Molecular characterizations of subcellular localization signals in the nucleocapsid protein of porcine epidemic diarrhea virus

Viruses. 2014 Mar 13;6(3):1253-73. doi: 10.3390/v6031253.

Abstract

The nucleolus is a dynamic subnuclear structure, which is crucial to the normal operation of the eukaryotic cell. The porcine epidemic diarrhea virus (PEDV), coronavirus nucleocapsid (N) protein, plays important roles in the process of virus replication and cellular infection. Virus infection and transfection showed that N protein was predominately localized in the cytoplasm, but also found in the nucleolus in Vero E6 cells. Furthermore, by utilizing fusion proteins with green fluorescent protein (GFP), deletion mutations or site-directed mutagenesis of PEDV N protein, coupled with live cell imaging and confocal microscopy, it was revealed that, a region spanning amino acids (aa), 71-90 in region 1 of the N protein was sufficient for nucleolar localization and R87 and R89 were critical for its function. We also identified two nuclear export signals (NES, aa221-236, and 325-364), however, only the nuclear export signal (aa325-364) was found to be functional in the context of the full-length N protein. Finally, the activity of this nuclear export signal (NES) was inhibited by the antibiotic Lepomycin B, suggesting that N is exported by a chromosome region maintenance 1-related export pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Nucleolus / chemistry
  • Chlorocebus aethiops
  • Coronavirus Nucleocapsid Proteins
  • Cytoplasm / chemistry
  • DNA Mutational Analysis
  • Nucleocapsid Proteins / genetics*
  • Nucleocapsid Proteins / metabolism
  • Porcine epidemic diarrhea virus / genetics*
  • Porcine epidemic diarrhea virus / physiology
  • Protein Sorting Signals*
  • Vero Cells

Substances

  • Coronavirus Nucleocapsid Proteins
  • Nucleocapsid Proteins
  • Protein Sorting Signals