Epstein-Barr Virus BBLF1 Mediates Secretory Vesicle Transport to Facilitate Mature Virion Release

Abstract

Enveloped viruses undergo a complex multistep process of assembly, maturation, and release into the extracellular space utilizing host secretory machinery. Several studies of the herpesvirus subfamily have shown that secretory vesicles derived from the trans-Golgi network (TGN) or endosomes transport virions into the extracellular space. However, the regulatory mechanism underlying the release of Epstein-Barr virus, a human oncovirus, remains unclear. We demonstrate that disruption of BBLF1, a tegument component, suppressed viral release and resulted in the accumulation of viral particles on the inner side of the vesicular membrane. Organelle separation revealed the accumulation of infectious viruses in fractions containing vesicles derived from the TGN and late endosomes. Deficiency of an acidic amino acid cluster in BBLF1 reduced viral secretion. Moreover, truncational deletion of the C-terminal region of BBLF1 increased infectious virus production. These findings suggest that BBLF1 regulates the viral release pathway and reveal a new aspect of tegument protein function. IMPORTANCE Several viruses have been linked to the development of cancer in humans. Epstein-Barr virus (EBV), the first identified human oncovirus, causes a wide range of cancers. Accumulating literature has demonstrated the role of viral reactivation in tumorigenesis. Elucidating the functions of viral lytic genes induced by reactivation, and the mechanisms of lytic infection, is essential to understanding pathogenesis. Progeny viral particles synthesized during lytic infection are released outside the cell after the assembly, maturation, and release steps, leading to further infection. Through functional analysis using BBLF1-knockout viruses, we demonstrated that BBLF1 promotes viral release. The acidic amino acid cluster in BBLF1 was also important for viral release. Conversely, mutants lacking the C terminus exhibited more efficient virus production, suggesting that BBLF1 is involved in the fine-tuning of progeny release during the EBV life cycle.

Keywords: BBLF1; EBV; lytic replication; secretory vesicles; viral release.

FIG 1

EBV BBLF1 is a late lytic protein. (A) Identification of endogenous BBLF1. Akata cells were treated with anti-human IgG antibody, and HEK293-EBV and AGS-EBV cells were transfected with the BZLF1 expression vector for lytic reactivation. Cells were harvested at 48 h after reactivation and then subjected to immunoblotting (IB) using a newly generated anti-BBLF1 antibody. (B) B95-8 cells were treated with T/A/B (12-O-tetradecanoylphorbol-13-acetate [TPA], A23187, and sodium butyrate) for lytic induction, collected at the indicated time points and monitored for viral protein expression via IB for BZLF1 (IE), BMRF1 (E), BALF4 (L), and BBLF1, with GAPDH as a housekeeping gene. (C) B95-8 cells were treated with T/A/B with or without phosphonoacetic acid (PAA), which is a viral DNA polymerase inhibitor. (D) HEK293 cells stably carrying the EBV WT or BDLF4 knockout viral genomes were lytically reactivated through BZLF1 transfection in the presence or absence of pBDLF4, harvested after 48 h, and subjected to IB with the indicated antibodies.

FIG 2

Generation of the BBLF1-knockout EBV mutant using the EBV-BAC system. (A) Diagram of the construction of BBLF1-knockout (dBBLF1) and -revertant (dBBLF1/R) viruses using the homologous recombination technique in E. coli. An intermediate of EBV-BAC (intermediate-1) was first created through insertion of a tandemly arranged neomycin-sensitive and streptomycin-resistance gene cassette (Neo^s/St^r) at the 61st nucleotide of the BBLF1 gene in the WT EBV-BAC. The cassette was then replaced with a sequence of BBLF1 carrying the G61 to T61 mutation, which introduces a stop codon into the BBLF1 gene. Similarly, a revertant virus (dBBLF1/R) was generated from the dBBLF1 virus with exactly the same genetic construct as the WT. (B) Electrophoresis of digested EBV-BAC DNA. Isolated EBV-BACs (WT, dBBLF1, and dBBLF1/R) were digested through treatment with BamHI or EcoRI and then electrophoresed. In the lane containing intermediates 1 and 2 (BamHI digests), the DNA fragment containing the BBLF1 gene (indicated with a white asterisk in the WT lane) migrated more slowly due to the size of the Neo^s/St^r cassette (indicated with a red asterisk). (C) Nucleotide sequencing of EBV-BAC DNA for the generation of the BBLF1-knockout and revertant viruses. (D) Clonal HEK293-EBV cells (WT, dBBLF1, and dBBLF1/R) were collected immediately or at 3 days post-BZLF1 transfection (dpt). The indicated expression levels of EBV and GAPDH proteins were examined through IB. (E) Expression vectors carrying WT and dBBLF1 sequences with a C-terminal HA-tag were constructed and transfected into HEK293 cells. Cells were subjected to IB using anti-HA and anti-tubulin antibodies.

FIG 3

BBLF1 promotes virion release during lytic infection. (A) Virus samples of three fractions as follows: cell-free, cell-associated, and total. At day 3 after reactivation, HEK293-EBV cells (WT, dBBLF1, and dBBLF1/R) and their culture media were harvested and separated into cell-free and cell-associated fractions through low-speed centrifugation. The total fraction was prepared without fractionation. Titers of cell-free (B), cell-associated (C), and total virions (D) are shown. (E) The titer of cell-free virions at various time points was monitored. (F) The titer of cell-associated virions at 96 h postreactivation. (G) Observation of the fate of accumulated viruses over a long incubation period. The cell-free viral titers of reactivated HEK293-EBV cells (WT, dBBLF1, and dBBLF1/R) were measured until 192 h. (H) The titer of cell-associated virions at 196 h after lytic induction. The mean ± standard deviation (SD) of three independent biological replicates is shown. Student's t test was performed to assess significance. No significant difference (ns), P > 0.05; *, P < 0.05; **, P < 0.01.

FIG 4

BBLF1 knockout does not affect infectivity. (A) Adjustment of virus fluid based on the amount of virus genome DNA. (B) Measurement of extracellular EBV genome DNA in DNase-treated culture supernatant by qPCR. (C) Infectivity was measured in the presence of equal amounts of virus DNA. Results are means ± SD of three independent biological replicates. Student's t test was performed to assess significance. No significant difference (ns), P > 0.05; *, P < 0.05; **, P < 0.01.

FIG 5

Transcomplementation of BBLF1 in knockout clones rescues the reduction in cell-free virions. BZLF1 expression vectors were expressed in HEK293-EBV WT, dBBLF1, and dBBLF1/R cells. dBBLF1 cells were transcomplemented with BBLF1-HA expression vectors. After 72 h of incubation, samples for cell-free and cell-associated virion titer and immunoblotting were collected. (A) Expression levels of transcomplemented BBLF1-HA, BZLF1, and tubulin obtained by IB using the indicated antibodies. (B) Cell-free virion. (C) Cell-associated virion. Results are means ± SD of three independent biological replicates. Student's t test was performed to assess significance. No significant difference (ns), P > 0.05; *, P < 0.05; **, P < 0.01.

FIG 6

Unreleased virions accumulate in secretory vesicles. Visualization via electron microscopy of the cellular localization of unreleased virions from WT and BBLF1-knockout virus-infected cells. (A) Ultrathin section of a reactivated HEK293-EBV WT cell; scale bar, 5 μm. (B to D) Ultrathin sections of the cytoplasmic area; scale bar, 0.5 μm for panel B and 0.1 μm for panel C. (D) Vesicles harboring single viruses or virus-like particles. (E) Ultrathin section of a reactivated HEK293-dBBLF1 cell; scale bar, 5 μm. (F to H) Ultrathin sections of the cytoplasmic area; scale bar, 0.5 μm for panel F and 0.1 μm for panel G. (H) Vesicles harboring single or multiple viruses or virus-like particles. (I) Diagram of subcellular fractionation of reactivated HEK293-EBV WT and dBBLF1 cell organelles. Reactivated HEK293-EBV cells were harvested at 72 h, disrupted with a Potter-Elvehjem grinder, and centrifuged to remove the nuclear fraction. The cytosolic fraction was poured into an iodixanol (OptiPrep) gradient tube and ultracentrifuged. The resulting fractions (F1 to F12) were collected, aliquoted, frozen, and used for further analysis. (J) The titer of infectious viruses in the subcellular fractions of reactivated HEK293-EBV WT and dBBLF1 cells. (K) Detection of organelle markers in the subcellular fractions. IB was performed using antibodies against calnexin, Rab5, Rab9, Rab11, GM130, TGN38, and CD63. (L) Transmission electron microscopy (TEM) image of a mature virus (white arrow) and a vesicle (black arrow); scale bar, 0.1 μm. The image is of subcellular fraction 6 of dBBLF1 cells.

FIG 7

An acidic amino acid cluster is required for virion release. (A) Construction of BBLF1 mutant expression vectors. Through site-directed mutagenesis, alanine substitution mutations were generated at the targeted amino acids. G2A and C8A mutations were generated to obtain BBLF1 myristoylation (Myr) and BBLF1 palmitoylation (Pal) mutants, respectively. The BBLF1 acidic amino acid motifs, NDE (amino acids 28 to 31), SDE (amino acids 58 to 65), and both NDE and SDE, were also substituted with alanine. (B) IB of HEK293T cells transfected with mutant vectors showing the expression of BBLF1 mutants. (C) IB of cells transfected with BBLF1 mutants, with or without the proteasome inhibitor MG132. (D) IB of reactivated HEK293-dBBLF1 cells transcomplemented with mutant vectors showing the expression of BZLF1, BBLF1 mutants, and tubulin. Cell-free (E) and cell-associated (F) infectious viral titer of reactivated HEK293-dBBLF1 cells transcomplemented with the constructed mutant vectors. Results are means ± SD of three independent biological replicates.

FIG 8

The C-terminal region of BBLF1 can partially suppress virion release. (A) Amino acid sequences of mutants with a BBLF1-C-terminal domain (CTD) mutation obtained through gene editing. (B) Akata-EBV cells were reactivated through treatment with anti-human IgG for 3 days and then subjected to IB to assess the expression of BZLF1, BMRF1, BALF4, and BBLF1. (C, D) Cell-free (C) and cell-associated (D) virion titers of Akata-EBV cells at 3 days postreactivation. Cell-free and cell-associated virion samples were obtained as described in Fig. 3A. (E) EBV genome copies in DNase-treated culture supernatant. (F to H) Transcomplementation of the BBLF1-knockout virus. HEK293-dBBLF1 cells were transfected with BBLF1-HA expression vectors (WT or BBLF1-CTD mutant) during lytic reactivation and their cell-free (F), cell-associated (G), and total (H) progeny virions were quantified. (I) IB of transcomplemented dBBLF1 cells showing the expression of BZLF1, BBLF1-WT-HA, BBLF1-CTD-HA, and tubulin as a housekeeping gene. (J) A similar experiment to that shown in Fig. 8H was conducted, except that WT HEK293-EBV cells were used. (K) IB of transcomplemented WT cells showing the expression of BZLF1, BBLF1-WT-HA, BBLF1-CTD-HA, and tubulin as a housekeeping gene. The data are means ± SD of three independent biological replicates. Student's t test was performed to assess significance. No significant difference (ns), P > 0.05; *, P < 0.05; **, P < 0.01.