Nucleolin is important for Epstein–Barr virus nuclear antigen 1-mediated episome binding, maintenance, and transcription

Abstract

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for EBV episome maintenance, replication, and transcription. These effects are mediated by EBNA1 binding to cognate oriP DNA, which comprise 20 imperfect copies of a 30-bp dyad symmetry enhancer and an origin for DNA replication. To identify cell proteins essential for these EBNA1 functions, EBNA1 associated cell proteins were immune precipitated and analyzed by liquid chromatography-tandem mass spectrometry. Nucleolin (NCL) was identified to be EBNA1 associated. EBNA1's N-terminal 100 aa and NCL's RNA-binding domains were critical for EBNA1/NCL interaction. Lentivirus shRNA-mediated NCL depletion substantially reduced EBNA1 recruitment to oriP DNA, EBNA1-dependent transcription of an EBV oriP luciferase reporter, and EBV genome maintenance in lymphoblastoid cell lines. NCL RNA-binding domain K 429 was critical for ATP and EBNA1 binding. NCL overexpression increased EBNA1 binding to oriP and transcription, whereas NCL K 429 A was deficient. Moreover, NCL silencing impaired lymphoblastoid cell line growth. These experiments reveal a surprisingly critical role for NCL K429 in EBNA1 episome maintenance and transcription, which may be a target for therapeutic intervention.lymphoma | chromatin | oncogenic herpesvirus | nasopharyngeal carcinoma

Ribosome Protein L4 is essential for Epstein–Barr Virus Nuclear Antigen 1 function

“…Despite a 2.4-Å resolution crystal structure of the EBNA1 DNA binding domain bound to its cognate DNA element (4), mechanistic insights into EBNA1 and oriP-mediated episome maintenance mainly come from genetic studies using EBV recombinants and biochemical studies of EBNA1's association with cell proteins, including CTCF, Bromodomain Protein 4 (BRD4), Nucleosome Assembly Protein 1 (NAP1), the cell Origin Recognition Complex, and the Mini Chromosome Maintenance complex (5-8). Recent studies indicate that EBNA1 may use complex strategies for episome maintenance (9-16).EBNA1-associated ribosome biogenesis factors Nucleophosmin (NPM1) and Nucleolin (NCL) have been implicated in EBNA1 and oriP-dependent functions (17,18). Other viruses also use ribosomal proteins (RPs), such as RPL4, RPS19, and RPL40, to enhance virus protein translation (19-21).…”

“…EBNA1-associated ribosome biogenesis factors Nucleophosmin (NPM1) and Nucleolin (NCL) have been implicated in EBNA1 and oriP-dependent functions (17,18). Other viruses also use ribosomal proteins (RPs), such as RPL4, RPS19, and RPL40, to enhance virus protein translation (19-21).…”

“…S1A). RPL4 was identified as a FLAG-EBNA1 (FEBNA1)-associated protein in BJAB B-lymphoma cells (BJAB-FEBNA1) by immune precipitation, liquid chromatography, and tandem mass spectrometry (LC-MS/MS) (18) (Fig. S1B).…”

“…2A). These cells were transiently transfected with an EBNA1 expression plasmid together with an oriP luciferase reporter, oriP-SV40-Luc (oriP-Luc) (18). Luciferase activities were determined 24 h after transfection.…”

“…RPL4 EBNA1-binding null mutants FYC-RPL4 Δ62-170 and Δ170-238 did not elicit BiFC signals (Fig. S4D) (18). Nuclear and cytoplasm fractionation followed by Western blotting were also used to determine the RPL4 nuclear localization.…”