Probing the nuclear import signal and nuclear transport molecular determinants of PRV ICP22

Abstract

Background: Herpes simplex virus 1 (HSV-1) ICP22 is a multifunctional protein and important for HSV-1 replication. Pseudorabies virus (PRV) ICP22 (P-ICP22) is a homologue of HSV-1 ICP22 and is reported to be able to selectively modify the transcription of different kinetic classes of PRV genes, however, the subcellular localization, localization signal and molecular determinants for its transport to execute this function is less well understood.

Results: In this study, by utilizing live cells fluorescent microscopy, P-ICP22 fused to enhanced yellow fluorescent protein (EYFP) gene was transient expressed in live cells and shown to exhibit a predominantly nucleus localization in the absence of other viral proteins. By transfection of a series of P-ICP22 deletion mutants fused to EYFP, a bona fide nuclear localization signal (NLS) and its key amino acids (aa) of P-ICP22 was, for the first time, determined and mapped to aa 41-60 (PASTPTPPKRGRYVVEHPEY) and aa 49-50 (KR), respectively. Besides, the P-ICP22 was demonstrated to be targeted to the nucleus via Ran-, importin α1-, and α7-mediated pathway.

Conclusions: Our findings reported herein disclose the NLS and molecular mechanism for nuclear transport of P-ICP22, these results will uncover new avenues for depicting the biological roles of P-ICP22 during PRV infection.

Keywords: Importin; Nuclear localization signal (NLS); Nuclear transport; PRV ICP22; Ran-GTP.

Fig. 1

Subcellular localization of P-ICP22 in transfected living cells. Subcellular distribution of P-ICP22-EYFP and EYFP-P-ICP22 in transfected-COS-7 cells. Cells were stained with DAPI to visualize the nuclei. All scale bars indicate 10 um

Fig. 2

Subcellular localization of the P-ICP22 deletion mutants. a Schematic representation of wild-type P-ICP22 protein and its N- and C-terminal deletion mutants fused with EYFP. b Subcellular localization of these P-ICP22 deletion mutants shown in (a). c Subcellular distribution of the negative control EYFP in transfected-COS-7 cells. Cells were stained with DAPI to visualize the nuclei. All scale bars indicate 10 um

Fig. 3

Ran-dependent nuclear import of P-ICP22. a Fluorescence microscopy of COS-7 cells co-transfected with plasmids pP-ICP22-EYFP and pRan-Q69L-ECFP. b Fluorescence microscopy of COS-7 cells only transfected with plasmid encoding DN kβ1-ECFP, DN kα1-ECFP, M9M-RFP or Bimax2-RFP. The white arrowhead showed the nuclei in a cell that co-transfected with two plasmids. All scale bars indicate 10 um

Fig. 4

The nuclear import mechanism of P-ICP22. a Fluorescence microscopy of COS-7 cells co-transfected with plasmid pP-ICP22-EYFP and plasmid encoding DN kβ1-ECFP, DN kα1-ECFP, M9M-RFP or Bimax2-RFP, respectively. b Fluorescence microscopy of COS-7 cells co-transfected with pP-ICP22-EYFP and pECFP-N1. The white arrowhead showed the nuclei in a cell that co-transfected with two plasmids. All scale bars indicate 10 um

Fig. 5

Investigation of the interactions between P-ICP22 and importin β1, transportin-1, importin α5, importin α1, importin α3 or importin α7. Co-IP of P-ICP22 with importin β1 (a), importin α5 (b), Transportin-1 (c), importin α1 (d), importin α3 (e) or importin α7 (f). HEK293T cells were co-transfected with pP-ICP22-EYFP and pCMV9-3 × Flag-importin β1 (3 × Flag-importin β1), pcDNA-Flag-kα1 (Flag-importin α5), pFLAG-CMV-transportin-1 (Flag-transportin-1), Flag-importin α1, Flag-importin α3 or Flag-importin α7. 24 h after transfection, cells were lysed and IP with anti-Flag or mouse IgG control. Immunoprecipitated proteins as well as the cell lysates were separated in denaturing 10 % SDS-PAGE, and analyzed by IB with anti-Flag mAb or anti-YFP pAb