Subcellular Distribution of BALF2 and the Role of Rab1 in the Formation of Epstein-Barr Virus Cytoplasmic Assembly Compartment and Virion Release

Abstract

Epstein-Barr virus (EBV) replicates its genome in the nucleus and undergoes tegumentation and envelopment in the cytoplasm. We are interested in how the single-stranded DNA binding protein BALF2, which executes its function and distributes predominantly in the nucleus, is packaged into the tegument of virions. At the mid-stage of virus replication in epithelial TW01-EBV cells, a small pool of BALF2 colocalizes with tegument protein BBLF1, BGLF4 protein kinase, and the cis-Golgi marker GM130 at the perinuclear viral assembly compartment (AC). A possible nuclear localization signal (NLS) between amino acids 1100 and 1128 (C29), which contains positive charged amino acid 1113RRKRR1117, is able to promote yellow fluorescent protein (YFP)-LacZ into the nucleus. In addition, BALF2 interacts with the nucleocapsid-associated protein BVRF1, suggesting that BALF2 may be transported into the cytoplasm with nucleocapsids in a nuclear egress complex (NEC)-dependent manner. A group of proteins involved in intracellular transport were identified to interact with BALF2 in a proteomic analysis. Among them, the small GTPase Rab1A functioning in bi-directional trafficking at the ER-Golgi interface is also a tegument component. In reactivated TW01-EBV cells, BALF2 colocalizes with Rab1A in the cytoplasmic AC. Expression of dominant-negative GFP-Rab1A(N124I) diminished the accumulation of BALF2 in the AC, coupling with attenuation of gp350/220 glycosylation. Virion release was significantly downregulated by expressing dominant-negative GFP-Rab1A(N124I). Overall, the subcellular distribution of BALF2 is regulated through its complex interaction with various proteins. Rab1 activity is required for proper gp350/220 glycosylation and the maturation of EBV.