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Clustered Basic Amino Acids of the Small Sendai Virus C Protein Y1 Are Critical to Its RAN GTPase-mediated Nuclear Localization

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Clustered Basic Amino Acids of the Small Sendai Virus C Protein Y1 Are Critical to Its RAN GTPase-mediated Nuclear Localization

Takashi Irie et al. PLoS One.

Abstract

The Sendai virus (SeV) C proteins are shown to exert multiple functions during the course of infection. Perhaps reflecting their many functions, they occur at multiple sites of the cell. In this study, we focused on the nuclear-localizing ability of the smaller C protein, Y1, and found that this translocation is mediated by Ran GTPase but not by passive diffusion, and that basic residues within the 149-157 amino acid region are critical for that. The mechanism of inhibition of interferon (IFN)-signaling seemed to differ between the C and Y1 proteins, since deletion of 12 C-terminal amino acids resulted in a loss of the function for the C but not for the Y1 protein. The ability of Y1 mutants to inhibit IFN-α-induced, ISRE-driven expression of a reporter gene almost paralleled with that to localize in the nucleus. These results suggest that nuclear localization of the Y1 protein might be important for the inhibitory effect on type-I IFN-stimulated gene expression.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Subcellular localization of the Y1 mutants possessing C-terminal deletions.
(A) Schematic representation of expression plasmids encoding C-WT, C-d2Y, Y1, Y1-d192, -d188, -d184, -d170, -d160, and -d150. (B) The C and Y1 proteins were expressed in 293T cells. At 24 h p.t., cells were fixed with 3% formaldehyde, permeabilized with 0.1% Triton X-100, and stained with anti-C pAb and Alexa 488-conjugated anti-rabbit IgG antibody as primary and secondary antibodies, respectively. Cells were observed under a Zeiss LSM5 confocal microscope.
Figure 2
Figure 2. Subcellular distribution of the EGFP-fused C and Y1 mutants.
(A) Schematic representation of expression plasmids encoding EGFP, EGFP-C, -C(1–23), -Y1, and -Y1-d150. (B) The EGFP-fused proteins were expressed in 293T cells. At 24 h p.t., cells were fixed with 3% formaldehyde and observed under a confocal microscope.
Figure 3
Figure 3. Subcellular distribution of EGFP (A) and the Y1 protein (B) in the presence of HA-Ran-DN.
293T cells co-transfected with Y1 and HA-Ran-DN were stained with anti-C (green) and anti-HA (red) antibodies at 24 h p.t., and observed under a Zeiss LSM5 confocal microscope.
Figure 4
Figure 4. Fractionation analysis of 293T cells co-transfected with the C and Y1 mutants, and HA-Ran-DN.
(A) Cytosolic (Cy) and nuclear (Nu) fractions were prepared as described in Materials and Methods at 24 h p.t., and equal amounts of each sample were analyzed by Western blotting using anti-C pAb. (B) The C and Y1 mutants in the cytosolic and nuclear fractions were quantitated using an LAS-1000 luminescent image analyzer. Ratios of the amounts each protein in the nuclear fractions to those in the cytosolic fractions are shown as bar graphs.
Figure 5
Figure 5. Subcellular distribution of the Y1 mutants possessing triple alanine substitutions.
(A) Schematic representation of expression plasmids encoding Y1-KMK149A3, -TER152A3, -WLR155A3, -TLI158A3, -RGE161A3, -KTK164A3, and -LKD167A3. (B) The indicated mutants were subjected to immunofluorescent microscopy as shown in Figure 1B.
Figure 6
Figure 6. Subcellular distribution of the Y1 mutants possessing single alanine substitutions.
(A) Schematic representation of expression plasmids encoding Y1-K149A, -M150A, -K151A, -W155A, -L156A, -R157A, -T158A, -L159A, and -I160A. (B) The indicated mutants were subjected to immunofluorescent microscopy as shown in Figure 1B.
Figure 7
Figure 7. Subcellular distribution of the Y1 mutant, Y1-T152R,TLI158A3.
(A) Schematic representation of Y1-T152R,TLI158A3. (B) Cells expressing the mutants were subjected to immunofluorescent microscopy as shown in Figure 1B.
Figure 8
Figure 8. ISRE reporter assay in the presence of the C and Y mutants.
293T cells were co-transfected with a series of deletion mutants of the C protein (A), deletion mutants of the Y1 protein (B), or the Y1 mutants possessing triple alanine substitutions (C) together with a reporter plasmid, pISRE-EGFP. At 18 h p.i., cells were treated with IFN-α (1,000 IU/ml) for 8 h, and then expression level of EGFP and the transfected C and Y1 proteins were analyzed by Western blotting using anti-GFP and anti-C antibodies.

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Grant support

This work was supported by JSPS KAKENHI Grant Number 23790505. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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