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

概要

主論文の要旨

Epstein-Barr Virus BBLF1 Mediates Secretory Vesicle
Transport to Facilitate Mature Virion Release
エ プ ス タ イ ン バ ー ウ イ ル ス 遺 伝 子 BBLF1は 分 泌 小 胞 輸 送 を
制御し成熟ウイルス粒子の放出を促進する

名古屋大学大学院医学系研究科
微生物・免疫学講座

総合医学専攻

ウイルス学分野

（指導：木村 宏

教授）

Md. Kamal Uddin

【 Background】
Epstein-Barr virus (EBV) is a double-stranded DNA virus belonging to the family
Herpesviridae. EBV infects >95% of the global population, often leads to asymptomatic
infections, and is involved in the development of various epithelial cancers, including
nasopharyngeal and gastric cancers, as well as B-cell, NK-cell, and T-cell lymphomas. There
are two types of EBV infection as follows: latent and lytic. In general, EBV establishes a
latent infection without viral production. The switch from latent to lytic infection is called
reactivation, which leads to the production of viral particles. Herpesviruses 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 EBV remains unclear.
In this study, we demonstrate that disruption of BBLF1, a tegument component of EBV,
suppressed viral release and resulted in the accumulation of viral particles inside the vesicles.
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.
【 Methods and Results】
After lytic reactivation of various cell lines stably carrying EBV genomes, we identified
BBLF1’s expression by using a newly generated antibody against BBLF1. The analysis of
expression kinetics showed that BBLF1 expressed at 36 hours post-lytic reactivation like other
late lytic proteins, and the expression was halted when cells were treated by an inhibitor of
viral DNA polymerase (Figure 1). These indicate that BBLF1 is an EBV lytic gene expressed
with late kinetics during the productive cycle.
To determine its biological role, we generated a BBLF1-knockout virus (B95-8 strain) using
the EBV bacterial artificial chromosome (BAC) system following the homologous recombination
technique. A BBLF1-deficient virus (dBBLF1) carrying a stop codon (TGA) near N-terminus
was generated from the wild type (WT), and a revertant virus (dBBLF1/R) was constructed
from dBBLF1. Through transfection of EBV-BAC DNA (WT, dBBLF1, or dBBLF1/R) into
HEK293 cells, we established clonal cell lines stably carrying EBV-BAC DNA. We then
analyzed the expression levels of viral proteins during the lytic cycle and found that BBLF1
knockout does not hamper the expression of representative viral proteins (Figure 2).
To examine the impact of BBLF1 knockout on infectious virus production from
HEK293EBV cells, we infected EBV-negative cells with virion derived from the culture
medium (cell-free virions) and cells (cell-associated virions) separately and monitored viral

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titers by FACS. Cell-free virion production in dBBLF1 was significantly lower than in the
WT or dBBLF1/R. In contrast, cell-associated virion levels were higher for the knockout virus.
When virions were collected without fractionation (total virions), no significant difference
was observed among cell lines. These data suggest that BBLF1 plays a role in the secretion
of viruses. When we measured the viral titer in the presence of an equal quantity of cell-free
viral DNA, there was no significant difference among the WT, dBBLF1, and dBBLF1/R clones
indicating that BBLF1 knockout does not impact infectivity. Moreover, exogenous expression
of BBLF1 in dBBLF1 cells significantly increased virion release to a level comparable to WT
and dBBLF1/R cells. Taken together, these data (Figure 3) indicate that BBLF1 expression
promotes virion release during lytic phase.
To determine where BBLF1-knockout viral particles accumulate, we observed lytically
induced HEK293-EBV WT and dBBLF1 cells by electron microscopy. Viral particles or viruslike particles accompanying vesicles were observed in both types of cells; however, more
virus particles accumulated in dBBLF1 cells than in WT cells. Due to the low number of
accumulated viral particles in the WT cells, we hypothesized that BBLF1 knockout affects
intracellular trafficking. Further investigation by subcellular fractionation showed that
BBLF1 deficiency causes an accumulation of infectious viruses in intracellular organelles
involved in intracellular trafficking, including secretory vesicles (Figure 4).
Through mutagenesis of BBLF1, several mutant vectors were generated targeting its posttranslational modification (myristoylation and palmitoylation) sites and distinctive acidic
amino acid-rich clusters, and the importance of these features was analyzed in the context of
virion release. We found that optimal shedding of virions required a cluster of acidic amino
acids but did not require posttranslational modifications (Figure 5).
To extend the previous results, we aimed to prepare a BBLF1-knockout EBV of the Akata
strain using the CRISPR/Cas9 system. We constructed mutant clones, in which the BBLF1 Cterminal domain (CTD), including the SDE domain, was disrupted. Subsequent analysis shows
that truncation of the C-terminal region of BBLF1 increased infectious virus production,
suggesting that the C terminus of BBLF1 regulates its production function (Figure 6).
【 Discussion】
Elucidating the functions of viral lytic genes induced by reactivation, and the mechanisms
of lytic infection, is essential to understanding pathogenesis. Through functional analysis
using BBLF1-knockout viruses, we demonstrated that BBLF1 promotes viral release. Based
on the common involvement of the BBLF1 homologs in secondary envelopment and the
previous finding that BBLF1 is located on the envelope as an outer tegument, we hypothesized
that BBLF1 facilitates cytoplasmic virion envelopment. One unanticipated result of this study
was that BBLF1 knockout reduced extracellular virion abundance, whereas total virion levels,
including both the intra- and extracellular fractions, were comparable to the WT. Furthermore,

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electron microscopy images showed no abnormal nucleocapsids in the nuclei of BBLF1knockout virus-infected cells. Thus, unlike its homologs, BBLF1 affects the transport-torelease phase of infectious virions after the acquisition of the final envelope but not nuclear
membrane transport or virion maturation.
We observed that BBLF1 knockout led to an accumulation of budding viral particles in the
vesicular membrane, and some viral particles appeared as multivesicular bodies containing
numerous internal small vesicles under the electron microscope. The mechanism through
which BBLF1 facilitates the transport-to-release process remains unclear. Further research is
required to address whether BBLF1 regulates the TGN-associated exocytosis pathway or
exosomal release pathway via the MBV. The increase of virus production by Akata-CTD
mutants suggests that BBLF1 might have an autoinhibitory effect. Certain viral genes have
conflicting functions depending on the intracellular environment and stage of the viral life
cycle, for example, EBNA1. When and how BBLF1 switches between promotion and
suppression of viral production remains unclear, but we predict that BBLF1 negatively
regulates virus production during primary infection, thereby increasing the rate of latent
infection. Alternatively, BBLF1 may promote an abortive lytic infection to avoid cell death.
The C-terminal region of BBLF1 has low sequence homology with other herpesviruses and is
assumed to represent a survival strategy unique to gammaherpesviruses; further analysis of
its effects on the viral life cycle is needed to clarify the biological significance of BBLF1.
【 Conclusion】
We performed initial functional analysis of BBLF1 using BBLF1-knockout viruses and
found that BBLF1 contributes to the viral release process. Furthermore, we found that an
acidic amino acid cluster enhances the efficiency of viral release. This is the first report
showing that the CTD of BBLF1 suppresses viral production, suggesting that the BBLF1
protein plays dual roles. The molecular mechanism through which BBLF1 suppresses viral
production remains unclear, and BBLF1 may fine-tune the secretion of progeny virions.

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