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• Research Paper Volume 12, Issue 7 pp 5751-5763

  Molecular anatomy of the subcellular localization and nuclear import mechanism of herpes simplex virus 1 UL6

  Relevance score: 7.3208857

  Mingsheng Cai, Xiaowen Ou, Yiwen Li, Xingmei Zou, Zuo Xu, Yuanfang Wang, Hao Peng, Yangxi Deng, Yingjie Guo, Manjiao Lu, Weidong Gan, Tao Peng, Meili Li

  Keywords: HSV-1, UL6, nuclear import, importin, Ran-GTP

  Published in Aging on April 1, 2020

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  As an indispensable structure protein, the herpes simplex virus 1 (HSV-1) UL6 has been described to exert numerous roles in viral proliferation. However, its exact subcellular localization and subcellular transport mechanism is not well known. In the present study, by utilizing confocal fluorescent microscopy, UL6 was shown to mainly locate in the nucleus in enhanced yellow fluorescent protein or Flag tag fused expression plasmid-transfected cells or HSV-1-infected cells, whereas its predicted nuclear localization signal was nonfunctional. In addition, by exploiting dominant negative mutant and inhibitor of different nuclear import receptors, as well as co-immunoprecipitation and RNA interference assays, UL6 was established to interact with importin α1, importin α7 and transportin-1 to mediate its nuclear translocation under the help of Ran-mediated GTP hydrolysis. Accordingly, these results will advance the knowledge of UL6-mediated biological significances in HSV-1 infection cycle.

  

  Subcellular distribution of UL6 in plasmid-transfected and HSV-1-infected cells. Subcellular distribution of EYFP-UL6 (A), EYFP (B) and FLAG-UL6 (C) in related plasmid transfected COS-7 cells. (D) Subcellular distribution of UL6 in HSV-1 infected Vero cells. Vero cells were infected with HSV-1 (F strain) at an MOI of 1. 8 h post-infection, Vero cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and incubated with the anti-UL6 pAb. Then, cells were incubated with FITC-conjugated goat anti-rabbit IgG (green) and stained with DAPI (blue) to visualize the nuclei. EYFP fusion proteins were shown in pseudocolor green. The image shown represents a great proportion of the cells with homogeneous subcellular distribution. All scale bars indicate 10 um. Statistical analysis of the fluorescence was shown in Table 1.

  
  
  

  

  Subcellular distribution of the UL6 deletion mutants. (A) Schematic representation of wild-type UL6 protein and its N- and C-terminus deletion mutants fused with the C-terminus of EYFP. (B) Subcellular distribution of these UL6 deletion mutants shown in (A). Cells were stained with DAPI to visualize the nuclei. All scale bars indicate 10 um. Statistical analysis of the fluorescence was shown in Table 2.

  
  
  

  

  Nuclear import mechanism of UL6. (A) Fluorescence microscopy of COS-7 cells co-transfected with plasmids pFLAG-UL6 and pRan-Q69L-mCherry. (B) Fluorescence microscopy of COS-7 cells co-transfected with plasmid pFLAG-UL6 and plasmid encoding Bimax2-RFP, M9M-RFP, DN kα1-mCherry or DN kβ1-mCherry. (C) Fluorescence microscopy of COS-7 cells co-transfected with pFLAG-UL6 and pmCherry-N1. FITC-labeled proteins and mCherry fusion proteins were shown in its original color green and red, respectively, and the merged image was presented in yellow signal. All scale bars indicate 10 um, Statistical analysis of the fluorescence was shown in Table 3.

  
  
  

  

  UL6 binds transportin-1, importin α1 and importin α7. (A–F) Co-IP of UL6 with Transportin-1 (A), importin α1 (B), importin α3 (C), importin α5 (D), importin α7 (E) or importin β1 (F). pEYFP-UL6 was co-transfected with plasmid expressing pFLAG-CMV-transportin-1 (A), Flag-kα2 (importin α1) (B), Flag-kα4 (importin α3) (C), Flag-kα1 (importin α5) (D), Flag-kα6 (importin α7) (E) or pCMV9-3×Flag-importin β1 (F) into HEK293T cells. 24 h post-transfection, cells were lysed and Co-IPed with anti-Flag mAb or mouse IgG control. Cell lysates and the Co-IPed proteins were separated in denaturing 10% SDS-PAGE, and analyzed by IB with anti-Flag mAb or anti-YFP pAb.

  
  
  

  

  Subcellular distribution of UL6 in presence of different shRNA expression plasmids. (A) Verification of knock down efficiency of the constructed shRNA expression plasmids for importin α1, importin α7 and transportin-1. HEK293T cells were co-transfected with the plasmids combination of Flag-kα2 (importin α1)/pSuper, Flag-kα2/shRandom, Flag-kα2/shImportin-α1, Flag-kα6 (importin α7)/pSuper, Flag-kα6/shRandom, Flag-kα6/shImportin-α7, pFLAG-CMV-transportin-1/pSuper, pFLAG-CMV-transportin-1/shRandom or pFLAG-CMV-transportin-1/shTransportin-1 for 24 h. Then, cells were lysed and IB was performed with anti-Flag mAb. β-actin was used as a loading control. (B) One or two or three plasmids of shImportin-α1, shImportin-α7 and shTransportin-1 were co-transfected with pFLAG-UL6 into COS-7 cells for 24 h, then IFA was carried out using confocal fluorescence microscopy. Statistical analysis of the fluorescence was shown in Table 4.