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. Author manuscript; available in PMC: 2014 Nov 13.
Published in final edited form as: Cell Host Microbe. 2013 Nov 13;14(5):10.1016/j.chom.2013.10.012. doi: 10.1016/j.chom.2013.10.012

Figure 7. Kif4 mediates MT stabilization and early HIV-1infection.

Figure 7

(A) Kif4 regulates AC MT levels in human cells. 293A or NHDF cells were transfected with control or Kif4 siRNAs then analyzed by WB with the indicated antibodies. (B-C) TIRF microscopy images of infected 293A (B) and CHME3 cells (C). Cells were infected with HIV-1-VSV then fixed and stained for Kif4 and Glu-MTs. Kif4 present on Glu-MTs at the periphery of HIV-1 infected cells is indicated by arrows. (B-C, right panels) Higher magnification images of Kif4 on Glu-MTs from samples in B and C. Boxed regions in the merged image are shown at higher magnification in the lower right panels. (D) Expression of Gag or MA induces MT acetylation in human cells. CHME3 or NHDF cells were transfected with control pcDNA, pcDNA-Gag or pcDNA-MA then analyzed by WB using anti-HIV-1 Gag, anti-Kif4 and anti-AC-tubulin antibodies. (E-F) An N-terminal region of HIV-1 MA mediates binding to Kif4 and MT stabilization. (E) HeLa cells were transfected with HA-tagged forms of Gag and immunoprecipitated using anti-Kif4 antibody. WB using anti-Kif4 or anti-HA antibodies shows the levels of Kif4 or HIV-1 Gag polyprotein in immune complexes and input levels of Gag. (F) WB analysis of AC-MT levels in HeLa cells 48h after transfection with HA-tagged forms of Gag. eIF4E served as loading control. (G) Kif4 is required for HIV-1-induced MT stabilization. NHDF cells were transfected with control or Kif4 siRNAs then infected with HIV-1-VSV for 6h. Fixed samples were stained for AC-MTs (green) and Tyr-MTs (red). Nuclei were stained with Hoechst. Scale bar, 10μm. (H-I) Kif4 is required for HIV-1 infection. NHDFs were transfected with control or Kif4 siRNAs then infected with HIV-1-VSV-luc (H) or HIV-1-Ampho-luc (I). Infectivity was determined by measurement of luciferase activity 48 h.p.i. (J-K) Effects of Kif4 depletion on HIV-1 DNA synthesis. Control or Kif4 siRNA treated cells were infected with HIV-1-VSV-puro and levels of viral MSS-DNA (J) and total viral DNA (K) in samples were measured by qPCR. Data in H-K are represented as mean +/− SEM. See also Figure S4.