Lili Wang, Haitao Guo, Nichole Reyes, Shaoying Lee, Eric
Bortz, Fengli Guo, Ren Sun, Liang Tong and Hongyu Deng
J. Virol. 2012, 86(3):1348. DOI: 10.1128/JVI.05497-11.
Published Ahead of Print 16 November 2011.
Updated information and services can be found at:
http://jvi.asm.org/content/86/3/1348
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Distinct Domains in ORF52 Tegument
Protein Mediate Essential Functions in
Murine Gammaherpesvirus 68 Virion
Tegumentation and Secondary
Envelopment
Lili Wang,a,b Haitao Guo,a,b Nichole Reyes,c Shaoying Lee,d Eric Bortz,c Fengli Guo,e Ren Sun,c Liang Tong,f and Hongyu Denga,d
Chinese Academy of Sciences Key Laboratory of Infection and Immunity, Institute of Biophysics, Beijing,a and Graduate School of the Chinese Academy of Sciences,
Beijing,b People’s Republic of China; Department of Molecular and Medical Pharmacology, David Geffen School of Medicinec and School of Dentistry,d University of
California Los Angeles, Los Angeles, California, USA; Stowers Institute for Medical Research, Kansas City, Missouri, USAe; and Department of Biological Sciences, Northeast
Structural Genomics Consortium, Columbia University, New York, New York, USAf
Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus are etiologically associated with several types of human malignancies. However, as these two human gammaherpesviruses do not replicate efficiently in cultured cells, the morphogenesis of
gammaherpesvirus virions is poorly understood. Murine gammaherpesvirus 68 (MHV-68) provides a tractable model to define
common, conserved features of gammaherpesvirus biology. ORF52 of MHV-68 is conserved among gammaherpesviruses. We
have previously shown that this tegument protein is essential for the envelopment and egress of viral particles and solved the
crystal structure of ORF52 dimers. To more closely examine its role in virion maturation, we performed immunoelectron microscopy of MHV-68-infected cells and found that ORF52 localized to both mature, extracellular virions and immature viral particles in the cytoplasm. ORF52 consists of three ␣-helices followed by one ␤-strand. To understand the structural requirements
for ORF52 function, we constructed mutants of ORF52 and examined their ability to complement an ORF52-null MHV-68 virus.
Mutations in conserved residues in the N-terminal ␣1-helix and C terminus, or deletion of the ␣2-helix, resulted in a loss-offunction phenotype. Furthermore, the ␣1-helix was crucial for the predominantly punctate cytoplasmic localization of ORF52,
while the ␣2-helix was a key domain for ORF52 dimerization. Immunoprecipitation experiments demonstrated that ORF52 interacts with another MHV-68 tegument protein, ORF42; however, a single point mutation in R95 in the C terminus of ORF52 led
to the loss of this interaction. Moreover, the homologues of MHV-68 ORF52 in Kaposi’s sarcoma-associated herpesvirus and
Epstein-Barr virus complement the defect in ORF52-null MHV-68 and interact with MHV-68 ORF52. Taken together, these data
uncover the relationship between the ␣-helical structure and the molecular basis for ORF52 function. This is the first structurebased functional domain mapping study for an essential gammaherpesvirus tegument protein.
H
erpesviruses constitute an ancient virus family consisting of
three subfamilies, Alpha-, Beta-, and Gammaherpesvirinae.
The herpesvirus virion consists of four morphologically distinct
components: the double-stranded DNA genome in the core, an
icosahedral capsid shell, the outer lipid-glycoprotein envelope,
and an electron-dense tegument between the capsid and the envelope (18). Virion morphogenesis for herpesviruses is a multistep process that is generally classified into four distinct stages:
nucleocapsid assembly in the nucleus, primary envelopment at
the nuclear membrane followed by de-envelopment and egress
into the cytoplasm, secondary envelopment, and egress from the
cell (15). Most studies on herpesvirus morphogenesis have focused on alphaherpesviruses, including herpes simplex virus 1
(HSV-1) and pseudorabiesvirus (PrV). In contrast, relatively little
is known about the morphogenesis of beta- and gammaherpesviruses. Productive replication (i.e., the complete lytic phase) of two
human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), is limited in cultured cells. Murine gammaherpesvirus-68 (MHV-68) is closely
related to KSHV and EBV (8, 20, 23); approximately 90% of
MHV-68 genes have homologous counterparts in KSHV and
EBV, particularly lytic genes (26). MHV-68 can establish productive infections in a variety of fibroblast and epithelial cell lines, so
it provides an excellent model for investigating the basic biology of
gammaherpesviruses (20). By using MHV-68 genome cloned as a
bacterial artificial chromosome (BAC), the functions of MHV-68
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proteins have been studied in the context of viral infection via a
genetics approach (1, 4, 22, 28).
Compared with capsids and glycoproteins, little is known
about the structure and composition of the virion tegument proteins. Tegument proteins are also less conserved, as a herpesvirus
in each subfamily encodes a number of tegument proteins that are
absent in other subfamilies. In recent years, emerging evidence has
suggested that the tegument is an organized structure built
through specific protein-protein interactions (7, 13, 19, 27). Furthermore, tegumentation is a key step for virion maturation, during which nascent nucleocapsids are wrapped and bud into the
lumen of cytoplasmic compartments, forming a nearly complete
virion within the lumen. Mature virions are then released into the
extracellular space in a manner resembling exocytosis (10). Although specific tegument proteins apparently play an important
role in the herpesvirus tegumentation and envelopment process,
the molecular mechanisms responsible for the assembly of tegu-
Received 23 June 2011 Accepted 24 October 2011
Published ahead of print 16 November 2011
Address correspondence to Hongyu Deng, [email protected].
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.05497-11
0022-538X/12/$12.00
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Distinct Domains in ORF52 Tegument Protein Mediate Essential
Functions in Murine Gammaherpesvirus 68 Virion Tegumentation
and Secondary Envelopment
MHV-68 ORF52 Tegument Protein
MATERIALS AND METHODS
Viruses and cells. Wild-type (WT) MHV-68 was originally obtained from
the American Type Culture Collection (ATCC; VR1465), and the working
stock was generated by infecting BHK-21 cells at a multiplicity of infection
(MOI) of 0.02 PFU per cell. Viral titer was determined by plaque assay in
BHK-21 cells as previously described (measured in PFU) (29). BHK-21
and 293T cells were cultured in complete Dulbecco’s modified Eagle’s
February 2012 Volume 86 Number 3
medium (DMEM) supplemented with 10% fetal bovine serum, penicillin,
and streptomycin. The Flp-In-FRT293-FLAG ORF52 cell line was generated based on the Flp-In system manual (Invitrogen). Briefly, Flp-In-293
cells were maintained in 15 ␮g/ml blasticidin and 100 ␮g/ml zeocin.
pcDNA5-FRT-TO-FLAG ORF52 plasmid and pOG44 plasmid (a 5.8-kb
Flp recombinase expression vector designed for use with the Flp-In system) were cotransfected into Flp-In 293 cells with Lipofectamine 2000
transfection reagent (Invitrogen) and screened with 200 ␮g/ml hygromycin for cells stably expressing FLAG-ORF52.
Plasmid construction. To facilitate the manipulation of MHV-68
genes in the context of the viral genome, the whole genome of MHV-68
was cloned into a bacterial artificial chromosome (BAC). The construction of MHV-68 BAC plasmid (WT BAC) and the ORF52-null MHV-68
BAC plasmid (52S BAC) has been described previously (4, 28). Expression
plasmids of FLAG-tagged ORF52 were generated by PCR amplification
from genomic MHV-68 DNA and cloning in frame into pFLAG-CMV2
(Sigma) at BglII and XbaI sites. Hemagglutinin (HA)-tagged ORF52 was
expressed using the pCMV-HA vector (Clontech). Coding sequences for
full-length ORF52 and a mutant with deletion of the N-terminal 33 amino
acids (aa) (N33del-ORF52) were amplified from wild-type MHV-68 BAC
by PCR. Plasmids expressing the single-site mutants (R95A, L20A, E23A,
N24A, L27A) and ␣2-helix-deleted ORF52 (Mdel-ORF52) were created
using a two-step oligonucleotide-directed PCR mutagenesis method. The
coding sequence for KSHV-ORF52 or EBV BLRF2 was amplified using
total cellular DNA extracted from the latently infected cell line BCBL-1 or
B95-8 as the template. The PCR fragments were cloned into the EcoRI and
KpnI sites of pCMV-HA and verified by sequencing. The sequences of
primers used for plasmid construction are available upon request.
Production of monoclonal anti-ORF52 antibodies. Rabbit cells were
infected with MHV-68 at an MOI of 2 and collected at 48 h postinfection.
Viral antigens were then provided to Epitomics, Inc. (Burlingame, CA) to
generate monoclonal rabbit hybridomas against a panel of MHV-68 lytic
antigens. His-tagged ORF52 was expressed in 293T cells and purified by
use of a histidine-Ni column. Purified His-ORF52 protein was then used
to screen hybridoma cell lines that produced anti-ORF52 antibodies. Rabbit hybridoma cells were further selected for single clones that produced
high-affinity anti-ORF52 antibodies and maintained with RPMI medium
supplemented with 1% 2-mercaptoethanol, 10% fetal bovine serum, and
10% Epitomics rabbit hybridoma supplement A under hypoxanthineaminopterin-thymidine (HAT) selection. Rabbit hybridoma cells were
proliferated in Epitomics hybridoma growth medium, and the antibodies
were prepared in Epitomics serum-free medium.
Immunoprecipitation and immunoblotting. 293T cells seeded onto
a 6-cm plate (0.8 ⫻ 105 per plate) were transfected with 4.5 ␮g of total
DNA by the calcium phosphate method. Thirty-six hours after transfection, cells were washed once with ice-cold phosphate-buffered saline
(PBS) and then solubilized in EBC buffer (50 mM Tris-Cl [pH 7.4], 120
mM NaCl, 1% NP-40, 0.25% deoxycholic sodium, 1 mM EDTA) with
protease inhibitors. Lysates were clarified by centrifugation (13,000 rpm,
15 min ⫻ 2). Ten percent of the supernatant was used as an input control.
Soluble proteins were mixed with 10 ␮l anti-FLAG M2 agarose (Sigma)
and rotated at 4°C overnight. Beads were washed five times with EBC
buffer before use. Immune complexes were washed five times in NETN
buffer (20 mM Tris-Cl [pH 8.0], 1 mM EDTA, 0.5% NP-40, 120 mM
NaCl), and supernatant was depleted. Bound proteins were recovered by
boiling in SDS sample buffer for 10 min. The protein samples were separated on a 12% SDS-polyacrylamide gel, and proteins were transferred
onto nitrocellulose membrane (Millipore). The nitrocellulose was blocked
for 1 h at room temperature in PBS supplemented with 5% nonfat dry milk.
Anti-HA monoclonal antibody (Sigma) was diluted to 1:5,000 in blocking
buffer. The diluted antibodies were reacted with the blocked nitrocellulose for
2 h at room temperature, washed, and reacted with goat anti-mouse horseradish peroxidase (HRP)-conjugated IgG secondary antibody diluted at
1:20,000 for 1 h. The bound IgGs were revealed by enhanced chemiluminescence (Pierce).
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ment proteins into nascent herpesvirus particles are poorly understood.
MHV-68 ORF52 encodes a tegument protein that is abundantly present in virions (5). ORF52 has no homologue in the
alpha- or betaherpesviruses and thus is unique to gammaherpesviruses. By constructing an ORF52-null MHV-68/BAC (52S BAC)
genome, we have previously shown that ORF52 encodes a highly
expressed late protein, with an essential function after viral genome replication, viral DNA cleavage/packaging, and nucleocapsid assembly in the nucleus but prior to complete virion tegumentation and envelopment in the cytoplasm and egress of infectious
virions from the cell (4). Without the ORF52 protein, cytoplasmic
viral particles cannot form mature virions and be released from
the cell. Partially tegumented capsids produced by the ORF52-null
mutant contain all of the capsid proteins and some inner tegument proteins (e.g., ORF64 and ORF67) but fail to acquire outer
tegument proteins (e.g., ORF45). In addition, ORF52 localizes in
the cytoplasm to a distinct compartment that is reminiscent of the
secretory pathway (4). Collectively, these findings indicate that
ORF52 is essential for the morphogenesis of infectious MHV-68
particles in the cytoplasm and likely acts via the cellular secretory
pathway.
The MHV-68 ORF52 gene encodes a 135-amino-acid protein.
To gain insight into the mechanism of ORF52 function, we have
previously solved its protein crystal structure, which contains
three ␣-helices and one ␤-strand (3). Among these domains, the
N-terminal ␣1-helix is extended as an arm in the dimer form,
while the ␣2-helix, a very small ␣3-helix, and the ␤-strand form a
hydrophobic core. There are five strictly conserved sites within
ORF52. One of them (Arg95) is localized in the middle of the
␤-strand and is likely to be critical for ORF52 protein function; the
other four (Leu20, Glu23, Asn24, and Leu27) are localized close to
each other in the N-terminal ␣1-helix. Besides the four strictly
conserved sites, there are other relatively conserved sites within
the ␣1-helix that may form hydrophobic patches capable of interacting with other proteins. According to structural analysis,
ORF52 is likely to function as a dimer, although the protein could
also self-associate as an asymmetric tetramer (3). ORF52 dimers
form independently of the ␣1-helix and the conserved site Arg95.
The ␣2-helix was predicted to play a central role in dimerization,
and the ␣3-helix served as a linker to connect the ␣2-helix and
␤-strand. The ␣2- and ␣3-helices and the ␤1-strands from two
molecules form a scaffold, with the ␣1-helices extending away
from this scaffold (3).
In this work, we determined that MHV-68 tegument protein
ORF52 associates with viral particles undergoing the tegumentation and secondary envelopment stage of virion morphogenesis.
In order to further understand the molecular functions of
MHV-68 ORF52, we have specifically dissected the role of ORF52
domains and critical amino acids based on structural information
and identified domains important for localization and dimerization as well as interaction with another tegument protein, ORF42.
Wang et al.
or WT BAC (C). Approximately 70-nm-thin sections were examined 6 days postinfection by TEM. Putative A-capsids (a), B-capsids (b), and C-capsids (c) were
found in the nuclei of either 52S BAC- or WT BAC-transfected cells. Virion that finished secondary envelopment (¡) and putative released virions (v) could be
found in WT BAC-transfected cells (panel C). Bars: A, 0.2 ␮m; B and C, 0.5 ␮m. Nu, nucleus; Cy, cytoplasm.
Immunofluorescence assay. 293T cells were seeded onto cover glasses
in 24-well plates the day before transfection. HA-ORF52 or its mutants
were transfected into 293T cells with jet-PEI (Polyplus transfection). Six
hours after transfection, the cells were infected with wild-type MHV-68 at
an MOI of 10. Eighteen hours after infection, the cells were washed twice
with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. After another wash, the cells were permeabilized with 0.1%
TritonX-100 in PBS for 15 min. Then, the cells were blocked with 2%
bovine serum albumin (BSA) and 0.05% Tween 20 in PBS for 30 min. The
cells were immunolabeled with anti-HA primary antibody and Cy5conjugated secondary antibody. Before the cover glass was sealed, the cells
were treated with DAPI (4=,6-diamidino-2-phenylindole) to stain the nuclei. Finally, the glass with cells was covered onto a slide and sealed with a
drop of Fluoromount (Sigma). Fluorescent photos were taken with a
Nikon Eclipse TE2000-E fluorescence microscope.
Complementation assay. 293T cells plated in 24-well plates were
transfected with 600 ng of BAC DNA plus 200 ng of plasmid DNA expressing ORF52 or ORF52 mutant, by using Lipofectamine 2000 (Invitrogen). Cultures were observed at 4 to 5 days posttransfection for cytopathic effect (CPE). When the CPE was obvious, supernatants were
collected for quantifying released viral DNA by real-time PCR.
Real-time PCR. Supernatant from the complementation assay was
collected and viral genomic DNA was extracted as follows: 200 ␮l of supernatant was incubated with 100 mg/ml proteinase K and 20 mg/ml
RNase A at 37°C for 15 min; then, the reaction was stopped by adding 1/10
volume of 0.5 M EDTA (pH 8.0), and the mixture was incubated at 70°C
for 10 min to inactivate the enzymes. An equal volume of 2⫻ lysis buffer
(200 mM NaCl, 20 mM Tris-Cl [pH 8.0], 50 mM EDTA [pH 8.0]) was
added, and samples were incubated with shaking at 50°C for 12 to 18 h.
The samples were extracted twice by phenol-chloroform/isoamyl alcohol
(25:24:1) and once by chloroform and then precipitated. Extracted
genomic DNA preparation was dissolved in 15 ␮l of Tris-EDTA (TE)
buffer. Real-time PCR was performed to determine viral genome copies in
1 ␮l of such sample by using SYBR green and primers that amplify a
fragment in MHV-68 ORF65 coding region (sense, 5=-GTCAGGGCCCA
GTCCGTA-3=; antisense, 5=-TGGCCCTCTACCTTCTGTTGA-3=). All
real-time PCR was performed on an i-Cycler (Bio-Rad), and viral genome
copy numbers were analyzed with MyiQ software calibrated to standards
ranging from 103 to 108 MHV-68 BAC genome copies.
Electron microscopy. For thin-section transmission electron microscopy (TEM), 293T cells were transfected with MHV-68 BAC or 52S, collected at 6 days posttransfection in phosphate-buffered saline, fixed in 2%
electron microscopy-grade glutaraldehyde (Ted Pella, Redding, CA) in
phosphate-buffered saline at 4°C for 12 h, postfixed in 1% OsO4, dehy-
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drated, and embedded in Epon. Approximately 60- to 70-nm-thin sections were stained with 2% uranyl acetate and 0.3% lead citrate and examined at 200 kV on an FEI Tecnai 20 transmission electron microscope
according to published protocols (25).
Immunogold TEM. BHK cells were seeded a day before infection with
MHV-68 at 50% confluence in 10-cm plates. The cells were infected at an
MOI of 3. Sample preparation and immunostaining were performed as
described previously (21). Briefly, at 18 h after infection, the cells were
fixed with 2% paraformaldehyde plus 0.01% glutaraldehyde in 0.1 M
phosphate buffer (PB) (pH 7.2) for 2 h at 4°C on the plate and embedded
in 12% gelatin in 0.1 M PB. Small blocks were infiltrated in 2.3 M sucrose
plus 20% polyvinylpolypyrrolidone (PVP) in PB overnight at 4°C and
quickly plunged into liquid nitrogen. Sections approximately 60 nm thick
were cut with a Leica UC6/FC6 ultramicrotome and picked up with 2.3 M
sucrose. Immunostaining was performed as follows: the sections were
washed in BSA buffer (1% BSA and 0.15% glycine in PBS) followed by
blocking in normal rabbit serum (1:20 dilution in BSA buffer) for 30 min.
Subsequently, the sections were reacted for 2 h at room temperature with
rabbit-derived anti-ORF52 monoclonal antibody (or BSA buffer as negative controls) and then for 1 h at room temperature with goat anti-rabbit
IgG conjugated with 15-nm colloidal gold particles (Sigma). After a brief
wash in BSA buffer and PB, the sections were treated with 2.5% glutaraldehyde for 5 min. Finally, the sections were stained by 2% neutral uranyl
acetate and 4% uranyl acetate and sealed with methyl cellulose. The sections were examined with an FEI Tecnai Spirit operated at 80 kV.
RESULTS
ORF52 is involved in tegumentation and secondary envelopment. Our previous studies indicated that, in the absence of
ORF52, although MHV-68 viral DNA replication in the nucleus
was normal, virion morphogenesis and egress were affected. Statistical data showed that the proportions of capsids in the nuclei to
total particles were similar in WT BAC- and 52S BAC (ORF52null virus)-transfected cells, but an obvious block of viral particle
morphogenesis was observed in the cytoplasm (4). To observe this
blockage in greater detail, we collected 52S BAC- or WT BACtransfected cells 6 days posttransfection and examined the subcellular localization of viral particles in these cells through thinsection TEM. In the cytoplasm of 52S BAC-transfected cells, large
numbers of immature viral particles surrounded cytoplasmic vesicles without entry, forming a garland-like structure. Moreover,
no viruses were found outside the cells (Fig. 1A). However, DNA
Journal of Virology
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FIG 1 ORF52 is involved in the tegumentation and secondary envelopment stage of MHV-68 maturation. 293T cells were transfected with 52S BAC (A and B)
MHV-68 ORF52 Tegument Protein
February 2012 Volume 86 Number 3
FIG 2 Immunolabeling of MHV-68 ORF52 in infected BHK cells. BHK cells
were infected with wild-type MHV-68 at an MOI of 3. (A to D) Ultrathin
sections were immunolabeled with anti-ORF52 primary antibody and 15-nm
gold particles conjugated with secondary antibody. ORF52 was found on released viruses (1) outside the cells (A). In the cytoplasm (Cy) of infected cells,
ORF52 was detected on the membrane of vesicles (B) and viral particles inside
the vesicles (‘) (C). ORF52 was also found on Golgi complex (GC) and immature viral particles outside the vesicles (’) (D). (E and F) No gold particle
was found on images from negative control samples. Bars: A, B, and C, 100 nm;
D, E, and F, 200 nm.
N33del-ORF52 and the R95A-ORF52 mutations exerted a dominant negative effect on wild-type ORF52 expressed from the viral
genome (Fig. 3A). To confirm this result, N33del-ORF52 or
R95A-ORF52 was cotransfected with 52S BAC into a cell line (FlpIn-293-FLAG-ORF52) that constitutively expresses wild-type
ORF52. Similarly, viruses released into the supernatant were reduced to approximately 33% (N33del) or 50% (R95A) of those of
52S BAC transfectant (Fig. 3B). The mild differences in values
between these assays may be due to constitutive expression of
ORF52 in the stable cell line, which may take part in virus assembly before a high level of mutant ORF52 synthesis. These results
indicate that N33del-ORF52 and R95A-ORF52 display a dominant negative effect on wild-type ORF52 and that the ␣1-helix and
Arg95 may exert independent effects on ORF52 function.
MHV-68 ORF52 and its homologous proteins in KSHV and
EBV share similar functions. As a model of gammaherpesviruses,
MHV-68 is phylogenetically related to KSHV and EBV, both of
which are human pathogens. The homologues of MHV-68 ORF52
in KSHV and EBV are KSHV ORF52 (KORF52) and BLRF2, respectively. According to the amino acid alignment (Fig. 4A),
KORF52 and BLRF2 share a high degree of similarity to MHV-68
ORF52. KORF52 and BLRF2 share 28% identity and 40% identity,
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replication and capsid assembly in the nucleus appeared to be
normal. All 3 types of intracellular capsids were identified: A capsids (empty), B capsids (containing scaffold proteins but no
DNA), and C capsids (containing packaged genomic DNA) (Fig.
1B). Compared with 52S BAC-transfected cells, few immature viruses were retained in the cytoplasm in WT BAC-transfected cells.
Viruses could be found inside intracytoplamsic vesicles; some virions were fixed at the point of envelopment within vesicles. An
electron-dense tegument layer was found between the virus and
the vesicle, suggesting active protein-protein interactions (Fig.
1C). This result, together with our previous data (4), strongly
suggested that ORF52 is involved in tegumentation and/or secondary envelopment.
The subcellular localization of tegument proteins is closely related to their functions (14, 15). Previous immunofluorescence
studies have shown that transfected ORF52 protein localized predominantly to distinct puncta in the cytoplasm. ORF52 partially
colocalized with p115, a cytoplasmic marker for Golgi-derived
compartments and vesicles in the secretory pathway (4); however,
the exact localization of ORF52 in relation to vesicles involved in
secondary envelopment could not be ascertained by immunofluorescence. We therefore performed immunogold TEM to acquire
detailed localization data on ORF52 in wild-type MHV-68infected cells. By using a primary anti-ORF52 monoclonal antibody, gold particles were observed on extracellular virions (Fig.
2A), confirming that ORF52 was a virion tegument protein.
ORF52 was also found on the membrane of cytoplasmic vesicles
(Fig. 2B), viruses that had entered vesicles (Fig. 2C), and immature viral particles (Fig. 2D), suggesting that ORF52 localizes to
nascent viral particles prior to or during tegumentation and secondary envelopment. In addition, gold particles were also localized to the Golgi complex (Fig. 2D), consistent with the colocalization of ORF52 with p115 (4). No gold particle was found on
negative control images (Fig. 2E and F). Quantitation and statistical analysis of the distribution of gold particles (n ⫽ 96; enumerated from samples treated with anti-ORF52 primary antibody)
from 20 immuno-EM images revealed that ORF52 was localized
to intracellular immature virions (43%) and extracellular virions
(19%) as well as the membrane of cytoplasmic vesicles (24%),
consistent with ORF52’s suggested role in tegumentation and the
secondary envelopment of immature viral particles (no gold particles were found in 8 negative control images).
ORF52 mutants display dominant negative effects. Previously, we examined ORF52 deletion mutants, including a 33amino acid deletion of the N-terminal ␣1-helix (N33del-ORF52)
and a point mutation at amino acid 95 from arginine to alanine
(R95A-ORF52), in complementation assays. Our findings suggested that the ␣1-helix and Arg95 are both essential for the function of ORF52 (3). Considering the structural independence of
different domains, we hypothesized that ORF52 function may require coordination of multiple domains; thus, some mutants of
ORF52 might have a dominant negative effect on wild-type
ORF52. WT BAC and plasmids encoding ORF52 mutants were
cotransfected into cells to examine whether these mutants affect
the lytic replication and/or release of the MHV-68 virus. Four days
posttransfection, supernatants were analyzed by real-time PCR to
measure MHV-68 genomes in released viral particles. For the
ORF52 mutants, viruses released into the supernatant were significantly reduced to approximately 17% (N33del) or 25% (R95A)
compared to WT BAC transfectant, suggesting that both the
Wang et al.
respectively, with MHV-68 ORF52 (12, 30). To investigate
whether these two proteins possess functions similar to those of
MHV-68 ORF52, we tested their ability to complement the null
mutation of ORF52 in the 52S BAC MHV-68 genome. We performed a complementation assay in 293T cells by cotransfecting
52S BAC genome and KORF52 or BLRF2 expression plasmid.
Both proteins rescued the virus propagation to nearly the same
level as MHV-68 ORF52 (Fig. 4B). Our previous data demonstrated that MHV-68 ORF52 can self-associate as a homodimer or
tetramer. To determine whether this feature was conserved
among gammaherpesviruses, we performed coimmunoprecipitation (co-IP) experiments and examined the ability of KORF52 and
BLRF2 to form hybrid dimers with MHV-68 ORF52. Both
KORF52 and BLRF2 interacted with MHV-68 ORF52 (Fig. 4C),
possibly as a hybrid dimer. Taken together, these experiments
suggest that KORF52 and BLRF2 share sequence homology that
translates to a common structure and function.
The N-terminal ␣1-helix determines the localization of
ORF52. In order to further evaluate the role of ORF52 in tegumentation and secondary envelopment, we explored functions of
important domains identified in our previous ORF52 structural
study (3). There are five strictly conserved sites within the
MHV-68 ORF52 protein (Fig. 4A). Four of the five residues
(Leu20, Glu23, Asn24, and Leu27) are located in the ␣1-helix. As
these residues are conserved among gammaherpesviruses, we reasoned that these residues may play significant roles in ORF52
function. To explore the importance of these residues, four plas-
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FIG 3 Mutants of ORF52 have a dominant negative effect on normal ORF52
function. (A) 293T cells were cotransfected with WT BAC and ORF52 mutant
plasmid or empty vector. (B) Flp-In-FRT293-FLAG ORF52 cells were cotransfected with 52S BAC and ORF52 mutant plasmid or empty vector. Four days
after transfection, viral DNA was extracted from the supernatant, and genome
copy numbers were analyzed by real-time PCR. The results are representative
of three independent experiments.
mids expressing point mutants of ORF52, HA-L20A-ORF52
(L20A), HA-E23A-ORF52 (E23A), HA-N24A-ORF52 (N24A),
and HA-L27A-ORF52 (L27A), were constructed (Fig. 5A). Complementation assays were performed by cotransfecting 52S BAC
and each ORF52 mutant plasmid individually into 293T cells. A
single point mutation in any of these conserved sites was sufficient
to abolish the function of ORF52 (Fig. 5B). Thus, all four
N-terminal conserved sites (Leu20, Glu23, Asn24, and Leu27) are
important for the function of ORF52. Because these sites are located within the ␣1-helix of ORF52, we next examined the localization of the ORF52 point mutants in the context of infection. As
shown in Fig. 5C, wild-type ORF52 was distributed predominantly in a distinct perinuclear, punctate pattern in the cytoplasm.
When the ␣1-helix was deleted, ORF52 displayed a dispersed distribution in the cytoplasm and prominent nuclear localization.
Interestingly, a single point mutation altered the ORF52 protein
distribution severely: L20A-ORF52 was found both in the cytoplasm and in the nuclei; N24A-ORF52 and L27A-ORF52 were still
retained in the cytoplasm but in a dispersed pattern; and E23AORF52 retained a distinct punctate pattern in the cytoplasm, but it
also showed some dispersal from the mainly perinuclear pattern of
the wild-type ORF52 protein. Taken together, our data show that
the ␣1-helix, particularly the four conserved sites (Leu20, Glu23,
Asn24, and Leu27) play an important role in ORF52 protein localization.
␣2-Helix contributes to the dimerization of ORF52. Our previous structural study indicated that ORF52 could form a dimer,
and ␣2-helix appeared to be a key domain responsible for
dimerization (3). Purification of His-tagged ORF52 expressed in
Escherichia coli also showed that ORF52 could dimerize independently in vitro (data not shown). To evaluate the importance of the
␣2-helix in mediating protein dimerization, we constructed a
plasmid expressing an ORF52 mutant with amino acids 48 to 69
deleted (Mdel-ORF52), as shown in Fig. 6A. Using this mutant, we
performed a co-IP experiment to see if the ability to dimerize was
dependent on the ␣2-helix. While both wild-type ORF52 and
N33del-ORF52 formed dimers with wild-type ORF52, the MdelORF52 failed to do so (Fig. 6B). In addition, previous studies have
shown that the point mutant R95A can also form dimers (3).
These results indicate that the ␣2-helix of ORF52 is a key domain
for dimerization. Immunofluorescence assay shows that the localization of the Mdel-ORF52 mutant is similar to that of wild-type
ORF52 (Fig. 6D). To evaluate the effect of ORF52 dimerization on
viral proliferation, we performed a complementation assay to determine whether this mutant rescues the ORF52-null (52S BAC)
virus. Mdel-ORF52 was unable to complement the dysfunction of
52S BAC (Fig. 6C). These results indicate that ␣2-helix is responsible for ORF52 dimerization, which, in turn, is critical to
MHV-68 ORF52 function.
ORF52 interacts with ORF42, and the C-terminal conserved
R95 is critical for this interaction. As the tegumentation of nascent virions is likely driven by specific protein-protein interactions, it is possible that ORF52 interacts with other viral proteins
during secondary envelopment. We thus tested a panel of structural proteins for their interactions with ORF52, with an emphasis
on tegument proteins. This panel included ORF19, ORF33,
ORF38, ORF42, ORF63, ORF64, ORF67, ORF75a, ORF75b,
ORF75c, ORF25, ORF26, ORF43, ORF62, ORF65, ORF39, and
ORF53. Some of the large proteins were broken into two or several
domains to achieve better expression. Overall, expression of pro-
MHV-68 ORF52 Tegument Protein
tein or protein domains was detected from approximately 75% of
the clones (data not shown). We further examined the physical
interaction between ORF52 and each of these expressed proteins/
protein domains by using co-IP and found that ORF52 interacts
with ORF42 (Fig. 7A, lane 1). ORF42 has also been suggested to be
a tegument protein of MHV-68 (2). To examine which domains of
ORF52 are responsible for ORF42 interaction, we cotransfected
3FLAG-tagged ORF42 and HA-tagged ORF52 mutants into 293T
cells. Under the same conditions described above, ORF42 was
found to interact with Mdel-ORF52 (Fig. 7A, lane 2) and N33delORF52 (Fig. 7A, lane 3) but not R95A-ORF52 (Fig. 7A, lane 4).
These results indicate that Arg95 was an essential site for ORF52 to
interact with ORF42. According to the analysis of ORF52 quaternary structure, Arg95 is localized to the surface of the ␤1-strand
and is highly hydrophilic. While our complementation assay results indicate that the R95A point mutation affects ORF52 function (3), there is no effect on dimerization (3) or localization (Fig.
7B). Furthermore, the interaction between Mdel-ORF52 and
ORF42 was severely attenuated in comparison to that observed
between wild-type ORF52 and ORF42, suggesting that the
February 2012 Volume 86 Number 3
C-terminal domain and the dimerization state of ORF52 affect the
interaction with ORF42.
DISCUSSION
ORF52 is a tegument protein conserved in gammaherpesviruses. It exists in abundance in MHV-68 virions and plays an
essential role in viral proliferation (5). However, because
ORF52 has no homologue in alpha- or betaherpesviruses, the
mechanisms of ORF52 function cannot be inferred from studies on known tegument proteins of alpha- or betaherpesviruses. In this report, we provide evidence of MHV-68 ORF52
involvement in virion tegumentation and secondary envelopment and gained detailed functions of different domains of
ORF52 in the MHV-68 model. We show that ␣1-helix is crucial
for the localization of ORF52. Single point mutation of the four
conserved sites inside the ␣1-helix severely disrupted ORF52
localization. The ␣2-helix is indeed the key domain mediating
ORF52 dimerization, and Arg95 is important for the interaction
of ORF52 with another tegument protein, ORF42 (Fig. 8). To
our knowledge, this is the first structure-based functional do-
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FIG 4 ORF52 homologues from EBV and KSHV can substitute for the function of MHV-68 ORF52. (A) Alignment of amino acid sequences of MHV-68
ORF52, KORF52, and BLRF2. (B) The dysfunction of 52S BAC could be rescued by EBV BLRF2 and KSHV-ORF52 (KORF52). 293T cells were
cotransfected with 52S BAC and corresponding complement plasmid or empty vector. Four days after transfection, viral DNA was extracted from the
supernatant, and genome copy numbers were analyzed by real-time PCR. The results were representative of two independent experiments. (C) BLRF2 and
KORF52 interacted with MHV-68 ORF52. 293T cells were cotransfected with FLAG-tagged MHV-68 ORF52 and HA-tagged KORF52 or BLRF2.
Thirty-six hours after transfection, the cells were harvested and the lysates were immunoprecipitated with anti-FLAG beads. The samples were analyzed
by Western blotting (WB) with an anti-HA antibody.
Wang et al.
main mapping study for an essential gammaherpesvirus tegument protein, and it may provide a deeper insight into the
process of herpesvirus assembly and egress.
The morphogenesis of herpesviruses is very complex,
following an envelopment– de-envelopment–reenvelopment
process (14, 16, 17). TEM imagery of MHV-68 virion morphogenesis in the cytoplasm (4) primed us to explore the function
of ORF52 in tegumentation and secondary envelopment. In
ORF52-null (52S BAC-transfected) cells, garland-like clusters
of immature viral particles accumulated in the cytoplasm in
close proximity to intracellular vesicles (Fig. 1). In agreement
with our previous observations for MHV-68 infection in the
absence of ORF52 (4), we noticed occasional single viral particles in the concave recess of a vesicle membrane partly surrounded by an electron-dense, tegument-like layer but no fully
enveloped virions (Fig. 1A). In 52S BAC-transfected cells
where a significant number of viral particles had completed
primary envelopment– de-envelopment and entered the cytoplasm, although the cytoplasmic face of some vesicles was occupied by these immature virions, no concave recess was
found, suggesting that these immature virions might compete
for the vesicle membrane (Fig. 1A). However, without ORF52,
the formation of a complete tegument and envelope could not
be achieved, indicating specific vesicle compositions and/or
morphologies might facilitate tegumentation and secondary
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envelopment in an ORF52-mediated manner. Meanwhile, in
WT BAC-transfected cells, at the interacting surface of immature virions and vesicles in the cytoplasm, an electron-dense
proteinaceous structure was found, suggesting that that active
protein-protein interactions occur during tegumentation and
secondary envelopment. These interactions may contribute to
the fission of fully enveloped virions into the vesicle. By immunoelectron microscopy, we detected ORF52 both within the
tegument of mature virions and in this electron-dense proteinaceous material proximal to nascent viral particles (Fig. 2). A
prior cryoelectron tomography study showed that the MHV-68
virion contains inner and outer tegument layers (7), thereby
providing details regarding tegument interactions and suggesting a complex tegument organization in which ORF52 plays a
vital role bridging tegument proteins and/or host proteins.
Previous immunofluorescence experiments have demonstrated that ectopically expressed ORF52 colocalized partially
with Golgi-derived vesicles (4). In this study, immunoelectron
microscopy also showed that native ORF52 expressed from viral genome is found on the Golgi complex and cytoplasmic
vesicles in the context of infection (Fig. 2). Moreover, statistical
analysis of ORF52 distribution revealed that, in addition to
association with immature and mature virions, a significant
proportion of ORF52 (24%) was localized to the membrane of
cytoplasmic vesicles (see above), suggesting that these vesicles
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FIG 5 N-terminal conserved sites of ORF52 are essential for its function and determine the protein localization. (A) Schematic presentation of ORF52 and its
mutants: the conserved sites L20, E23, N24, and L27 were individually substituted with alanine. (B) Point mutations of ORF52 resulted in a loss of function. 293T
cells were cotransfected with 52S BAC and corresponding ORF52 mutant complement plasmids. Cotransfection of 52S BAC and a pCMV-HA vector was used
as a negative control. Four days after transfection, viral DNA was extracted from the supernatant and genome copy numbers were analyzed by real-time PCR. The
results were representative of three independent experiments. (C) Localization of ORF52 mutant proteins in Vero cells. Vero cells were plated on coverslips the
day before transfection. Plasmids expressing ORF52 or ORF52 mutants were transfected individually into Vero cells with transfection reagent jet-PEI. Six hours
after transfection, the cells were infected with wild-type MHV-68 at an MOI of 10. Eighteen hours after infection, the cells were fixed and stained with an anti-HA
primary antibody followed by labeling with a Cy5-conjugated secondary antibody. Before the coverslips were sealed, the cells were treated with DAPI to stain the
nuclei.
MHV-68 ORF52 Tegument Protein
could be a major site for the secondary envelopment of
MHV-68 virions. Collectively, these results suggest that
MHV-68 virion morphogenesis may involve the Golgi complex
and related components of the host secretory pathway.
Building on previous structural studies (3), we found that
two disparate mutants of ORF52 had a dominant negative effect on wild-type ORF52 (Fig. 3). This suggests that different
domains of ORF52 may exert distinct or independent func-
FIG 7 ORF52interactswithORF42,andaminoacidR95iscrucialforthisinteraction.
FIG 8 Summary of ORF52 domains and their functions. The figure depicts a
(A) 293T cells were cotransfected with 3FLAG-tagged ORF42 and HA-tagged ORF52
construct. Thirty-six hours after transfection, cell lysates were collected and immunoprecipitated with anti-FLAG beads. The samples were analyzed by Western blotting
(WB) with anti-HA antibody. (B) The localization of R95A-ORF52 was normal. Vero
cells on coverslips were transfected with R95A-ORF52 plasmid by using jet-PEI. The
cells were treated as described for Fig. 5C.
dimer of MHV-68 ORF52 (3). Based on its crystal structure and the results
herein, the functions of ORF52 domains are summarized as follows: the
N-terminal ␣1-helix is required for correct protein localization, the ␣2-helix
plays a crucial role in ORF52 dimerization, and the conserved site Arg95 in the
C terminus is critical for mediating ORF52 interaction with MHV-68 tegument protein ORF42.
February 2012 Volume 86 Number 3
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FIG 6 ␣2-Helix is crucial for ORF52 dimerization. (A) Schematic presentation of ORF52 and Mdel-ORF52. Mdel-ORF52 is an ORF52 mutant with the ␣2-helix
(aa 48 to 69) deleted. (B) Mdel-ORF52 could not form dimer. 293T cells were cotransfected with FLAG-ORF52 and HA-tagged ORF52 or ORF52 mutants. After
36 h, the cells were solubilized and the lysates were immunoprecipitated with anti-FLAG beads. The samples were analyzed by Western blotting (WB) with
anti-HA antibody. (C) Mdel-ORF52 could not substitute for the function of wild-type ORF52. 293T cells were cotransfected with 52sBAC and Mdel-ORF52 or
empty vector. Four days after transfection, viral DNA was extracted from the supernatant and genome copy numbers were analyzed by real-time PCR. The results
were representative of three independent experiments. (D) The localization of Mdel-ORF52 was normal. Vero cells on coverslips were transfected with
Mdel-ORF52 plasmid by using jet-PEI. The cells were treated as described for Fig. 5C.
Wang et al.
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ACKNOWLEDGMENTS
We thank Lei Sun (Center for Electron Microscope, Institute of Biophysics, CAS) for help with EM sample preparation and members of the Deng
Laboratory for kind help and discussions.
This study was supported by the “Hundred Talent Program” from the
Chinese Academy of Sciences and grants from the Chinese Ministry of
Science and Technology (2006CB910901 and 2007DFC30190) to H.D.,
grant U54 GM074958 from the Protein Structure Initiative of the NIGMS
at the U.S. National Institutes of Health (NIH) to L.T., and grants
DE019085 and CA091791 from the NIH to R.S.
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tions. We thus investigated the functions of MHV-68 ORF52 in
detail by domain mapping. The results of these analyses are
summarized in Fig. 8: the N-terminal ␣1-helix is required for
the correct subcellular localization of ORF52; the central ␣2helix plays a critical role in ORF52 dimerization; and the conserved site Arg95 in the C-terminal ␤-strand of ORF52 is essential for interaction with another MHV-68 protein, ORF42.
Thus, the domains of ORF52 are relatively independent structures that mediate critical aspects of ORF52 function. For example, deleting the N-terminal 33 amino acids led to a drastic
change in localization of the ORF52 protein (Fig. 5C) and abolished its function in virion assembly and egress (Fig. 5B). However, this N33del-ORF52 retained the ability to dimerize with
WT ORF52 (3) and interact with ORF42 (Fig. 7A). Thus,
N33del-ORF52 would compete with WT ORF52 in binding to
ORF52 or ORF42, exert a dominant negative effect on WT
ORF52, and as a result, drastically inhibit viral egress (Fig. 3).
Similarly, the R95A mutant, although incapable of interacting
with ORF42 (Fig. 7A), retained normal localization (Fig. 7B)
and the ability to associate with WT ORF52 (3) and hence
interfered with the normal function of WT ORF52 (Fig. 3). It is
noted that four strictly conserved sites in the ␣1-helix are critical for the essential function of ORF52 in MHV-68 replication
(Fig. 5B), thereby highlighting the importance of this domain.
Several other hydrophobic residues in the ␣1-helix are also
conserved. According to the structural model (Fig. 8), as the
␣1-helix extends away from the rest of the dimer, several highly
hydrophobic patches will be exposed. The four strictly conserved sites and several other amino acids within the ␣1-helix
may thus provide a hydrophobic cluster ideal for interaction
with additional viral and/or cellular proteins, as suggested by
yeast two-hybrid analysis of KSHV ORF52 (19, 24). Interestingly, the point mutants result in subcellular distributions different from those seen for the ␣1-helix deletion mutant,
thereby suggesting other interacting partners for the ␣1-helix.
However, the identity of cellular proteins interacting with
ORF52 needs further exploration.
In this study, we find that in MHV-68, ORF52 interacts with
another tegument protein, ORF42. Until now, little research
has been performed on MHV-68 ORF42. Thus, the interaction
between ORF42 and ORF52 provides a clue for studying
MHV-68 virion assembly in the cytoplasm. For ORF42, we
have found an interacting cellular protein involved in the secretory pathway and recycling (data not shown). Through interacting with tegument protein ORF42, ORF52 may serve as
one bridge between tegument proteins and cellular proteins
that facilitate secondary envelopment. By studying interacting
cellular and viral partners of ORF42 and ORF52, we can work
toward building a proteome to help explain the mechanism(s)
of tegumentation and secondary envelopment of gammaherpesvirus virions. Considering that the lytic phase of gammaherpesvirus infection is crucial for the spread of virus from
initially infected tissues to B cells or lymphatic epithelial cells
(6, 11) and that the lytic phase has been implicated in tumor
establishment and progression (9), the mapping of a protein
interaction network involved in virion assembly and egress,
particularly virus-host protein interactions, may shed light on
the regulation of virion assembly and provide insights into
developing therapeutic treatments in EBV- and KSHVassociated diseases.
MHV-68 ORF52 Tegument Protein
February 2012 Volume 86 Number 3
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