EBNA1-Mediated Recruitment of a Histone H2B Deubiquitylating Complex to the Epstein-Barr Virus Latent Origin of DNA Replication

Abstract

The EBNA1 protein of Epstein-Barr virus (EBV) plays essential roles in enabling the replication and persistence of EBV genomes in latently infected cells and activating EBV latent gene expression, in all cases by binding to specific recognition sites in the latent origin of replication, oriP. Here we show that EBNA1 binding to its recognition sites in vitro is greatly stimulated by binding to the cellular deubiquitylating enzyme, USP7, and that USP7 can form a ternary complex with DNA-bound EBNA1. Consistent with the in vitro effects, the assembly of EBNA1 on oriP elements in human cells was decreased by USP7 silencing, whereas assembly of an EBNA1 mutant defective in USP7 binding was unaffected. USP7 affinity column profiling identified a complex between USP7 and human GMP synthetase (GMPS), which was shown to stimulate the ability of USP7 to cleave monoubiquitin from histone H2B in vitro. Accordingly, silencing of USP7 in human cells resulted in a consistent increase in the level of monoubquitylated H2B. The USP7-GMPS complex formed a quaternary complex with DNA-bound EBNA1 in vitro and, in EBV infected cells, was preferentially detected at the oriP functional element, FR, along with EBNA1. Down-regulation of USP7 reduced the level of GMPS at the FR, increased the level of monoubiquitylated H2B in this region of the origin and decreased the ability of EBNA1, but not an EBNA1 USP7-binding mutant, to activate transcription from the FR. The results indicate that USP7 can stimulate EBNA1-DNA interactions and that EBNA1 can alter histone modification at oriP through recruitment of USP7.

Replication of Epstein-Barr Viral DNA

“…Inhibiting Tim leads to the accumulation of linear EBV DNA in cells, consistent with these proteins stabilizing replication forks at oriP (Dheekollu and Lieberman 2011). The deubiquitylating enzyme, USP7, binds EBNA1 in vitro and localizes to oriP in vivo (Sarkari et al 2009). This association affects the levels of monoubiquitylated histone H2B in the vicinity of oriP and is thought to affect transactivation of transcription by EBNA1 bound to FR (Sarkari et al 2009).…”

“…The deubiquitylating enzyme, USP7, binds EBNA1 in vitro and localizes to oriP in vivo (Sarkari et al 2009). This association affects the levels of monoubiquitylated histone H2B in the vicinity of oriP and is thought to affect transactivation of transcription by EBNA1 bound to FR (Sarkari et al 2009). …”

Ubiquitin-specific Protease 7 Is a Regulator of Ubiquitin-conjugating Enzyme UbE2E1

“…U2OS cells were transfected three times with siRNA for USP7 (5Ј-CCCAAAUUAUUCCGCGGCAAA as described in Tang et al (29) or a negative control siRNA (provided by GenePharma) as outlined previously (30). siRNA-transfected U2OS cells and untransfected HCT116 cells (WT and USP7 Ϫ/Ϫ ) were then treated with 10 g/ml cycloheximide (Sigma) and harvested at various times after treatment.…”

Early adipogenesis is regulated through USP7-mediated deubiquitination of the histone acetyltransferase TIP60

“…Two molecular mechanisms, which are not mutually exclusive, have been described that can regulate USP7 activity: interaction with specific proteins and PTM. Studies in D. melanogaster and human cells found the metabolic enzyme GMP synthase to stimulate the ability of USP7 to deubiquitinate histone H2B [30][31][32] , whereas the TSPYL5 protein can inhibit the activity of USP7 towards p53 in mammalian cells 33 . Besides these two proteins, Sowa et al 34 identified 430 additional USP7-interacting proteins using a global proteomic analysis.…”