or absence (black bars) of doxycycline. Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments.

Figure 8—figure supplement 3. Effect of ARFAPTIN2 fusion protein expression on lentivirus and HIV-1 CA mutant infection.

(A) Infection of HeLa cells stably transduced with doxycycline-inducible arfaptin2 myc-tagged fusion proteins with various GFP reporter viruses in the presence (white bars) or absence (black bars) of doxycycline. Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. (B) Infectivity of HIV-1 N57S mutant GFP reporter virus in HT1080 cells stably transduced with arfaptin2 fusion proteins in the presence (white bars) or absence (black bars) of doxycycline and the presence or absence of CsA. Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. (C) Infectivity of HIV-1-GFP reporter virus in dividing or non-dividing (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible arfaptin2 or MX2(N91)-arfaptin2 in the presence (open circles) or absence (filled circles) of doxycycline. Titers are mean ± sem, n = 3 technical replicates and are representative of three independent experiments. (D) Infectivity of HIV-1 GFP reporter virus in growth arrested (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible MX2(N91)-ARFAPTIN2 in the presence (open circles) or absence (filled circles) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments.

Figure 8—figure supplement 4. Effect of Nup and NTR depletion on primate lentivirus infection and MX2 sensitivity.

Infectivity of (A) HIV-2 or (B) SIVmac GFP reporter virus in HeLa cells stably transduced with doxycycline-inducible MX2 in the presence (open circles) or absence (filled circles) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments.

Figure 8—figure supplement 5. Effect of Nup and NTR depletion on non-primate lentivirus infection and MX2 sensitivity.

Infectivity of (A) EIAV or (B-D) FIV GFP reporter virus in (A-B) HeLa or (C) HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of doxycycline, or (D) HOS cells. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).