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Journal of Virology, December 2001, p. 12431-12438, Vol. 75, No. 24
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.24.12431-12438.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Glycoprotein K Specified by Herpes Simplex Virus
Type 1 Is Expressed on Virions as a Golgi Complex-Dependent
Glycosylated Species and Functions in Virion Entry
Timothy P.
Foster,
Galena V.
Rybachuk, and
Konstantin G.
Kousoulas*
Department of Pathobiological Sciences,
School of Veterinary Medicine, Louisiana State University, Baton
Rouge, Louisiana 70803
Received 3 July 2001/Accepted 5 September 2001
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ABSTRACT |
To facilitate detection of glycoprotein K (gK) specified by herpes
simplex virus, a 12-amino-acid epitope tag was inserted within gK
domain III. Recombinant virus gKprotC-DIII, expressing the tagged gK,
was isolated. This virus formed wild-type plaques and replicated as
efficiently as the wild-type KOS virus in Vero cells. Anti-protein C
MAb detected high-mannose and Golgi complex-dependent glycosylated gK
within cells as well as on purified virions. The gK-null virus 
gK
(gK
/) entered Vero cells substantially more slowly than
the wild-type KOS (gK+/+), while gK virus grown in
complementing VK302 cells (gK/+) entered with entry
kinetics similar to those of the KOS virus.
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TEXT |
Herpes simplex virus type 1 (HSV-1)
specifies at least 12 glycoproteins, gB, gC, gD, gE, gG, gH, gI, gJ,
gK, gL, gM, and gN, that are expressed during a productive viral
infection. These glycoproteins function in pH-independent virus entry
via fusion of the viral envelope with cellular membranes, cell-to-cell
spread, egress of infectious virion particles, and virus-induced
cell-to-cell fusion. (26, 37, 42, 43). Mutations that
cause extensive virus-induced cell fusion, syncytia, can arise in at
least four different regions of the viral genome, including the UL20
gene (1, 24), the UL24 gene (20, 39), the
UL27 gene encoding glycoprotein B (gB) (5, 30), and the
UL53 gene coding for glycoprotein K (gK) (2, 7, 32, 38).
However, syncytial mutations (syn) in the UL53 (gK) gene are
more frequently isolated than syncytium mutations in any other genes
(2, 3, 7, 8, 32, 33, 35, 38).
HSV-1 gK is encoded by the UL53 open reading frame (ORF) (7,
25), and it has characteristics of a glycosylated membrane protein, including an N-terminal signal sequence, two potential sites
for N-glycosylation 10 amino acids apart (7, 33), and several hydrophobic domains (7). Antisera generated in
rabbits against gK-specific peptides indicated that gK exists as a
single 40-kDa protein species in infected cells (17). In
contrast, gK translated in vitro had an apparent molecular mass of 36 kDa, and N-linked glycosylation occurred in the first 112 residues of
the protein, consistent with glycosylation at amino acids 48 and 58 (34). Initially, gK was predicted to have four
transmembrane regions (7); however, experiments with in
vitro-translated gK in the presence of microsomal membranes suggested
that gK contained three instead of four membrane-spanning regions
(27). This topological orientation placed all
syn mutations within the proposed ectodomains of gK
(10, 27).
Mutants that are deficient in gK expression have been isolated and
characterized for several alphaherpesviruses. These studies have
indicated that gK is important in virion morphogenesis and egress
(10, 18, 21, 22, 28). Deletion of gK resulted in a
small-plaque phenotype, reduced virus yield, and a restriction in the
ability of virus to be translocated from the cytoplasm to the
extracellular space. Moreover, at least for pseudorabies virus (PRV),
there appeared to be a role for gK in preventing reinfection of cells
(22).
Despite the established role of gK in membrane fusion, initial
characterization of gK localization indicated that HSV-1 gK was
retained within the endoplasmic reticulum (ER) and nuclear membranes
(19). The inability of gK to be transported to the Golgi
complex and, subsequently, to cell surfaces complicated the elucidation
of the role gK could play in mediating cell-to-cell fusion.
Furthermore, contrary to studies with other alphaherpesviruses, including PRV and varicella-zoster virus (VZV) (22, 28),
HSV-1 gK was thought not to be a structural component of virion
particles (19). The absence of gK from virions was
difficult to reconcile with studies that had indicated that HSV-1
virions which specified mutations in gK exhibited delayed entry
kinetics (31). Due to limitations in the ability of gK
peptide antisera to detect HSV-1 gK, we generated recombinant viruses
that specified protein C (protC) epitope tags within gK. Here we show
that HSV-1 gK, like gK of other alphaherpesviruses, is a structural
component of purified virions. Moreover, gK exists on purified virions
and within cells as a Golgi complex-dependent glycosylated species.
Construction and characterization of HSV-1 recombinant virus
gKprotC-DIII expressing gK containing an in-frame insertion of a
12-amino-acid protC tag.
Over the past 20 years, different
laboratories have attempted to generate monospecific antibodies and
monoclonal antibodies (MAbs) against gK to facilitate its detection. To
improve detection of gK, the 12-amino-acid protein C-derived epitope
tag was inserted in-frame within gK domain III at a site predicted not
to significantly affect the secondary structure of gK (Fig.
1D). The recombinant gK
gene coding for the epitope-tagged gK was constructed using PCR-based
splice-overlap extension methodology as described previously (6,
10, 11) and cloned into plasmid pSJ1723, generating plasmid
pTF9301 (Fig. 1C). This plasmid contained gK-flanking sequences
corresponding to the UL52 and UL54 (ICP27) genes to facilitate
homologous recombination with viral DNA (21). Recombinant virus gKprotC-DIII was constructed by rescuing the mutant virus d27-1(KOS) (36), which has a lethal deletion
within the UL54 gene specifying the immediate-early protein ICP27 as
shown previously (9, 10, 21) (Fig. 1A to C). Putative
recombinant virus isolates were plaque purified and tested by PCR and
DNA sequencing for the presence of contaminating d27-1 virus
and the engineered epitope-tagged gK (not shown).
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FIG. 1.
Construction of recombinant virus gKprotC-DIII,
specifying gK containing a protein C epitope tag. (A) The top line
represents the prototypic arrangement of the HSV-1 genome with the
unique long (UL) and unique short (US) regions
flanked by the terminal repeat (TR) and internal repeat (IR) regions.
(B) Shown below is the region of the mutant virus HSV-1
d27-1 genome (between map units 0.7 and 0.8) containing
the UL52, UL53, and the partially deleted UL54 open reading frames
(shaded and boxed regions of the UL54 gene pointed to by an arrow) with
relevant restriction endonuclease sites. (C) Plasmid construct,
pTF9301, containing the recombinant gK-protC gene and flanking UL52 and
UL54 sequences used to generate recombinant virus gKprotC-DIII. (D)
Schematic model of the predicted secondary structure of gK
(10). The predicted putative hydrophobic domains (hpd) of
gK that transverse the membrane are shown embedded within the membrane.
The arrow points to the site of the protC epitope tag insertion. The
primary structure of the epitope tag is shown. Known syncytial
mutations are denoted by asterisks.
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For analysis of virus spread and replication, Vero cells were infected
with serial dilutions of wild-type HSV-1 (KOS) and
gKprotC-DIII
viruses, overlaid with medium containing 1% methyl
cellulose, and
incubated at 37°C for 48 h. Viral plaques were
visualized by
phase-contrast microscopy and photographed. Recombinant
virus
gKprotC-DIII produced viral plaques that were morphologically
similar
to KOS plaques (Fig.
2A and B).
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FIG. 2.
Comparison of plaque morphology and replication
characteristics of gKprotC-DIII and KOS viruses. Comparison of virus
plaque morphologies formed on Vero cells at 48 h postinfection.
(A) KOS. (B) gKprotC-DIII. (C) Time-dependent kinetics of infectious
virus production after infection of Vero cells at an MOI of 5 and
incubation at 37°C. The graph depicts one of three separate
experiments with similar results. Each separate experiment was repeated
in triplicate to obtain standard deviations.
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For analysis of one-step growth kinetics, each virus at a multiplicity
of infection (MOI) of 5 was adsorbed to approximately
8 × 10
5 Vero cells at 4°C for 1 h. Thereafter,
warm medium was added,
and virus was allowed to penetrate for 2 h
at 37°C. Any remaining
extracellular virus was inactivated by low-pH
treatment (0.1 M
glycine, pH 3.0). Cells and supernatants were
harvested immediately
(0 h) or after 4, 8, 12, 20, 30, or 48 h of
incubation. Virus
titers were determined by titration on Vero cells.
The gKprotC-DIII
virus replicated as efficiently as the KOS parental
strain in
Vero cells (Fig.
2C). These results indicated that insertion
of
the 12-amino-acid protC epitope tag within gK domain III did not
adversely affect the structure and function of gK with regard
to virus
replication and cell-to-cell
spread.
Detection and characterization of gK specified by gKprotC-DIII
using anti-protC MAb.
Subconfluent Vero cell monolayers were
infected with either gKprotC-DIII or KOS at an MOI of 5. Forty-eight
hours postinfection, cells were collected by low-speed centrifugation,
washed with Tris-buffered saline (TBS), and lysed at room temperature
for 15 min in mammalian protein extraction reagent (MPER) supplemented with a cocktail of protease inhibitors (Invitrogen-Life Technologies, Carlsbad, Calif.). Insoluble cell debris was pelleted, and samples were
electrophoretically separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to
nitrocellulose membranes (9). Blots were probed overnight with anti-protC MAb HPC-4 at 1:50 dilution (ATCC CRL HB-9892). Subsequently, blots were incubated for 1 h with a
peroxidase-conjugated secondary antibody at a 1:50,000 dilution and
visualized on x-ray film by chemiluminescence (Pierce Chemical,
Rockford, Ill.) (9, 12). All antibody dilutions and buffer
washes were performed in TBS supplemented with 0.135 M
CaCl2 and 0.11 M MgCl2.
MAb HPC-4 detected gK protein species ranging from approximately 36 to
43 kDa, with the predominant species migrating with
an apparent
molecular mass of approximately 36 kDa. Additional
minor protein
species were detected with molecular masses ranging
from 36 to 43 kDa,
as well as a protein species with an apparent
molecular mass of 25 kDa.
In contrast, antibody HPC-4 did not
react against extracts derived from
KOS-infected cells (Fig.
3A).
Similar to
other herpesvirus glycoproteins, these results show
that the
protC-tagged gK exists as multiple protein species in
virus-infected
cell extracts.
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FIG. 3.
Characterization of synthesis and processing of
protC-tagged gK specified by gKprotC-DIII. Immunoblots of gKprotC-DIII-
(lanes 1 to 3) and KOS (lanes 4 to 6)-infected cell extracts reacted
with either anti-protC MAb HPC-4 (A) or anti-gD MAb 1103 (B). Cellular
extracts were treated with Endo-H (lanes 2 and 5), PNGase-F (lanes 3 and 6), or mock treated (lanes 1 and 4). (C) Cellular extracts obtained
from Vero cells infected with gKprotC-DIII in the presence (lanes 2 and
4) or absence (lanes 1 and 3) of TM were probed with either anti-protC
MAb (lanes 1 and 2) or anti-gD MAb (lanes 3 and 4).
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The predicted molecular mass of the unglycosylated gK protein backbone
(308 amino acids after signal peptide cleavage) is
approximately 31 kDa; however, after addition of the protC tag,
the molecular mass
should be approximately 32.5 kDa. Thus, the
gK-related species with
molecular masses of 36 to 43 kDa represent
glycosylated gK derivatives.
The 25-kDa protein could be either
a proteolytically processed peptide
or a previously predicted
gK-related protein, which may be produced by
an alternate transcript
utilizing an initiation codon within the UL53
ORF (
29).
Previous investigations have shown that HSV-1 gK contained N-linked
carbohydrate species (
17,
34). To characterize the
carbohydrate character of gK, Vero cell monolayers were infected
with
gKprotC-DIII at an MOI of 5 and incubated in the presence
or absence of
5 µg of tunicamycin (TM) per ml. Cellular extracts
were prepared at
48 h postinfection and analyzed by SDS-PAGE and
immunoblot
analysis using either anti-protC antibody, HPC-4, or
anti-gD antibody
1103 (Rumbaugh-Goodwin Institute, Plantation,
Fla.) (Fig.
3C). In the
presence of TM, gK had an apparent molecular
mass of approximately 32 kDa, which corresponded to the predicted
molecular mass of the
unglycosylated gK after removal of the signal
peptide (Fig.
3C, lanes 1 and 2). As a control, a parallel blot
was incubated with anti-gD
antibody (1:2,000). As expected, TM
reduced the apparent molecular mass
of gD from 59 to 50 kDa (Fig.
3C, lanes 3 and 4) (
41).
To further investigate the glycosylation of gK, infected cellular
extracts were mock treated or incubated in the presence
of either
endoglycosidase H (Endo-H) or peptide:N-glycosidase
F (PNGase F).
N-linked carbohydrates were removed from proteins
contained within
infected cell lysates by a modified protocol
of the manufacturer's
instructions and essentially as described
previously (
22).
Briefly, virally infected cells were lysed
at 42°C for 1 h in
MPER that contained a protease inhibitor cocktail,
0.5% SDS, and 1%
-mercaptoethanol. Samples were subsequently
incubated for 18 h
at 42°C either with 50 mM sodium citrate (pH
5.5) and 100 U of Endo-H
(NEB Labs, Beverly, Mass.) for Endo-H
digestions, with 50 mM sodium
phosphate (pH 7.5), 1% NP-40, and
20 U of PNGase-F (NEB Labs) for
PNGase-F digestions, or without
enzyme prior to SDS-PAGE and Western
blot
analysis.
PNGase-F treatment, which cleaves off all N-linked carbohydrate chains
from the protein backbone, reduced the apparent molecular
mass of gK
from 36 to 32 kDa (Fig.
3A, lane 3), in agreement with
gK produced in
the presence of TM (Fig.
3C, lane 2). Endo-H treatment,
which is
specific for digestion of high-mannose carbohydrate chains,
resulted in
the production of multiple protein species migrating
with apparent
molecular masses of 36, 34, 32, and 25 kDa. The
34-kDa band was the
predominant species, indicating that the majority
of gK expressed in
infected cells contains high-mannose carbohydrate
chains (Fig.
3A, lane
2). As controls for the PNGase F and Endo-H
activities, the effect of
these enzymes on gD specified by either
gKprotC-DIII or KOS was also
assessed. Both enzymes reduced the
apparent molecular mass of gD
similar to the reduction exhibited
by gK, confirming that gD contains
N-linked carbohydrates and
that gD is present as both Endo-H-sensitive
and Endo-H-resistant
forms (Fig.
3B). These results are consistent with
previous reports
that suggested gK is expressed predominantly as a
high-mannose
glycosylated species (
19). However, a portion
of the expressed
gK was Endo-H resistant even after an 18-h incubation
with a high
concentration of Endo-H, demonstrating that gK is
transported
to the Golgi apparatus and undergoes further processing of
the
high-mannose precursor carbohydrate
moieties.
gK is a structural component of purified virions.
Previous
reports suggested that HSV-1 gK might not be a structural component of
virions (19). However, studies with PRV and VZV
demonstrated that gK was present in purified virions. To ascertain
whether gK was a structural component of HSV-1 virions, we investigated
whether gK specified by gKprotC-DIII could be detected in purified virions.
KOS and gKprotC-DIII virions derived from infected Vero cell
supernatants were purified by two consecutive sucrose density
gradients
as described previously (
15,
16,
23). Specifically,
roller
bottles of Vero cells were infected at an MOI of 0.1 PFU/cell.
Forty-eight hours postinfection, supernatants containing extracellular
virus were harvested, and cellular debris was removed by centrifugation
at 10,000 ×
g for 15 min. Virus was concentrated by
pelleting
through a 10% sucrose-TBS cushion at 100,000 ×
g for 1.5 h. Virus
pellets were resuspended in TBS,
layered onto a 60 to 10% continuous
sucrose gradient, and sedimented
at 100,000 ×
g for 2 h. Purified
virus was
collected from the light-scattering phase midway through
the gradient,
diluted threefold in TBS, and layered on a 10%-30%-60%
sucrose-TBS discontinuous step gradient. Purified virion preparations
were collected by side puncture at the 30%-60% interface, diluted
in
30 ml of TBS, layered onto a 10% sucrose-TBS cushion, and centrifuged
at 100,000 ×
g for 1.5 h to concentrate the
virion preparations.
Viral proteins were extracted and processed in
MPER-protease inhibitor
cocktail as described for cell
lysates.
Antibody HPC-4 detected gK in gKprotC-DIII purified virions, while it
did not react with KOS purified virions (Fig.
4A). Treatment
of purified virions with
PNGase-F caused the appearance of a gK
species migrating as a 32-kDa
band similar to that observed in
PNGase-F-treated cellular extracts
(compare Fig.
3A, lane 3, and
Fig.
4A, lane 3). The 25-kDa species
observed in cellular extracts
was not detected in immunoblots of
purified virion preparations.
In contrast to the results obtained with
Endo-H treatment of infected
cellular extracts, the majority of gK
detected in purified virions
was resistant to enzymatic digestion,
indicating that it contained
peripheral sugars added at the Golgi
apparatus (Fig.
4A, lane
2).
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FIG. 4.
Detection and characterization of protC-tagged gK
expressed on purified virions. Immunoblots of gKprotC-DIII (lanes 1 to
3) or KOS (lanes 4 to 6) purified virion preparations reacted with
anti-protC MAb HPC-4 (A), anti-gD MAb 1103 (B), and anti-ICP27 MAb 1113 (C). Purified virion preparations were treated with Endo-H (lanes 2 and
5), PNGase-F (lanes 3 and 6), or mock treated (lanes 1 and 4). (D)
Cellular extracts from Vero cells infected with either gKprotC-DIII
(lanes 1 to 3) or KOS (lanes 4 to 6) were reacted with anti-ICP27 MAb.
Cellular extracts were treated with Endo-H (lanes 2 and 5), PNGase-F
(lanes 3 and 6), or mock treated (lanes 1 and 4).
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For control purposes, parallel immunoblots were assayed for the
presence of gD and ICP27. As expected, gD specified by KOS
and
gKprotC-DIII virions was found to contain N-linked, Golgi
complex-dependent sugars (Fig.
4B). HSV-1 (UL54) ICP27 is a known
nonstructural protein, which is expressed in infected cells but
is not
present in purified virions (
45). As expected, ICP27
was
detected in gKprotC-DIII- and KOS-infected cell extracts using
anti-ICP27 antibody 1113 (Rumbaugh-Goodwin Institute); however,
it was
not present in purified virions (Fig.
4C and
D).
HSV-1 gK enhances virion entry.
Based on the finding that gK
was present in purified virions and the fact that syncytium mutations
in gK alter virus entry kinetics (31) as well as cause
substantial virus-induced membrane fusion (2, 8), we
examined the penetration kinetics of the gK virus in comparison to
its parental strain, KOS. Subconfluent monolayers of VK302 cells in
six-well culture dishes were infected at 4°C for 1 h with
approximately 300 PFU of KOS or gK grown in either Vero
(gK/) or VK302 (gK/+)
cells. The inoculum was subsequently removed, warm medium (34°C) was
added, and the cultures were shifted to 34°C to allow virus penetration. Immediately thereafter (0 h) and at 30, 60, 120, and 180 min, remaining extracellular virus was inactivated by treatment with
low-pH buffer (0.1 M glycine, pH 3.0). Cells were washed three times
with phosphate-buffered saline (PBS) and overlaid with 1% methyl
cellulose in Dulbecco's modified Eagle's medium. Virus plaques were
counted at 48 h postinfection, and the percentage of PFU surviving
low-pH inactivation compared to PBS-treated controls was calculated.
The kinetics of gK (gK/) virion entry was
substantially reduced in comparison to the KOS
(gK+/+) strain. Furthermore, gK virions
produced in the complementing cell line, VK302
(gK/+), which is transformed with the gK gene
(18), entered substantially faster, exhibiting entry
kinetics similar to that of the KOS virus (Fig.
5).
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FIG. 5.
Penetration kinetics of KOS and gK viruses into VK302
(Vero) cells. The penetration kinetics of KOS virus grown on Vero cells
and gK virus grown on either Vero or VK302 cells were obtained by
determining the percentage of PFU surviving low-pH treatment relative
to PBS-treated controls at different times postadsorption. Mean values
and standard deviations of three independent experiments are shown.
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Considering the importance of gK in membrane fusion phenomena, it is
not surprising that gK was found to be glycosylated by
the Golgi
apparatus as well as being a structural component of
the virion that
functions to enhance virion entry. In this regard,
gK localizes where
it is capable of participating in multiple
membrane fusion events
during virion morphogenesis, virus-induced
cell fusion, and virion
penetration.
gK virions have a major
defect in virion egress, which
causes the accumulation of virions
within vesicles in the cytoplasm of
infected cells. The origin
of these vesicles is not yet known; however,
morphological data
suggest that they may be derived from the Golgi
complex. If this
is true, it may mean that gK functions in a Golgi
complex-dependent
pathway involved in either reenvelopment at the Golgi
complex
according to the deenvelopment/reenvelopment model (
4,
13,
14,
40,
44) or post-Golgi complex transport of virions to
extracellular
spaces.
It is important to note that lack of gK is not absolutely lethal for
virus replication in cell culture, although it does reduce
virus titers
by more than 100-fold and substantially reduced the
kinetics of virus
entry into Vero cells. Importantly, experimental
infections in mice
using the eye route indicate that gK may be
required for virus
replication, spread, and neurovirulence in
vivo (unpublished data).
Therefore, it is possible that the true
functions of gK may not be
discernible in cell culture systems,
such as Vero cells, that have been
selected for the optimum replication
of HSV virions. In this regard,
more sensitive cell culture systems
and experimental animal studies may
be required to delineate the
functions of
gK.
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ACKNOWLEDGMENTS |
This work was supported by a grant from the National Institute of
Allergy and Infectious Diseases (AI43000) to K.G.K. We acknowledge support from the School of Veterinary Medicine, Louisiana State University.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pathobiological Sciences, School of Veterinary Medicine, Louisiana
State University, Baton Rouge, LA 70803. Phone: (225) 578-9682. Fax: (225) 578-9701. E-mail: vtgusk{at}lsu.edu.
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Journal of Virology, December 2001, p. 12431-12438, Vol. 75, No. 24
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.24.12431-12438.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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