Importin α7 Is Essential for Zygotic Genome Activation and Early Mouse Development

Importin α is involved in the nuclear import of proteins. It also contributes to spindle assembly and nuclear membrane formation, however, the underlying mechanisms are poorly understood. Here, we studied the function of importin α7 by gene targeting in mice and show that it is essential for early embryonic development. Embryos lacking importin α7 display a reduced ability for the first cleavage and arrest completely at the two-cell stage. We show that the zygotic genome activation is severely disturbed in these embryos. Our findings indicate that importin α7 is a new member of the small group of maternal effect genes

Cellular Importin-α3 Expression Dynamics in the Lung Regulate Antiviral Response Pathways against Influenza A Virus Infection.

Importin-α adaptor proteins orchestrate dynamic nuclear transport processes involved in cellular homeostasis. Here, we show that importin-α3, one of the main NF-κB transporters, is the most abundantly expressed classical nuclear transport factor in the mammalian respiratory tract. Importin-α3 promoter activity is regulated by TNF-α-induced NF-κB in a concentration-dependent manner. High-level TNF-α-inducing highly pathogenic avian influenza A viruses (HPAIVs) isolated from fatal human cases harboring human-type polymerase signatures (PB2 627K, 701N) significantly downregulate importin-α3 mRNA expression in primary lung cells. Importin-α3 depletion is restored upon back-mutating the HPAIV polymerase into an avian-type signature (PB2 627E, 701D) that can no longer induce high TNF-α levels. Importin-α3-deficient mice show reduced NF-κB-activated antiviral gene expression and increased influenza lethality. Thus, importin-α3 plays a key role in antiviral immunity against influenza. Lifting the bottleneck in importin-α3 availability in the lung might provide a new strategy to combat respiratory virus infections

Importin α1 is required for nuclear import of herpes simplex virus proteins and capsid assembly in fibroblasts and neurons

<div><p>Herpesviruses are large DNA viruses which depend on many nuclear functions, and therefore on host transport factors to ensure specific nuclear import of viral and host components. While some import cargoes bind directly to certain transport factors, most recruit importin β1 via importin α. We identified importin α1 in a small targeted siRNA screen to be important for herpes simplex virus (HSV-1) gene expression. Production of infectious virions was delayed in the absence of importin α1, but not in cells lacking importin α3 or importin α4. While nuclear targeting of the incoming capsids, of the HSV-1 transcription activator VP16, and of the viral genomes were not affected, the nuclear import of the HSV-1 proteins ICP4 and ICP0, required for efficient viral transcription, and of ICP8 and pUL42, necessary for DNA replication, were reduced. Furthermore, quantitative electron microscopy showed that fibroblasts lacking importin α1 contained overall fewer nuclear capsids, but an increased proportion of mature nuclear capsids indicating that capsid formation and capsid egress into the cytoplasm were impaired. In neurons, importin α1 was also not required for nuclear targeting of incoming capsids, but for nuclear import of ICP4 and for the formation of nuclear capsid assembly compartments. Our data suggest that importin α1 is specifically required for the nuclear localization of several important HSV1 proteins, capsid assembly, and capsid egress into the cytoplasm, and may become rate limiting <i>in situ</i> upon infection at low multiplicity or in terminally differentiated cells such as neurons.</p></div

Importin α1 is not required for nuclear targeting of incoming HSV-1 capsids in neurons.

<p>(A-C) Primary cells from DRG cultured on cover slips were transduced with scr shRNA (A,B) or shRNA targeting importin α1 (C). At 7 dpt, the cells were infected with HSV1(17⁺)Lox (2.5 x 10⁷ pfu/mL) in the presence of cycloheximide (A, C) or of nocodazole (ND) and cycloheximide (B). At 2.5 hpi, the cells were fixed and permeabilized with PHEMO-fix, stained with DAPI (i), labelled with antibodies against capsid (ii) or β-III-tubulin (iii), and analyzed by confocal fluorescence microscopy. (D-E) DRG cells cultured in microfluidic devices were transduced with scr shRNA (D) or a shRNA against importin α1 (E). At 7 dpt, neurons were selectively inoculated from the axonal side with HSV1(17⁺)Lox-CheVP26 (1.3 x 10⁸ pfu/mL) in the presence of cycloheximide. At 4 hpi, cells were fixed with PFA, stained with DAPI (i), labelled with anti-capsid antibodies (ii), and analyzed by confocal microscopy. Scale bars: 10 μm</p

Importin α1 is important for nuclear egress of HSV-1 capsids.

<p>MEF^wt (A) or MEF-Impα1^-/- (B) were infected with HSV1(17⁺)Lox with an MOI of 10 pfu/cell at 2.5 x 10⁶ pfu/mL, fixed at 12 hpi, and analyzed by electron microscopy. In both cell lines, all stages of virus assembly could be identified: nuclear A-capsids (labeled with A in Ai and Bi), B-capsids (labeled with B in Ai and Bi), and C-capsids (labeled with C in Ai and Bi); primary enveloped virions (Ai, white star), cytosolic capsids (Aii, Bii, white arrowheads), wrapping intermediates with capsids being closely associated with cytoplasmic membranes (Aiii, black arrowhead), virions after complete secondary envelopment (Aii, iii, iv, Biii, black stars), and extracellular virions (Aiv, Biv, arrows). Scale bar is 500 nm.</p

Importin α1 is required for productive HSV-1 infection.

<p><b>(A-E)</b> MEF^wt (A), nocodazole treated MEF^wt (wt + ND, B), MEF-Impα1^-/- (C), MEF-Impα3^-/- (D), or MEF-Impα4^-/- (E) were infected with HSV1(17⁺)Lox-CheVP26 (0.5 to 1.25 x 10⁶ pfu/mL, MOI of 2 to 5), fixed with 3% PFA at 8 hpi, permeabilized with TX-100, labeled with antibodies directed against capsid (pAb SY4563), and analyzed by confocal fluorescence microscopy. Scale bar 20 μm. (F) The mean fluorescence intensities were measured in the nuclear profiles for the capsids in more than 150 randomly selected cells per condition, and are shown as box plots with medians and whiskers representing the 10 to 90% percentile. The p values were determined with a Kruskal-Wallis test followed by Dunn’s multiple comparison testing. (G) MEF^wt transduced with scr shRNA or shRNAs targeting importin α1, α3 or α4 were infected, labeled with anti-capsid antibodies, and analyzed by confocal microscopy as described for (F). (H-I) MEF^wt, MEF-Impα1^-/-, MEF-Impα3^-/-, or MEF-Impα4^-/- were infected with HSV1(17⁺)Lox (2.5 × 10⁶ pfu/mL, MOI of 10), and cell-associated (H) and extracellular (I) virions were harvested at the indicated time points, and titrated on Vero cells.</p

Importins α1 and α3 but to a lesser extent α4 are required for HSV-1 gene expression in neurons.

<p>(A) DRG cells cultured in 12-well plates were transduced with scr shRNA or shRNAs against importin α1, α3 or α4 as indicated. At 7 dpt, cells were infected with HSV1(17⁺)Lox-GFP (5 x 10⁶ pfu/mL) for 5 h. Cell lysates were analyzed by immunoblot using antibodies against p150^Glued, importin α1, α3, α4 or several structural HSV-1 proteins including VP22 (pAb Remus V). (B-F) DRG cells cultured on cover slips were transduced with scrambled shRNA (B, C) shRNA targeting importin α1 (D), α3 (E), or α4 (F). At 7 dpt, the neurons were infected with HSV1(17⁺)Lox-GFP (5 x 10⁶ pfu/mL) in the absence (B, D-F) or presence of 10 μM nocodazole (C; ND). At 4 hpi, the cells were fixed and permeabilized with PHEMO-fix, stained with DAPI (i), and analyzed by confocal fluorescence microscopy. GFP was detected by its intrinsic fluorescence (ii). Scale bar: 10 μm (G) The intra-nuclear GFP signals were quantified with a CellProfiler pipeline using 108 to 129 neurons per condition, and are shown as box plots with medians and whiskers representing the 10 to 90% percentile.</p

Importin α1 is required for efficient nuclear egress of progeny capsids.

<p>Importin α1 is required for efficient nuclear egress of progeny capsids.</p

Importin α1 is required for HSV-1 gene expression in neurons.

<p>Primary cells from dorsal root ganglia cultured on cover slips were transduced with a scrambled shRNA (A, B) or an shRNA targeting importin α1 (C). At 7 dpt, the neurons were infected with HSV1(17⁺)Lox-GFP (5 x 10⁶ pfu/mL) in the absence (A, C) or presence of 10 μM nocodazole (B). At 4 hpi, the cells were fixed and permeabilized with PHEMO-fix, stained with DAPI (i), labelled with antibodies directed against ICP4 (ii) or capsids (iv), and analyzed by confocal fluorescence microscopy. GFP was detected by its intrinsic fluorescence (iii). Scale bar: 10 μm. The intra-nuclear ICP4 (D), GFP (E) and VP5 (F) signals were quantified with a CellProfiler pipeline using 109 (sh scr), 51 (sh scr + ND), and 106 (sh Imp α1) neurons and are shown as box plots with medians and whiskers representing the 10 to 90% percentile.</p