Fig 2: A subset of BALF2 colocalizes with Rab1A in the nuclear concave region in Rta-reactivated TW01-EBV cells. (A) TW01-EBV cells were transfected with pSG5-Rta or pSG5. The distribution of BALF2 and endogenous Rab1A in TW01-EBV cells was detected with mouse anti-BALF2 monoclonal antibody (OT13B) and rabbit anti-Rab1A monoclonal antibody (Cell Signaling, D3X9S) and observed by confocal microscopy at 48 hpt. (B) The distribution of BALF2 and GFP-Rab1A was detected with specific antibodies and observed by confocal microscopy at 48 h post-Rta transfection in TW01-EBV cells. Cells with patterns of BALF2 localized in both the nucleus and viral assembly compartment were 25 out of 30 of BALF2-positive cells. (C) Coimmunoprecipitation of BALF2 and GFP-Rab1A using antibodies against BALF2, GFP, and GST was carried out using TW01-EBVcell lysates harvested at 48 h posttransfection of the Rta-expressing plasmid. Immunoblotting was carried out with BALF2 antibody or GFP antibody. The GFP-Rab1A band is indicated with a black arrowhead. (D) GFP-Rab1A and Flag-BALF2 were coexpressed in HeLa cells. The distribution of Flag-BALF2 and GFP-Rab1A was detected with Flag antibody and observed under confocal microscopy.

Fig 3: Expression of GFP-Rab1(N124I) in TW01-EBV and 293/rM81 cells attenuates EBV release and infectivity. (A) TW01-EBV cells were transfected with pSG5-Rta or pSG5 plus GFP, GFP-Rab1A, GFP-Rab1A(N124I), or GFP-Rab1A(Q70L) using Lipofectamine 2000. The cell lysates were harvested for immunoblotting at 48 hpt. The expression levels of Rta, GM130, endogenous Rab1A, BALF2, and wild-type, dominant-active, or constitutive GFP-Rab1A in TW01-EBV cells were detected by GFP antibody, BRLF1 antibody, BMRF1 antibody, and Rab1A antibody. Gp110 and gp350/220 were analyzed by 8% SDS-PAGE (bottom panels). (B) The intracellular EBV genomic DNA copy numbers of TW01-EBV cells were analyzed by q-PCR at 48 hpt of Rta and GFP-C1 vector or GFP-Rab1A constructs. (C) The released EBV virion DNA in the medium in the same setting was analyzed by q-PCR at 48 hpt. The representative data from 4 independent experiments are displayed. (D) 293/rM81 cells were cotransfected with Zta and gp110 expression plasmids or pSG5 control plus GFP, GFP-Rab1A, GFP-Rab1A(N124I), or GFP-Rab1A(Q70L) using TransIT-LT1. Cell lysates were harvested for immunoblotting at 72 hpt. The expression levels of Zta, Rta, BMRF1, BALF2, and wild-type, dominant-active, or constitutive GFP-Rab1A in 293/rM81 cells were detected using specific antibodies. For detecting gp350/220, gp110, and BcLF1, lysates were displayed in nonreducing 8% SDS-PAGE. (E) The intracellular EBV genomic DNA copy number of 293/rM81 cells was analyzed by q-PCR at 72 hpt of Zta, gp110, and GFP-C1 vector or GFP-Rab1A constructs. (F) The released EBV virion DNA of the same setting was analyzed with q-PCR at 72 hpt. (G) Culture supernatants from the experiment in panel D were filtered through a 0.45-μm filter, and that same volume of filtered viral supernatant was added to Raji cells and incubated for 72 h in duplicated wells. The percentages of Raji cells expressing GFP were determined through flow cytometry. The results of one experiment are presented. The horizontal axis represents the forward scatter area (FSC-A), and the vertical axis indicates the intensity of GFP fluorescence (GFP-A). The GFP positivity threshold was determined according to the noninduction control (C1-pSG5). Experimental results are presented using the mean with standard deviation (SD) as the bar graph in panel H.

Fig 4: Expression of dominant-negative GFP-Rab1A(N124I) diminishes the clustering of BALF2 to the viral assembly compartment. (A) TW01-EBV cells were cotransfected with pSG5-Rta or pSG5 plus GFP-C1, GFP-Rab1A, GFP-Rab1A N124I, or GFP-Rab1A Q70L. The cell lysates were harvested at 48 hpt. The expression levels of viral proteins BALF2, BMRF1 (EA-D), BLLF1 (gp350/220), and BALF4 (gp110) and cellular proteins GM130 and Rab1A were detected with BRLF1 antibody, BALF2 antibody, BMRF1 antibody, gp350/220 antibody, gp110 antibody, GM130 antibody, and Rab1A antibody in immunoblotting. Transfected GFP, GFP-Rab1A, and BRLF1 (Rta) were also detected in the blotting. GAPDH served as the loading control. Proteins in the top panel were displayed in regular 10% SDS-PAGE, whereas gp350/220 and gp110 were detected in immunoblotting after nonreducing 8% SDS-PAGE. Open arrowheads indicate mature gp350 or gp220, while the solid arrowhead indicates the precursor of gp350/220. (B) The distribution of BALF2 with different GFP-Rab1A constructs was observed by immunostaining and confocal microscopy at 48 h post cotransfection of Rta and GFP, GFP-Rab1A, GFP-Rab1A(N124I), or GFP-Rab1A(Q70L). The percentages listed next to each group were calculated to indicate cells expressing BALF2 localized to the viral assembly compartment or not over GFP+ GM130+ cells.

Fig 5: A hypothetical diagram of the BALF2 subcellular distribution during virus replication and the role of Rab1A in the assembly of EBV viral particles. The top part, above the dotted line, indicates early steps of EBV reactivation. (a) BALF2 is translated from the ER and transported into the nucleus through a canonical importin-β-dependent mechanism. (b) At the mid- to late stage of EBV replication, viral DNAs are encapsidated into procapsids, and the nucleocapsids are egressed from the nucleus through either a primary envelopment and deenvelopement process (b) or a nuclear egress complex and ESCRT-dependent mechanisms (b′). (c) After synthesis on the ER membrane, gp350/220 was transported through Rab1-mediated vesicle transport to the cis-Golgi apparatus for proper glycosylation. (d) Fragmented Golgi and Golgi-derived vesicles with membrane-associated gp350/220 may target the viral assembly compartment. (e) Rab1 interacts with GM130 and anchors on the cis-Golgi membranes and Golgi-derived vesicles. The membrane-associated Rab1 helps the engulfment of nucleocapsids or nucleocapsids containing vesicles through interaction with a BALF2-associated protein-protein interaction. (f) The mature virion with tegument (yellow regions) may be released through exocytosis.