The nuclear chaperone nucleophosmin escorts an Epstein-Barr Virus nuclear antigen to establish transcriptional cascades for latent infection in human B cells

PLoS Pathog. 2012;8(12):e1003084. doi: 10.1371/journal.ppat.1003084. Epub 2012 Dec 13.

Abstract

Epstein-Barr Virus (EBV) is an oncogenic γ-herpesvirus that capably establishes both latent and lytic modes of infection in host cells and causes malignant diseases in humans. Nuclear antigen 2 (EBNA2)-mediated transcription of both cellular and viral genes is essential for the establishment and maintenance of the EBV latency program in B lymphocytes. Here, we employed a protein affinity pull-down and LC-MS/MS analysis to identify nucleophosmin (NPM1) as one of the cellular proteins bound to EBNA2. Additionally, the specific domains that are responsible for protein-protein interactions were characterized as EBNA2 residues 300 to 360 and the oligomerization domain (OD) of NPM1. As in c-MYC, dramatic NPM1 expression was induced in EBV positively infected B cells after three days of viral infection, and both EBNA2 and EBNALP were implicated in the transactivation of the NPM1 promoter. Depletion of NPM1 with the lentivirus-expressed short-hairpin RNAs (shRNAs) effectively abrogated EBNA2-dependent transcription and transformation outgrowth of lymphoblastoid cells. Notably, the ATP-bound state of NPM1 was required to induce assembly of a protein complex containing EBNA2, RBP-Jκ, and NPM1 by stabilizing the interaction of EBNA2 with RBP-Jκ. In a NPM1-knockdown cell line, we demonstrated that an EBNA2-mediated transcription defect was fully restored by the ectopic expression of NPM1. Our findings highlight the essential role of NPM1 in chaperoning EBNA2 onto the latency-associated membrane protein 1 (LMP1) promoters, which is coordinated with the subsequent activation of transcriptional cascades through RBP-Jκ during EBV infection. These data advance our understanding of EBV pathology and further imply that NPM1 can be exploited as a therapeutic target for EBV-associated diseases.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • B-Lymphocytes / metabolism*
  • B-Lymphocytes / virology
  • Cell Line, Tumor
  • Epstein-Barr Virus Nuclear Antigens / genetics
  • Epstein-Barr Virus Nuclear Antigens / metabolism*
  • Herpesvirus 4, Human / physiology*
  • Humans
  • Molecular Chaperones / genetics
  • Molecular Chaperones / metabolism*
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins c-myc
  • Transcription, Genetic*
  • Viral Matrix Proteins / biosynthesis
  • Viral Matrix Proteins / genetics
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*
  • Virus Assembly / physiology
  • Virus Latency / physiology*

Substances

  • EBNA-2 protein, Human herpesvirus 4
  • EBV-associated membrane antigen, Epstein-Barr virus
  • Epstein-Barr Virus Nuclear Antigens
  • MYC protein, human
  • Molecular Chaperones
  • Nuclear Proteins
  • Proto-Oncogene Proteins c-myc
  • Viral Matrix Proteins
  • Viral Proteins
  • nucleophosmin