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

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 co-expressed 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.

Identification and Expression Pattern of EZH2 in Pig Developing Fetuses

“…Wang et al used WoLF PSORT to predict subcellular localization of Bv1 fxre and Bv6 nyuw and subsequently validated the prediction by lab experiments [ 44 ]. A similar validation was reported in VP1 protein [ 45 ]. Therefore, computational analysis can be applied to predict the subcellular localization of protein.…”

A GFP-tagged version of the pseudorabies virus protein UL56 localizes to the Golgi and trans-Golgi network through a predicted C-terminal leucine-rich helix in transfected cells

“…To achieve this goal, we fuse a GFP tag at the N terminus of pUL56 due to a predicted transmembrane is present at the C terminus. Utilizing GFP as an indicator for investigating subcellular localization related signals in virus or host proteins of interest has been successfully applied in previous reports [20–24].…”

Types of nuclear localization signals and mechanisms of protein import into the nucleus

“…Additionally, viral protein1 (VP1) of chicken anemia virus (CAV) was found to contain a functional NLS motif necessary for its import into the nucleus. It spans the amino acids 3 RRARRPR G 10 , which makes it a classical MP NLS motif [ 21 ].…”