Identification of nuclear localization signal and nuclear export signal of VP1 from the chicken anemia virus and effects on VP2 shuttling in cells

Cheng et al. Virology Journal (2019) 16:45 https://doi.org/10.1186/s12985-019-1153-5 RESEARCH Open Access Identification of nuclear localization signal and nuclear export signal of VP1 from the chicken anemia virus and effects on VP2 shuttling in cells Jai-Hong Cheng1* , Guan-Hua Lai2, Yi-Yang Lien3,4, Fang-Chun Sun5, Shan-Ling Hsu6, Pei-Chin Chuang1 and Meng-Shiou Lee7* Abstract Background: VP1 of the chicken anemia virus (CAV) is a structural protein that is required for virus encapsulation. VP1 proteins are present both in the nucleus and cytoplasm; however, the functional nuclear localization signal (NLS) and nuclear export signal (NES) of VP1 are still unknown. This study aimed to characterize the NLS and NES motifs of VP1 using bioinformatics methods and multiple-site fragment deletions, and investigate shuttling of VP2 from nucleus to cytoplasm by co-transfection with VP1. Methods: Two putative NLS motifs were predicted by the WoLF PSORT and NLStradamus programs from the amino acid sequence of VP1. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. All mutants were created by multiple-site fragment deletion mutagenesis. VP1 and VP2 were coexpressed in cells using plasmid transfection. Results: A functional NLS motif was identified at amino acid residues 3 to 10 (RRARRPRG) of VP1. Critical amino acids 3 to 10 were significantly involved in nuclear import in cells and were evaluated using systematic deletion mutagenesis. Three NES motifs of VP1 were predicted by the NetNES 1.1 Server and ELM server programs. A functional NES was identified at amino acid residues 375 to 388 (ELDTNFFTLYVAQ). Leptomycin B (LMB) treatment demonstrated that VP1 export from nucleus to cytoplasm occurred through a chromosome region maintenance 1 (CRM1)-dependent pathway. With co-expression of VP1 and VP2 in cells, we observed that VP1 may transport VP2 from nucleus to cytoplasm. Conclusion: Our data showed that VP1 of CAV contained functional NLS and NES motifs that modulated nuclear import and export through a CRM1-dependent pathway. Further, VP1 may play a role in the transport of VP2 from nucleus to cytoplasm. Keywords: Chicken anemia virus, VP1, VP2, Nuclear localization signal, Nuclear export signal, CRM1-dependent pathway * Correspondence: ; 1 Center for Shockwave Medicine and Tissue Engineering, Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, 123 Tai-Pei Road, Niao Sung District, Kaohsiung, Taiwan833 7 Department of Chinese Pharmaceutical Science and Chinese Medicine Resources, China Medical University, 91, Hsueh-Shih Road, Taichung, Taiwan Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cheng et al. Virology Journal (2019) 16:45 Background The chicken anemia virus (CAV) is a small virus of 23 to 25 nm in size; it is an icosahedral, non-enveloped virus, resistant to heat, lipid solvents and disinfectants [1]. CAV belongs to the genus Gyrovirus of the Anelloviridae family. The viral genome is a single-stranded DNA that is circular and covalently linked, consisting of approximately 2300 base pairs in its replicative form [2]. In 1979, Dr. Yuasa reported that vaccines contaminated with CAV had been distributed worldwide, causing spread of the disease [3]. CAV infection results in large economic damage, as the clinical disease contributes to vertical transmission and immune dysfunction in combination with other pathogens [4]. The CAV genome replicates through the rolling circle method [5]. The genomic DNA encodes three viral proteins (VP1, VP2 and VP3) from a 2.1-kb transcript [6]. Among the viral proteins, VP1 is a capsid protein (51 kDa) and VP2 is a non-structural protein (24 kDa), containing a dual-specific phosphatase activity and acting as a scaffold protein in the capsid assembly. VP1 and VP2 are protective proteins that induce neutralizing antibodies [7]. VP3 is called “apoptin”; it is the smallest of the three viral proteins at 13 kDa, with a unique apoptotic-inducing property [6, 8]. In the life cycle of the virus, the subcellular localization of viral proteins, especially in the nucleus, may contribute to cell apoptosis, viral replication or host cell proliferation [9, 10]. Thus, determination of the nuclear localization signal (NLS) and nuclear exporting signal (NES) of a viral protein is critical, and is required in order to reveal the importance of viral proteins in virus replication in cells. Recently, three CAV viral proteins, VP1, VP2 and VP3, have been found to possess nuclear localization activity in cells [11–14]. When GFP-VP1 and GFP-VP2 are transiently expressed in cells, VP1 and VP2 are observed to be present throughout the nucleoplasm [11]. VP3 has been reported to trigger cell apoptosis, and has been found to aggregate within the nucleus by phosphorylation of Threonine 108 [13, 14]. These results suggested that a functional NLS may be present in all three CAV viral proteins. To date, the functional NLSs of VP2 and VP3 have been demonstrated via bioinformatics and biochemical experiments [12–15]. VP3 contains a bipartite-type NLS and NES, suggesting potential shuttling of the protein between nucleus and cytoplasm [14, 15]. VP2 is a CRM1-independent nuclear protein with a simple NLS spanning amino acid residues from 133 to 138, and may not have a NES motif [12]. Moreover, VP2 and VP3 expressions have indicated protein–protein interaction in the cell [16]. Further, the apoptosis ability of VP3 is down-regulated directly by VP2 phosphatase through de-phosphorylation of Threonine 108 of VP3 [14]. VP1 is the only structural protein of CAV, and is Page 2 of 9 required theoretically for packaging and formation of infectious particles [17]. A polyclonal chicken anti-CAV sera recognized all strains that have been tested, but only monoclonal antibody 2A9 has been mapped with VP1 [18–20]. The formation of epitopes recognized by polyclonal virus-neutralizing (VN) chicken antibodies requires co-expression of VP1 and VP2, implying that VP2 acts as a scaffold protein with VP1 [7, 21]. This finding was subsequently proved by analysis of VP1– VP2 protein–protein interaction [16]. In spite of this finding, the possible functional domain of VP1, especially in terms of the cellular localization, is not well understood, although some researche (...truncated)