J Virol. 2014 Oct 1. pii: JVI.01895-14. [Epub ahead of print] Viral Reprogramming of the Daxx-Histone H3.3 Chaperone During EBV Early Infection. Tsai K1, Chan L2, Gibeault R3, Conn K3, Dheekollu J2, Domsic J1, Marmorstein R1, Schang LM3, Lieberman PM4. Author information Abstract Host chromatin assembly can function as a barrier to viral infection. Epstein-Barr virus (EBV) establishes latent infection as chromatin-assembled episomes in which all but a few viral genes are transcriptionally silent. The factors that control chromatin assembly and guide transcription regulation during the establishment of latency are not well understood. Here, we demonstrate that the EBV tegument protein BNRF1 binds the histone H3.3 chaperone Daxx to modulate histone mobility and chromatin assembly on the EBV genome during the early stages of primary infection. We demonstrate that BNRF1 substitutes for the repressive co-chaperone ATRX to form a ternary complex of BNRF1-Daxx-H3.3-H4, using co-immunoprecipitation and size exclusion chromatography with highly purified components. Fluorescence recovery after photobleaching (FRAP) assays were used to demonstrate that BNRF1 promotes global mobilization of cellular histone H3.3. Mutation of putative nucleotide binding motifs on BNRF1 attenuates the displacement of ATRX from Daxx. We also show by immunofluorescence (IF) combined with fluorescence in situ hybridization (FISH) that BNRF1 is important for the dissociation of ATRX and Daxx from nuclear bodies during de novo infection of primary B-lymphocytes. Virion-delivered BNRF1 suppresses Daxx-ATRX-mediated H3.3 loading on viral chromatin as measured by chromatin immunoprecipitation (ChIP) assays and enhances viral gene expression during early infection. We propose that EBV tegument protein BNRF1 replaces ATRX to reprogram Daxx-mediated H3.3 loading, in turn generating chromatin suitable for latent gene expression. IMPORTANCE: Epstein-Barr Virus (EBV) is a human herpesvirus that efficiently establishes latent infection in primary B-lymphocytes. Cellular chromatin assembly plays an important role in regulating the establishment of EBV latency. We show that the EBV tegument protein BNRF1 functions to regulate chromatin assembly on the viral genome during early infection. BNRF1 alters the host cellular chromatin assembly to prevent anti-viral repressive chromatin, and establish chromatin structure permissive for viral gene expression and the establishment of latent infection.