EB病毒出核複合體之研究

Abstract

Epstein-Barr virus (EBV) is an ubiquitous virus that infects most people and is the first human virus directly implicated in carcinogenesis. After the viral DNA replication and encapsidation, the nuclear egress complex composed of BFRF1 and BFLF2 can facilitate the budding of nucleocapsids through the nuclear envelope and EBV BVRF1 is recognized as a capsid-associated tegument protein that interacts with the nuclear egress complex. It is reported that BFRF1 recruits ESCRT (endosomal sorting complex required for transport) machinery to nucleus-associated membrane and induces the formation of perinuclear vesicles for promoting nuclear egress. In our previous observation, BVRF1 was co-immunoprecipitated with BFRF1 during EBV lytic cycle. In this study, the biological function and interacting protein network with nuclear egress-associated proteins were investigated. The transiently transfected Flag-BFLF2 was co-immunoprecipitated with HA-BVRF1 during EBV lytic cycle and colocalized around the nuclear rim in immunostaining. In the functional domain mapping of BFLF2, deletion of a.a. 103-149, 150-209, 209-286 or 277-318 enhanced the interaction of BFLF2 deletion mutants with HA-BVRF1. Previously, HA-BVRF1 was found co-immunoprecipitated with TSG101, a component of ESCRT machinery, in EBV reactivated cells. In this study, HA-BVRF1 and TSG101 colocalized at nuclear rim and putative EBV cytoplasmic assembly compartment in immunofluorescence assay, suggesting that BVRF1 participates in nuclear egress and maturation process of EBV through interaction with TSG101. We also established 293TetEZ cells carrying ∆BVRF1 bacmid to investigate the biological function of BVRF1 in EBV replication. Moreover, in functional domain mapping, a.a. 2-102 and a.a. 209-286 of BFLF2 was found to mediate its import into nucleus. Besides, deletion of a.a. 150-209 or a.a. 209-286 in BFLF2 influenced the vesicle formation ability of BFRF1, thereby BFRF1 accumulated as aggregates at the nuclear membrane in co-expressing cells. Overall, data here indicate multiple consequent protein-protein interactions are involved in the EBV nuclear egress process.