Probing the nuclear import signal and nuclear transport molecular determinants of PRV ICP22
© Cai et al. 2016
Received: 4 August 2015
Accepted: 10 January 2016
Published: 25 January 2016
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.
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.
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.
KeywordsPRV ICP22 Nuclear transport Nuclear localization signal (NLS) Importin Ran-GTP
Pseudorabies virus (PRV), a member of genus Varicellovirus of the subfamily alphaherpesvirinae, is a pathogen of Aujeszky’s disease that can infect most mammals except for the higher-order primates. However, the neurotropic nature of PRV makes it a useful tool of neuronal connections and a useful model organism for the research of herpesvirus molecular pathomechanisms . PRV is reported to be closely related to human pathogens varicella-zoster virus (VZV), herpes simplex virus 1 and 2 (HSV-1 and HSV-2) , and the animal herpesvirus bovine herpesvirus type 1 (BHV-1) . To analyze the fundamental mechanisms underlying PRV spreading and pathogenesis, it is critical to gain a comprehensive understanding of the roles of each gene and their products during viral replication. In the course of lytic cycle of infection, PRV genes are expressed as other herpesviruses in a sequential cascade manner: immediate-early (IE), early (E) and late (L). PRV ICP22 (P-ICP22), the gene product of US1, does not express as an IE protein in the PRV life cycle, whereas its homologues HSV-1 ICP22 and VZV ORF63 are demonstrated to act as IE proteins , and BHV-1 BICP22 exhibits IE and L transcription kinetics . As important proteins for herpesviruses replication, P-ICP22 homologues play many roles in various aspects during infection, such as BHV-1 BICP22 is documented to act as a transrepressor protein on viral promoters of different kinetic classes . Moreover, HSV-1 ICP22 is implicated in ensuring proper virion morphology , inducing the formation of discrete nuclear foci containing cellular chaperone proteins known as VICE domains , modulating viral and cellular genes expressions or their activity [9, 10] and phosphorylated by viral and cellular kinases and nucleotidylylated by casein kinase II [11, 12]. Besides, VZV ORF63 is also involved in modulating viral and cellular genes expressions [13, 14], inhibiting apoptosis of primary human neurons  and the efficient establishment of latency . Recently, a paper reported that P-ICP22 is able to selectively modify the transcription of different kinetic classes of PRV genes, this may probably happen in the nucleus and/or the cytoplasm , however, the subcellular localization, transport signal and nucleo-cytoplasmic transport mechanism utilized by P-ICP22 to execute this function is less well understood.
In this work, live cells fluorescence microscopy and co-immunoprecipitation (Co-IP), which are widely applied and developed in our group [17–24], were employed to characterize the exact subcellular localization, localization signal and transport mechanism of P-ICP22. We showed that P-ICP22 localized predominantly to the nucleus in the absence of other viral proteins in transient transfected live cells. By sequence analysis and constructing a series of deletion mutants of P-ICP22 fused to enhanced yellow fluorescent protein (EYFP), the precise nuclear localization signal (NLS) and its key amino acids (aa) of P-ICP22 was identified and mapped to aa 41–60 and aa 49–50, respectively. Besides, the molecular mechanisms of nuclear import, which are crucial for understanding P-ICP22-mediated biological effects in PRV infection cycle, were characterized to target to the nucleus through a Ran-, importin α1- and α7-dependent nuclear import pathway.
Results and discussion
Subcellular localization of P-ICP22 in the transfected live cells
Although P-ICP22 was predicted to be localized mainly in the cytoplasm , it was substantiated here that it predominantly accumulates in nucleus, it’s well known that protein phosphorylation is one of the most normal and essential types of protein modification, and certain aspects of cell process modulation are regulated by protein phosphorylation. Signal transduction, proliferation, differentiation and metabolism are all controlled by the balance of activity of protein kinases and protein phosphatases upon pivotal target proteins. Phosphorylation site prediction revealed there is 22 potential phosphorylation sites in P-ICP22, including 15 serine, 5 threonine and 2 tyrosine residues . Tyrosine phosphorylation is reported to be involved in the replication of several herpesviruses [27, 28] and in shifting protein translocation from the cytoplasm to the nucleus during productive virus infection . Phosphorylation of VZV ORF63 is also associated with its subcellular localization and transcriptional regulatory properties [30, 31]. Therefore, the subcellular localization differences of P-ICP22 between the predicted result and experiment result may be due to the presence of potential phosphorylation sites especially the tyrosine residues, and P-ICP22 phosphorylation may also play an important role during PRV infection, perhaps in modulating its subcellular localization or other uncharacterized functions, such as transcriptional regulation.
In an attempt to examine whether the location of EYFP influences the localization of P-ICP22 in cells, a plasmid expressing P-ICP22 fused to the C terminus of EYFP (EYFP-P-ICP22) was constructed, and fluorescence microscopy showed identical subcellular distribution patterns of P-ICP22-EYFP with EYFP-P-ICP22 (Fig. 1). Therefore, subsequent experiments were done using pP-ICP22-EYFP.
Characterization of the NLS and its key aa of P-ICP22
To finally define the minimum region that has the ability to direct EYFP to the nucleus, plasmids encoding EYFP fused to aa 41–60 and aa 47–54 were constructed (Fig. 2a) and tested in COS-7 cells. As shown in Fig. 2b, fluorescence of aa47-54-EYFP was identical to that of EYFP, whereas fluorescence of aa41-60-EYFP showed predominant nuclear localization, suggesting the predicted NLS (47PPKRGRYV54) is not a genuine NLS, and the functional NLS in P-ICP22 is a 20-residue peptide 41PASTPTPPKRGRYVVEHPEY60, and its key aa may be 49KR50.
To further verify this deduction, basic residues within the 49KR50 sequences of P-ICP22 was mutated with neutral alanine residues to yield 49AA50, and fused with EYFP to create pP-ICP22-49-50mut-EYFP (Fig. 2a). As shown in Fig. 2b, mutation of aa 49–50 within P-ICP22 abrogated the nuclear localization of P-ICP22, showing pan-cytoplasm subcellular localization pattern, confirming aa 49–50 is the key aa of NLS for the nuclear localization of P-ICP22.
Characterization of the nuclear import mechanisms of P-ICP22
P-ICP22 binds importin α1 and importin α7
In conclusion, the subcellular localization, localization signal and nuclear transport mechanism of P-ICP22 were identified in this study. P-ICP22 is shown to principally localized to the nucleus. By constructing a series of deletion mutants fused with EYFP and fluorescence microscopy analysis, we identified an N-terminal arginine-rich functional NLS (41PASTPTPPKR GRYVVEHPEY60) in P-ICP22, and 49KR50 is the crucial aa for its nuclear localization. In addition, P-ICP22 was characterized to import into the nucleus via Ran-, importin α1- and α7- mediated pathway, which would expand its list of the host targets, and P-ICP22 probably transports into the nucleus via Ran-, importin α1- and α7- mediated pathway to selectively modify the transcription of different kinetic classes of PRV genes. Therefore, this study reveals the molecular mechanisms for the nuclear transport of P-ICP22, and will advance the understanding of P-ICP22-mediated biological functions in PRV replication.
All enzymes used for cloning processes were purchased from Takara (Dalian, China) except DNA polymerase KOD FX from TOYOBO and T4 DNA ligase from New England Biolabs (MA, USA). PRV BAC pBecker2 was a generous gift from Dr. Lynn W. Enquist . The P-ICP22 ORF (composed of 1047 bp) was amplified via PCR from the genomic DNA of PRV Becker strain pBecker2. After the PCR amplified product was validated as the intended product, it was purified using a PCR gel purification kit (Qiagen) according to the manufacturer’s instructions. The product was then digested with EcoRI and BamHI and inserted into the correspondingly digested green fluorescent protein variant mammalian expression vector pEYFP-N1 (Clontech) encoding EYFP at 16 °C overnight using T4 DNA ligase, to create the recombinant plasmid named pP-ICP22-EYFP, as described previously [3, 26, 50–53]. After mini-scale isolation of plasmid DNA using Qiagen plasmid Mini kits (Qiagen), the presence of the appropriate insert in the obtained plasmid was confirmed by PCR, restriction analysis and sequencing. Subsequently, the P-ICP22 ORF was subcloned into pEYFP-C1 (Clontech) to yield pEYFP-P-ICP22. The series of mutant deletions were generated by PCR-ligation-PCR mutagenesis  using appropriate primers and cloned into pEYFP-N1 as described above, all the N terminal deleted fusion proteins were inserted with an artificial ATG start codon starting at internal positions in P-ICP22 of the fusion constructs. Finally, all constructs were verified by PCR, restriction analysis and sequencing. Dominant negative (DN) mutant RanGTP , DN kα1  and DN kβ1  were subcloned into pECFP-N1 (Clontech) to generate pRan-Q69L-ECFP, pDN kα1-ECFP and pDN kβ1-ECFP, respectively. RFP-Bimax2 and RFP-M9M were generous gifts from Dr. Nobuyuki Nukina . pcDNA-Flag-kα1 (importin α5), Flag-kα6 (importin α7) , Flag-kα2 (importin α1), Flag-kα4 (importin α3)  and pCMV9-3 × Flag-importinβ1 were the generous gifts from Drs. Yoshihiro Yoneda, Reinhard Depping and Ben Margolis, respectively. The transportin-1 ORF was amplified from pGEX-tev-kapbeta2  and subcloned into pFLAG-CMV-2 (Sigma) to produce pFLAG-CMV-transportin-1. All the primers used in this study are available upon request.
Transfection and fluorescence microscopy
Transfection and fluorescence microscopy assays were fulfilled as shown in our previous studies [23, 53]. Briefly, to express the proteins in vitro, COS-7 cells were plated onto six-well plates (Corning) in DMEM with 10 % FBS at a density of ~2.5 × 105 cells per well overnight to 60–80 % confluency before transfection. The next day, monolayer of COS-7 cells were transfected with 1.0–1.5 μg of indicated plasmid DNA mixed with Thermo Scientific TurboFect Transfection Reagent following the manufacturer’s instructions. The transfection efficiency (~60 %) was determined by EYFP. 24 h post transfection, cells were washed with fresh growth medium and subjected to fluorescence microscopy with a Zeiss Axiovert 200 M microscope (Germany). In the same experiment, each transfection was performed for at least three times. Data shown were from one representative experiment. All the photomicrographs were taken under a magnification of 400×. Each photomicrograph represents an enormous majority of the cells with similar subcellular localization. Both fluorescent images of EYFP and ECFP fusion proteins were presented in pseudocolor, green and red, respectively, and the merged images were shown to prove the colocalization and appeared in yellow signals. All scale bars indicate 10 um. Images were processed by Adobe Photoshop.
Co-IP and western-blot
Co-IP and western-blot were manipulated as described previously [23, 53]. Briefly, HEK293T cells (~2.5 × 106) were co-transfected with 5 μg of each of the indicated expression plasmids bearing EYFP or Flag tag. Transfected cells were harvested 24 h post transfection and lysed on ice with 1 mL of lysis buffer. For each IP, a 0.5 mL aliquot of lysate was incubated with 0.5 μg of the anti-Flag monoclonal antibody (mAb) or nonspecific control mouse antibody (IgG) and 30 μL of 1:1 slurry of Protein A/G PLUS-Agarose (Santa Cruz Biotechnology) for at least 4 h or overnight at 4 °C. The Sepharose beads were washed three times with 1 mL of lysis buffer containing 500 mM NaCl. Subsequently, the immunoprecipitated proteins, as well as the cell lysates, were subjected to immunoblotting (IB) analysis with anti-Flag mAb (Sigma) and anti-YFP polyclonal antibody (pAb) (Santa Cruz Biotechnology). All Co-IP were repeated at least two times, and similar data were obtained.
herpes simplex virus 1
bovine herpesvirus type 1
enhanced yellow fluorescent protein
nuclear localization signal
enhanced cyan fluorescent protein
Conceived and designed the experiments: MSC and MLL. Performed the experiments and analyzed the data: MSC, CCM, ZCZ, SJ, XWL, YJY, CKC, PPX, YB, JLW, JLH, DXC, TP and MLL. Wrote the paper: MSC and MLL. All authors contributed to and have approved the final manuscript.
We thank Drs. Lynn W. Enquist, Yoshinari Yasuda, Haitao Guo, Nobuyuki Nukina, Yoshihiro Yoneda, Ben Margolis, Christopher F. Basler, Donna D. Zhang, Christoph Kaether and Reinhard Depping for the generous gifts of pBecker2, pGEX-Ran-Q69L, DN kα1, DN kβ1, RFP-M9M/RFP-Bimax2, pcDNA-Flag-kα1 (importin α5), pCMV9-3 × FLAG-importin β1, Flag-kα6 (importin α7), Flag-kα2 (importin α1) and Flag-kα4 (importin α3), respectively. We also thank Dr. Chunfu Zheng (Institutes of Biology and Medical Sciences, Soochow University) for the technical assistance and supply of COS-7 and HEK293T cells.
The authors declare that they have no competing interests.
This work was supported by grants from the National Natural Science Foundation of China (31400150 and 31200120); the Natural Science Foundation of Guangdong Province (2015A030313473 and S2013040016596); the Foundation for Distinguished Young Talents in Higher Education of Guangdong, China (2013LYM_0096); the Pearl River S&T Nova Program of Guangzhou (2013J2200018); the Science and Technology Program of Guangzhou, China (201504291022514); the Scientific Research Projects in Colleges and universities of Guangzhou (1201430024); the Scientific Research Foundation for the Ph.D., Guangzhou Medical University (2014C02); the Medical Scientific Research Foundation of Guangdong Province, China (B2012165); the Training Program for Outstanding Young Teachers in Universities of Guangdong Province (YQ2015132); the Thousand Hundred Ten Projects of Guangzhou Medical University, Guangdong; the Science and Technology Plan Projects of Guangdong Province, China (2013B031800022); and the Students’ extracurricular scientific and technological activities in Guangzhou Medical University (2015A003, 2015A008, 2015A019 and 2015B020).
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