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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. IMPORTANCE Upon EBV lytic reactivation, the virus-encoded DNA replication machinery functions in the nucleus, while the newly synthesized DNA is encapsidated and transported to the cytoplasm for final virus assembly. The single-stranded DNA binding protein BALF2 executing functions within the nucleus was also identified in the tegument layer of mature virions. Here, we studied the functional domain of BALF2 that contributes to the nuclear targeting and used a proteomic approach to identify novel BALF2-interacting cellular proteins that may contribute to virion morphogenesis. The GTPase Rab1, a master regulator of anterograde and retrograde endoplasmic reticulum (ER)-Golgi trafficking, colocalizes with BALF2 in the juxtanuclear concave region at the midstage of EBV reactivation. Rab1 activity is required for BALF2 targeting to the cytoplasmic assembly compartment (AC) and for gp350/220 targeting to cis-Golgi for proper glycosylation and virion release. Our study hints that EBV hijacks the bi-directional ER-Golgi trafficking machinery to complete virus assembly.