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Tegument Protein ORF45 Plays an Essential Role in Virion
Morphogenesis of Murine Gammaherpesvirus 68
Xing Jia,a,b Sheng Shen,a Ying Lv,a,b Ziwei Zhang,a,b Haitao Guo,a Hongyu Denga,c
Chinese Academy of Sciences Key Laboratory of Infection and Immunity, Institute of Biophysics, Beijing, People’s Republic of Chinaa; University of Chinese Academy of
Sciences, Beijing, People’s Republic of Chinab; CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People’s
Republic of Chinac
A
s a unique structure of herpesviruses, tegument functions in
multiple aspects of the viral life cycle, including modulation
of the host cellular environment and initiation of viral gene transcription at the immediate-early phase of virus infection, transport of nucleocapsids to the nuclear pore, and virion morphogenesis and egress (1–10). Virion morphogenesis is a complicated
event that takes place in the late phase of the herpesvirus lytic life
cycle. After acquisition of viral genomes in the nucleus, nucleocapsids bud at the inner nuclear membrane to form primary virions in the perinuclear space, followed by deenvelopment at the
outer nuclear membrane, releasing capsids into the cytoplasm.
Capsids then undergo tegumentation and reenvelopment to obtain tegument proteins and envelope glycoproteins (11, 12, 13).
Finally, mature virions are released from the cell.
ORF45 is a virion-associated protein that is conserved in gammaherpesviruses but has no homologue in alpha- or betaherpesviruses. The ORF45 homologues in Kaposi’s sarcoma-associated
herpesvirus (KSHV), murine gammaherpesvirus 68 (MHV-68),
Epstein-Barr virus (EBV), and rhesus rhadinovirus (RRV) are 407
amino acids (aa), 206 aa, 217 aa, and 353 aa, respectively (14, 15).
The homology mainly lies in the C-terminal region of the protein.
Among these homologues, KSHV ORF45 is the best studied. It is
virion associated (16, 17) and is a multifunctional protein. It interacts with interferon regulatory factor 7 and inhibits virus-induced interferon production, leading to viral immune evasion (2,
18). Characterization of a KSHV ORF45-null bacterial artificial
chromosome (BAC) showed that although ORF45 is not essential
for KSHV lytic replication, it plays roles in both early and late
stages of viral infection (19). Indeed, ORF45 was shown to interact
with kinesin-2 to mediate the transport of viral particles along
microtubules (4). ORF45 also interacts with another tegument
protein, ORF33, and plays a crucial role in efficient production of
KSHV viral particles (15). Most recently, ORF45 was shown to
mediate association of KSHV particles with internal lipid rafts for
viral assembly and egress (20).
MHV-68 ORF45 has also been identified as a virion-associated
protein (21, 22, 23). Biochemical analysis demonstrated that
ORF45 is a true tegument protein that is packaged into virions
(23). By construction and analysis of an ORF45-null MHV-68
mutant using a BAC system, Jia et al. demonstrated that the
ORF45-null MHV-68 BAC is incapable of virion production, in-
August 2016 Volume 90 Number 16
dicating that newly synthesized ORF45 is essential for MHV-68
lytic replication (3). However, this defect can be rescued in trans
by ORF45 fused to green fluorescent protein (GFP) to generate an
ORF45-null mutant virus, although the packaging efficiency of
GFP-ORF45 protein was significantly lower in the ORF45-null
mutant than in the wild-type (WT) ORF45 protein in WT virions
(3). Viral gene expression and DNA replication were blocked during de novo infection of the mutant virus, suggesting that the virion-associated GFP-ORF45 protein plays an important role in viral
gene expression. Kinetically, ORF45 is a highly expressed earlylate gene (24, 25). Thus, newly synthesized ORF45 is not expected
to regulate viral immediate-early or early gene expression in the
same infected cell. However, newly synthesized ORF45 is packaged into virions as tegument protein, and the virion-associated
ORF45 protein can then be delivered into new cells during the
next round of de novo infection and may modulate viral gene
expression in the new cells. Because virion morphogenesis takes
place following viral DNA replication and late gene expression,
the role of newly synthesized ORF45, if any, in virion morphogenesis could not be studied by such an approach. Therefore, we decided to directly address the role of newly synthesized ORF45 in
viral lytic replication under BAC transfection conditions and
within “single-round” viral replication, so that the effect of virionassociated ORF45 could not confound our data analysis.
We first examined the subcellular localization of MHV-68
ORF45 during virus infection, as the function of a protein is inevitably linked to its localization within the cell. Structured illumination microscopy (SIM) uses a grid to create several interference
patterns on the sample for each z plane and captures multiple
Received 26 December 2015 Accepted 17 May 2016
Accepted manuscript posted online 25 May 2016
Citation Jia X, Shen S, Lv Y, Zhang Z, Guo H, Deng H. 2016. Tegument protein
ORF45 plays an essential role in virion morphogenesis of murine
gammaherpesvirus 68. J Virol 90:7587–7592. doi:10.1128/JVI.03231-15.
Editor: R. M. Longnecker, Northwestern University
Address correspondence to Hongyu Deng, [email protected].
X.J. and S.S. contributed equally to this work.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Tegument proteins play critical roles in herpesvirus morphogenesis. ORF45 is a conserved tegument protein of gammaherpesviruses; however, its role in virion morphogenesis is largely unknown. In this work, we determined the ultrastructural localization
of murine gammaherpesvirus 68 (MHV-68) ORF45 and found that this protein was incorporated into virions around the site of
host-derived vesicles. Notably, the absence of ORF45 inhibited nucleocapsid egress and blocked cytoplasmic virion maturation,
demonstrating that ORF45 is essential for MHV-68 virion morphogenesis.
Jia et al.
infected with MHV-68 at a multiplicity of infection (MOI) of 3. At 24 h postinfection, cells were fixed and subjected to an indirect immunofluorescence assay using a
rabbit polyclonal anti-ORF45 antibody (Ab) or rabbit IgG as a negative control and observed with a three-dimensional structured illumination microscope (Delta Vision
OMX). The anti-ORF45 antibody and rabbit IgG were detected using an Alexa Fluor 488-conjugated secondary antibody (green channel). Nuclei were stained with DAPI
(4=,6-diamidino-2-phenylindole) (blue channel). (B) BHK-21 cells were infected with WT MHV-68 at an MOI of 5 and analyzed 11 h later by immunoelectron
microscopy with the anti-ORF45 antibody (B1 to B7 and B10) or rabbit IgG as a negative control (B8 and B9), followed by a secondary antibody conjugated with 5-nm
gold particles. Gold particles were found dispersed in the nucleus (Nu) around intranuclear capsids or with low occupancy on intranuclear capsids (B1, with black
arrowheads indicating intranuclear capsids labeled with gold particles), abundantly in the nuclear inclusion body (B2) and the nuclear membrane area (Nm) (B3), with
low occupancy on primary virions (B4, with white arrow indicating a primary virion labeled with a gold particle), abundantly in the tegument deposit (B5, B6, and B10),
on capsids undergoing tegumentation (B10, black arrowheads) and secondary envelopment in the cytoplasm (Cyto) (B6 and B10, white arrows), and on mature virions
in the cytoplasm (B7). Few gold particles were found in samples treated with rabbit IgG (B8 and B9).
cross sections of the specimen, producing three-dimensional
images with a high resolution (26). By taking advantage of this
technology, we found that ORF45 was localized in both the
nucleus and the cytoplasmic area (Fig. 1A). Such distribution is
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consistent with the subcellular distribution of its homologue in
KSHV (27, 28).
We next performed an immunogold labeling assay to examine
the ultrastructural localization of ORF45 and its association with
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FIG 1 ORF45 is localized in both the nucleus and the cytoplasm and is incorporated into MHV-68 virions around the site of host-derived vesicles. (A) BHK-21 cells were
MHV-68 ORF45 Is Essential for Virion Morphogenesis
August 2016 Volume 90 Number 16
individually transfected WT BAC, 45S BAC, or 45S.R BAC into
293T cells. At 90 h posttransfection, cells transfected with WT
BAC and 45S.R showed a severe cytopathic effect (CPE), whereas
no CPE was detected in cells transfected with 45S BAC. Two more
rounds of passage of the supernatants harvested from 45S BACtransfected cultures onto fresh cells did not result in development
of a CPE, indicating that no infectious virus was produced and
released into the supernatant (data not shown). Therefore, ORF45
is required for production of extracellular infectivity.
To identify the step of viral lytic replication in which newly
synthesized ORF45 may function under BAC transfection conditions, we first attempted to identify a near-single-round viral
replication time point through immunofluorescence assay
(IFA). Two structural proteins, ORF65 and ORF33, were
stained at different time points and examined. At 26 h posttransfection, fluorescence signals for ORF65 (Fig. 2A, left) or
ORF33 (data not shown) in all transfections were in isolated
fashion, suggesting no apparent virus spreading from cell to
cell at this time point. At 36 h posttransfection, in cells transfected with WT BAC, 45S.R BAC, or 45S BAC plus pFlag45, a
large agglomerate of ORF65 (Fig. 2A, right) or ORF33 (data not
shown) fluorescence signals, indicating progeny virions being
released from BAC-transfected cells and then infecting neighboring cells, was detected. However, the fluorescence signals
were still in isolated fashion in 45S BAC-transfected cells, again
confirming that ORF45 was required for completion of viral
lytic replication. We then examined the expression of a panel of
viral late genes at 26 h posttransfection (i.e., within singleround viral replication). As expected, ORF45 was not expressed
in 45S BAC-transfected cells. However, there was no apparent
decrease in the expression levels of capsid protein ORF65, tegument proteins ORF33 and ORF38, or ORF67 and ORF69 proteins (homologues of the nuclear egress complex in MHV-68),
compared to those of WT BAC- or 45S.R BAC-transfected cells
(Fig. 2B). Densitometric analysis confirmed that the absence of
ORF45 did not affect the expression levels of these viral late
genes (Fig. 2B). Expression of these late genes was also successfully detected in 45S BAC-transfected BHK-21 cells, although
at lower levels than those in 293T cells (data not shown). These
data indicated that the absence of newly synthesized ORF45 did
not affect the expression of viral late genes within single-round
replication.
The above-described findings, that newly synthesized ORF45
does not affect viral late gene expression but is required for production of progeny virions, suggested that, at later time points,
when multiple rounds of viral replication have occurred, the absence of ORF45 would have an effect on viral gene expression.
Indeed, at 90 h posttransfection in 293T cells, a panel of viral late
genes was successfully detected. However, the expression levels of
these genes were significantly lower in 45S BAC-transfected 293T
cells than in cells transfected with WT BAC or 45S.R BAC (Fig.
2C). These data are slightly different from the previous report
(Fig. 4B in reference 3), which may be caused by lower transfection
efficiencies in BHK-21 cells as well as inclusion of later time points
(5 days posttransfection, i.e., after more rounds of viral replication, which would further amplify the differences in viral gene
expression levels due to the absence of ORF45) in that study.
We next examined whether newly synthesized ORF45 may play
a role in viral immediate-early or early gene expression. Cotransfection of pGLRp6 (RTA promoter-driven luciferase reporter) or
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virions. BHK-21 cells were infected with MHV-68, fixed, embedded, and sectioned. The sections were immunolabeled with either
a rabbit anti-ORF45 polyclonal antibody or rabbit IgG as a negative control, followed by a secondary antibody conjugated with
5-nm gold particles. As shown in Fig. 1B, in the nucleus, dispersed
distribution of gold particles around intranuclear capsids (Fig. 1B,
B1), as well as accumulation of gold particles in inclusion bodies
formed during virion assembly and egress (29) (Fig. 1B, B2) and
around nuclear membrane areas (Fig. 1B, B3), were observed.
Despite abundant distribution of gold particles in the nucleus,
labeling of intranuclear capsids (Fig. 1B, B1, black arrowhead) or
primary virions in the perinuclear space (Fig. 1B, B4, white arrow)
occurred only infrequently. In the cytoplasm, gold particles accumulated in electron-dense areas, designated tegument deposits
(29), around host-derived vesicles (Fig. 1B, B5, B6, and B10), consistent with the cytoplasmic fluorescent signals revealed in the
SIM images (Fig. 1A). Furthermore, gold particles were detected
on mature virions (Fig. 1B, B7). In the negative-control sample
treated with rabbit IgG, very few gold particles were detected in
either the nucleus or the cytoplasm (Fig. 1B, B8 and B9), indicating the specificity of immunogold staining. Notably, tegument
deposits were very frequently observed and emerged in almost all
infected cells with an ongoing virion morphogenesis process. In
addition, accumulation of capsids, tegument deposits, host-derived vesicles, and virions undergoing secondary envelopment
formed representative structures in the cytoplasmic area (Fig. 1B,
B5, B6, and B10).
We further quantified labeling of capsids outside deposits
(n ⫽ 134) and virions undergoing secondary envelopment at
vesicles surrounded or semisurrounded by tegument deposits
(n ⫽ 51). Most capsids (76.1%) (Fig. 1B, B6, and B10, black
arrows, and images not shown) did not contain ORF45, but a
portion of labeled capsids (23.9%) (Fig. 1B, B10, black arrowheads, and images not shown) emerged around the tegument
deposit, suggesting that ORF45 was likely to be incorporated
into virions at the tegument deposit area. Most virions (88.2%)
(Fig. 1B, B6 and B10, white arrows, and data not shown) contained ORF45, again strongly indicating that ORF45 was incorporated into virions at the tegument deposit area. The portion
of virions (11.8%; data not shown) which was not labeled
might be due to several reasons. For example, depending on the
relative location of ORF45 protein within the embedded sample block, it may be difficult for the anti-ORF45 antibody to
gain access to ORF45 and/or for gold particles to penetrate the
medium used to embed samples for electron microscopy analysis. Collectively, these images revealed not only the distribution of ORF45 within the cell but also the sites where ORF45
was loaded into virions, suggesting multiple roles for the protein during virion assembly and egress.
To determine the function of newly synthesized ORF45 in virus lytic replication, we generated an ORF45-null (45S) BAC by
inserting triple-stop codons and a BglII site between nucleotide
(nt) 64156 and nt 64157 on the viral genome (116 nt downstream
of the translation initiation codon for ORF45) using a two-step
lambda Red recombination method, according to a previous report (3). A revertant of the ORF45-null mutant (45S.R) was also
generated by deleting the BglII site and triple-stop codons from
the 45S BAC using the same method. Restriction enzyme digestion analysis and sequencing results demonstrated that no other
sequences were introduced into 45S BAC or 45S.R BAC. We then
Jia et al.
WT BAC plus plasmid pcDNA3.1, 45S BAC plus pcDNA3.1, 45S.R BAC plus pcDNA3.1, or 45S BAC plus pFlag-45. At 26 h or 36 h posttransfection, cells were
analyzed by indirect immunofluorescence assay using a rabbit polyclonal anti-ORF65 antibody and observed with a fluorescence microscope (Nikon Eclipse Ti).
The anti-ORF65 antibody was detected using an Alexa Fluor 488-conjugated secondary antibody (green channel). Nuclei were stained with DAPI (blue channel).
(B) 293T cells were transfected with WT BAC, 45S BAC, or 45S.R BAC. At 26 h posttransfection, cells were collected and subjected to Western blotting. Viral
proteins were detected with specific antibodies, as indicated to the right. The expression level of each protein was quantitated by densitometric analysis,
normalized to that of actin in the same sample, and compared to that from WT BAC, with the expression level of each protein from WT BAC set to 1. (C) 293T
cells were transfected with WT BAC, 45S BAC, or 45S.R BAC. At 90 h posttransfection, cells were collected and subjected to Western blotting. Viral proteins, as
indicated to the right, were detected with specific antibodies. (D) Firefly luciferase reporter constructs driven by the RTA or ORF57 promoter were individually
cotransfected into 293T cells with MHV-68 BAC (WT, 45S, or 45S.R) and pRenilla-TK (as an internal control). At 18 h posttransfection, firefly luciferase activity
was measured and was normalized against Renilla luciferase activity from the same sample. Fold activation was calculated by comparing the normalized value of
45S or 45S.R sample to that of the WT sample. Results shown are means from one experiment representative of three independent experiments, with error bars
indicating standard deviation (SD).
pORF57pLUC (ORF57 promoter-driven luciferase reporter) with
MHV-68 BAC (WT, 45S, or 45S.R) and pRenilla-TK (as an internal control) showed that the absence of ORF45 did not affect the
activation of either viral promoter, suggesting that ORF45 is not
required for expression of viral immediate-early or early genes at
the transcription level (Fig. 2D). Taken together, our results demonstrated that newly synthesized ORF45 protein is not required
for expression of viral genes but is required for viral lytic replication under BAC transfection conditions. They also suggested that
ORF45 likely plays functional roles in the very late stage of viral
lytic replication, i.e., virion assembly and egress.
To determine the role of ORF45 in virion morphogenesis, we
examined the ultrastructure of cells transfected with WT BAC, 45S
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BAC, or 45S.R BAC by thin-section transmission electron microscopy (TEM) at 26 h posttransfection (i.e., within single-round
viral replication). Virion assembly and egress were examined by
noting the existence of nascent viral particles at each stage of virion maturation, according to the envelopment/deenvelopment/
reenvelopment model. As expected, virion morphogenesis took
place normally in WT BAC- or 45S.R BAC-transfected cells, with
formation and release of mature virions (Fig. 3, A1 to A3 and C1 to
C3, black arrows). In 45S BAC-transfected cells, empty or A capsids, B capsids containing scaffold proteins, and C capsids filled
with viral DNAs were visible (Fig. 3, B1), indicating that capsid
formation was not affected in the absence of ORF45. However,
only a small number of capsids (Fig. 3, B2, black triangles) and no
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FIG 2 ORF45 is not required for MHV-68 gene expression but is essential for production of infectious virions. (A) 293T cells were individually transfected with
MHV-68 ORF45 Is Essential for Virion Morphogenesis
fully enveloped virions were detected in the cytoplasm. Consequently, no mature virions were found at the cell surface (Fig. 3,
B3). These data demonstrated that ORF45 is required for virion
maturation in the cytoplasm. To quantify the block in virion assembly that occurs in the absence of ORF45, viral particles at various stages of maturation in electron micrographs (as shown in
Fig. 3) were classified and enumerated. In 45S BAC-transfected
cells, the proportion of nuclear capsids to total viral particles
(96.15%) is much higher than that in WT BAC (57.10%)- or 45S.R
BAC (55.84%)-transfected cells, whereas the proportion of cytoplasmic virions to total viral particles (3.85%) is remarkably lower
TABLE 1 Distribution of viral particles at 26 h posttransfection
BAC
No. of
particlesa
WT
45S
45S.R
697
832
693
Value (%) for:
Nucleusb
Cytoplasmc
Cell surfaced
398 (57.10)
800 (96.15)
387 (55.84)
292 (41.90)
32 (3.85)
302 (43.58)
7 (1.00)
0 (0)
4 (0.58)
a
Total viral and subviral particles enumerated in 10 selected TEM images that
contained viral particles.
b
Values are calculated as number of nucleocapsids/total number of viral particles.
c
Values are calculated as number of mature or immature virions (including capsids) in
the cytoplasm/total number of viral particles.
d
Values are calculated as number of fully mature extracellular virions/total number of
viral particles.
August 2016 Volume 90 Number 16
than that in WT BAC (41.90%)- or 45S.R BAC (43.58%)-transfected cells (Table 1). These results demonstrated that, in the absence of ORF45, nuclear egress of capsids was markedly inhibited
and maturation of virions was finally blocked in the cytoplasm.
In summary, we demonstrated that MHV-68 ORF45 is localized in both the nucleus and the cytoplasm during viral infection, plays functional roles in nuclear egress and cytoplasmic maturation of capsids, and is essential for MHV-68
morphogenesis. Therefore, the ORF45 protein plays dual roles
in MHV-68 lytic infection: the virion-associated ORF45 protein plays a critical role in the immediate-early phase of viral
replication during de novo infection (3), whereas the newly
synthesized ORF45 protein plays an essential role in virion
morphogenesis and egress (this study). The detailed mechanisms mediating ORF45’s involvement in virion assembly and
egress remain to be investigated.
ACKNOWLEDGMENTS
We are grateful to Lei Sun, Xixia Li, Yanxia Jia, Can Peng, and Shufeng
Sun at the Center for Biological Imaging (CBI), Institute of Biophysics,
and Jingnan Liang at the Institute of Microbiology for help with electron
microscopy sample preparation, Shuoguo Li at CBI for help with SIM
image analysis, and Ren Sun at the University of California, Los Angeles,
for providing the rabbit anti-ORF45 polyclonal antibody used in the
study. We thank the members of H.D.’s laboratory for helpful discussions.
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FIG 3 ORF45 is required for MHV-68 virion morphogenesis. 293T cells were transfected with WT BAC, 45S BAC, or 45S.R BAC. Approximately 70-nm
thin sections were prepared at 26 h posttransfection and examined by a 120-kV transmission electron microscope (FEI; Tecnai Spirit). Putative A capsids
(a), B capsids (b), and C capsids (c) were found in the nuclei (Nu) of WT BAC-, 45S BAC-, and 45S.R BAC-transfected cells (A1, B1, and C1). In the
cytoplasm (Cyto), viral particles, including tegumented-enveloped virions, were observed in WT BAC and 45S.R BAC transfectants (A2 and C2, black
arrowheads). Only capsids, not mature virions, were found in 45S BAC transfectant (B2, black triangles). In the extracellular space, WT BAC and 45S.R
BAC transfectants contained mature virions associated with the cellular plasma membrane (A3 and C3, black arrows), whereas no mature virions were
found in 45S BAC transfectant (B3).
Jia et al.
FUNDING INFORMATION
This work, including the efforts of Hongyu Deng, was funded by National
Natural Science Foundation of China (NSFC) (81171582 and 81325012).
This work, including the efforts of Sheng Shen, was funded by National
Natural Science Foundation of China (NSFC) (31500136).
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