Subcellular Localization of Epstein–Barr Virus BLLF2 and Its Underlying Mechanisms

Abstract

Epstein–Barr virus (EBV), the pathogen of several human malignancies, encodes many proteins required to be transported into the nucleus for viral DNA reproduction and nucleocapsids assembly in the lytic replication cycle. Here, fluorescence microscope, mutation analysis, interspecies heterokaryon assays, co-immunoprecipitation assay, RNA interference, and Western blot were performed to explore the nuclear import mechanism of EBV encoded BLLF2 protein. BLLF2 was shown to be a nucleocytoplasmic shuttling protein neither by a chromosomal region maintenance 1 (CRM1)- nor by a transporter associated with antigen processing (TAP)-dependent pathway. Yet, BLLF2’s two functional nuclear localization signals (NLSs), NLS1 (16KRQALETVPHPQNRGR31) and NLS2 (44RRPRPPVAKRRRFPR58), were identified, whereas the predicted NES was nonfunctional. Finally, BLLF2 was proven to transport into the nucleus via a Ran-dependent and importin β1-dependent pathway. This mechanism may contribute to a more extensive insight into the assembly and synthesis of EBV virions in the nucleus, thus affording a new direction for the treatment of viruses.

Epstein-Barr Virus Envelope Glycoprotein gp110 Inhibits IKKi-Mediated Activation of NF-κB and Promotes the Degradation of β-Catenin

“…Co-IP assays were performed as previously described ( 113 , 114 ). Hone1-EBV cells were treated with TPA (40 ng/mL) and NaB (3 mM) for 24 h to induce EBV lytic infection, or HEK293T cells were transfected with the indicated plasmid(s) for 24 h. After EBV infection or plasmid transfection, cells were collected and lysed in RIPA lysis buffer (Beyotime) on ice for 30 min.…”

Epstein–Barr virus envelope glycoprotein 110 inhibits NF-κB activation by interacting with NF-κB subunit p65

“…The co-IP assay was performed as previously described ( 74 , 75 , 76 , 77 ). In short, HEK293T cells were cotransfected with 1 μg of the indicated expression plasmids combination of gp110-HA/p65-FLAG, gp110-HA/MAVS-FLAG, gp110 truncated mutant/p65-Myc, IKKα-HA/p65-Myc, gp110-HA/p65 truncated mutant, gp110-HA/p65-EYFP, or gp110-HA/p65-Myc/p50-FLAG.…”