Fig 1: K579 acetylation of SMC1A causes mitotic catastrophe.(A) Top: Spindle morphologies of metaphase cells with multiple poles stained with a-tubulin (green) and DAPI (blue). Scale bar, 10 µm. KQ, K579Q; KQ-DD, K579Q-S957DS966D. Bottom: Bar graph illustrating the percentages of HCT116 cells with multipolar spindles (n > 100 cells). Data are means ± SEM. *P < 0.05; ***P < 0.001. (B) Top: Nuclear morphologies of cells are shown stained with a-tubulin (green) and DAPI (blue). Scale bar, 10 µm. Bottom: The histogram illustrates the percentage of multinuclear cells. Data from three independent experiments are presented (n > 300 cells). Data are means ± SEM. ***P < 0.001. (C) Representative images showing Giemsa staining of chromosome spread assays in HCT116-shSMC1 cells expressing SMC1 WT, K579Q, and K579Q-DD mutants (top). The graph shows chromosome number per cell (bottom). Scale bar, 10 µm. Data from three independent experiments are presented (n > 60 cells). Data are means ± SEM. *P < 0.05; **P < 0.005. (D) Flag/HA-tagged SMC1A WT, K579R, or K579Q plasmids were transfected into HCT116-shSMC1A cells. The interaction between Rae1 and SMC1A was determined by IP and Western blot analyses. (E) Flag-tagged SMC1A WT, K579Q, or K579Q-DD–mutant plasmid was transfected into HCT116-shSMC1A cells with or without H2O2 (500 µM, 1 hour). The cells were then stained with annexin V–fluorescein isothiocyanate (FITC) and PI, and analyzed by fluorescence-activated cell sorting (FACS). (F) Bar graph shows percentage of apoptotic cells. Data are means ± SEM from three independent experiments. *P < 0.05. (G and H) Cyclin B1, H3-p (Ser10), and SMC1A-p (Ser966) levels were determined in HCT116-shSMC1 cells with reconstituted expression of SMC1A WT, K579Q (Q) mutant, or K579Q-S957DS966D (Q-DD) mutant in normal medium or after H2O2 (500 µM, 1 hour) in the presence (G) or absence (H) of SIRT2.
Fig 2: ORF6 inhibits mRNA export to favour viral translation and supress innate signalling.(A) Incoming or expressed ORF6 (yellow rectangles) binds to the Rae1-Nup98 complex (grey/brown) blocking the export of cellular mRNA (black lines). This subsequently reduces the cellular mRNA pool, increasing the availability of cellular translational machinery for viral translation as well as decreasing the expression of cellular proteins. (B) Incoming or expressed ORF6 (yellow) binds to the Rae1-Nup98 complex (grey/brown), inhibiting export of cellular mRNA encoding IRF1 (orange). This prevents the translation of IRF1, blocking IRF1 regulation of the transcription of additional steady state antiviral factors, like RIG-I and BST-2. ORF6 also inhibits nuclear export of mRNA encoding RIG-I (green), preventing detection of viral dsRNA produced during coronavirus replication. This helps reduce IRF1/3/7 activity and subsequent transcription of IFNa/ß (purple), which prevents IFNa/ß inducing an antiviral state in an autocrine and paracrine manner.
Fig 3: ORF6 inhibits protein expression by interfering with Rae1.(A) HEK293T cells were co-transfected with plasmids encoding HIV-1 Gag-Pol, ORF6 or ORF9b and Rae1 or indicated Rae1 mutant. Mock cells were untransfected. After 48h, cell lysates were separated by SDS-PAGE and analysed for Gag(HIV-1), Rae1, ORF6 or ORF9b and HSP90 by immunoblotting. Gag expression was quantified, normalised to HSP90, and shown in numbers below the Gag panel. (B) HEK293T cells were transfected with plasmids encoding Twin-Strep-tagged GFP or ORF6 and Nup98 and/or HA-Rae1. After 24h, Twin-Strep-tagged proteins were immunoprecipitated with MagStrep beads and proteins eluted with biotin. Input lysates and eluate were separated by SDS-PAGE and analysed by immunoblotting for Strep, Nup98 and HA. (C) HEK293T cells were transfected with plasmids encoding Twin-Strep-tagged GFP or ORF6, Nup98 and either HA-Rae1(WT) or HA-Rae1(R305G). After 24h, Twin-Strep-tagged proteins were immunoprecipitated and analysed as in (C). (D) HEK293T cells were transfected with plasmids encoding Twin-Strep-tagged proteins; ORF6(CoV-2), ORF6(E55A), ORF6(M58A) or GFP and HA-Rae1(WT). After 24h, Twin-Strep-tagged proteins were immunoprecipitated with MagStep beads and input lysates and eluates separated by SDS-PAGE and analysed by immunoblotting for Strep and HA.
Fig 4: 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.
Fig 5: Effect of Nup and NTR depletion on HIV-1 CA mutant infection and MX2 sensitivity.Infectivity of HIV-1 CA mutant GFP reporter viruses in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of doxycycline, 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).
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