Replication of Herpes Simplex Virus: Egress of Progeny Virus at
Specialized Cell Membrane Sites
Rebecca M. Mingo,aJun Han,bWilliam W. Newcomb,aand Jay C. Browna
Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA,aand Department of Microbiology and Immunology,
Penn State University College of Medicine, Hershey, Pennsylvania, USAb
Mothes et al. ). As infection moves from cell to cell, it is more
advantageous for a virus to be released at locations adjacent to
sites of entry than to travel to entry sites after egress. Although
many viruses share common pathways in the process of directed
spread, each virus is unique in its approach (11, 20, 27, 43, 44,
Herpes simplex virus 1 (HSV-1) is a double-stranded DNA
teins that form the tegument, and a host-derived envelope incor-
porating the viral glycoproteins. During the process of virion for-
mation, capsids are assembled in the nucleus and then enveloped
encounters viral glycoproteins on a vesicle that is believed to be
derived from the trans-Golgi network (TGN) (30, 48, 54, 58).
Tegument proteins bind to the capsid and to the glycoprotein
tails. Negative curvature is induced, and the capsid buds into the
vesicle, acquiring an envelope and an outer vesicular membrane.
This virus-containing vesicle then moves to the plasma mem-
released virions remain cell associated until very late in infection
In a natural setting, HSV-1 infects epithelial cells of mucosal
surfaces and then moves on to establish latent infections in the
sensory neurons that innervate these areas. Mucosal tissues con-
sist of highly polarized epithelial cells in the apical layers of the
o ensure efficient transmission and replication, many viruses
have developed mechanisms for directed spread (reviewed by
different directions depending on a cell’s location in a tissue layer
and the cell’s differentiation status (35, 42, 67). The virus is re-
leased at cell junctions in highly polarized epithelial cells (cells
with tight junctions and high transepithelial resistance [TER])
cells (i.e., no tight junctions, low TER) of the epidermis has not
been extensively examined, although previous electron micros-
copy studies suggest that virus is randomly released (21). In neu-
rons, newly formed virions traffic down axon microtubules for
release at synaptic terminals and at virally induced varicosities (4,
6). Infection can then move swiftly through neural networks and
an area that is advantageous for host-to-host spread. In both po-
gE is a transmembrane protein that forms a dimer with gI (18,
Received 22 February 2012 Accepted 11 April 2012
Published ahead of print 24 April 2012
Address correspondence to Jay C. Brown, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.orgJournal of Virology p. 7084–7097July 2012 Volume 86 Number 13
19). Early during infection, gE colocalizes with TGN46 in the
TGN, while at late times gE moves to cell junctions in polarized
epithelial cells (9, 30). Although gE is not essential for HSV-1
as mutants produce the same amount of infectious virus as wild-
type (WT) HSV-1 (2, 7, 8, 29, 61). In highly differentiated, polar-
ized epithelial cells, virus lacking gE becomes randomly released
rather than specifically directed to cell junctions, and infection
eny virus is unable to be transferred from infected epithelial cells
to neurons, even though ?gE mutants are competent for neuron
entry (29, 61). Furthermore, in neurons infected with a ?gE mu-
traffic down axons to egress sites (28, 51, 60). Infection is, there-
fore, much more limited.
lial cells, such as those located in the basal layers of the epidermis.
cell types to fully understand the mechanism of HSV-1 egress. In
the study described here, we examined the process of HSV egress
as it occurs in Vero cells, a nonpolarized epithelial cell line that
that concentrated along the substrate-adherent surface of the cell
as well as at cell-cell contact areas despite the lack of mature junc-
tional complexes at these locations. Electron microscopy (EM),
confocal microscopy, and total internal reflection fluorescence
(TIRF) microscopy were employed to characterize the formation
of infected cells.
MATERIALS AND METHODS
Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) with 10% fetal
bovine serum (FBS; Atlanta Biologicals) on 75-cm2Nunc EasYFlasks.
Cells were incubated at 37°C in 7% CO2and passaged every 4 to 6 days
with a medium change at day 2. Viruses used were the green fluorescent
protein (GFP)-tagged VP26 (K26GFP) mutant developed by Prashant
Fred Homa (31). Both the K26GFP mutant and WT KOS HSV were pas-
saged on Vero cells. gE mutants, whose development is described below,
were passaged in a similar manner on Vero cells. ?UL25 mutant stocks
were made on the 8-1 complementing cell line (31).
kind gift from Gary Cohen and Roselyn Eisenberg. Each was used at a
1:1,500 dilution in 1% bovine serum albumin (BSA). The gE polyclonal
antibody used in Western blotting was kindly provided by Harvey Fried-
used to label major capsid protein VP5 at a 1:1,500 dilution. Commer-
cially available antibodies included monoclonal anti-?-tubulin (T6074;
1:1,000; Sigma) and monoclonal anti-vinculin (V-9131; 1:600; Sigma).
Actin was labeled with Texas Red-X phalloidin (T7471; Invitrogen) at
1:500 in phosphate-buffered saline (PBS). Rhodamine-labeled wheat
germ agglutinin (WGA; RL-1022) was obtained from Vector Laborato-
ries, Burlingame CA, and diluted to 2.5 ?g/ml in PBS. Alexa Fluor 594
mouse IgG (A11001; Invitrogen) were the secondary antibodies used
throughout. They were used at a 1:1,000 dilution.
Infection protocol. Cells were plated on glass coverslips in 6-well
plates at 3 ? 105cells/well. Twenty-four h later, cells were infected on ice
were warmed for 1 h at 37°C, after which they were acid washed (40 mM
citric acid, 10 mM KCl, 135 mM NaCl, pH 3) for 1 min to inactivate
extracellular virus (3). Acid was removed with 3 rinses in warm 1% FBS-
DMEM, and cells were returned to 37°C. Cells were fixed at noted times
after infection with 4% paraformaldehyde (PFA) for 10 min. Those that
X-100 and incubated in blocking buffer (10% goat serum and 1% BSA in
PBS) for 1 h at room temperature.
Electron microscopy. At 12 h postinfection (hpi), coverslip-grown
Vero cells infected as described above were fixed in 2.5% glutaraldehyde
for 12 h. Samples then were postfixed for 30 min in 1% (wt/vol) osmium
tetroxide, dehydrated in increasing concentrations of ethanol, and infil-
trated with EPON-812 (Electron Microscopy Sciences, Inc.). Embedding
To remove BEEM capsules with the embedded samples from coverslips,
80 nm) prepared on a Leica Ultracut UCT ultramicrotome using a
Diatome diamond knife were collected on 200-mesh copper grids and
contrast stained with lead citrate and uranyl acetate as described previ-
ously (39). Images were recorded on film using a Philips 400T transmis-
sion electron microscope operated at 80,000 eV.
inverted IX70 Olympus microscope with a 1.45-numeric-aperture (NA)
modular automation controller (Ludl Electronic Products) and operated
by MetaMorph software (Invitrogen). Images were acquired with a
charge-coupled device camera (Retiga Exi; Qimaging). Confocal images
of a Plan Apochromat 100?/1.4-NA oil immersion lens. Samples were
tube image detection.
were treated with either 1.7 ?g/ml cytochalasin B (C6762; Sigma), 10
?g/ml nocodazole (M1404; Sigma), or an equal volume of dimethyl sul-
were previously solubilized in DMSO. At 12 hpi, cells were fixed in 4%
paraformaldehyde for 10 min at room temperature and processed for
immunofluorescence microscopy. In a second experimental design, cells
2 rinses in PBS and 2 rinses in warm 1% DMEM. Incubation was contin-
aldehyde and processed for immunofluorescence microscopy.
Image analysis. TIRF images of 20 to 30 cells per sample set were
analyzed with ImageJ software. The pixel area was calculated for both the
total cell area and the patch areas as determined by WGA staining. Only
glycoprotein patches larger than 300 pixels in area were used for quanti-
fications. Virion density was determined by transferring the cell outlines
as determined on combined channel images onto the green channel pic-
ball algorithm (53) and setting the radius to 50 pixels. Integrated density
was then calculated and divided by the area in question to get the virion
density. P values were calculated using the t test for 2 samples assuming
Construction of recombinant HSV gE mutants. A bacterial artificial
chromosome (BAC) containing the HSV-1 KOS strain genome was used
codon ATG with stop codon TAA. The gE cytoplasmic tail deletion mu-
tant (gE?CT), generated by adding two copies of stop codon (TAATAA)
was previously reported (14). gE?CT rescue virus was generated by re-
moving the stop codons. Correct clones were verified by HindIII diges-
HSV Release Sites
July 2012 Volume 86 Number 13jvi.asm.org 7085
with Lipofectamine 2000. After the appearance of cytopathic effects (3 to
4 days), transfected cells were harvested and used to infect new Vero cell
monolayers to produce a viral stock.
Analysis of gE mutant expression and virion incorporation. Vero
cells were infected with wild-type HSV or gE mutants at a multiplicity of
at 16 to 20 h postinfection, cleared at 2,500 rpm for 10 min, and then
a 30% (wt/vol) sucrose cushion (1.7 ml). The virus-containing pellets
were dissolved in 100 ?l 1? SDS-PAGE sample buffer, and an approxi-
mately equal amount of virus normalized to VP5 was loaded into an
SDS-PAGE gel for Western blot analyses. The infected cell lysates were
also analyzed for the expression levels of gE and its mutants. Blots were
probed with ?-gE polyclonal antibody (see above) at a 1:6,000 dilution.
3 infected cells) was released from the cell surface into the supernatant by
12 hpi. Therefore, titer was determined for cell-associated virus only un-
less stated otherwise. To determine the amount of infectious, cell-associ-
ated virus, infected cells were harvested in PBS with the aid of a cell
scraper. Samples were frozen and thawed twice and placed in a bath son-
0.5 mg/ml thiazolyl blue tetrazolium bromide stain diluted in medium
cells. To study the process of HSV-1 egress in Vero cells, electron
microscopy was used to observe the pattern of progeny virus as-
sociation with the cell surface. Glass-grown Vero cells were in-
this time point, progeny virions that were not transferred to
nearby cells were found to be associated with the parental cell
surface with few virions released into the media. Micrographs
showed that at 12 hpi, the majority of virions were observed at
specific areas on the cell surface, rather than in a randomly dis-
persed release pattern (Fig. 1A to D). Virus-containing regions
were located at cell-cell contact sites and at areas along the adher-
ent cell surface. There were approximately 3-fold more virions at
these locations than on the nonadherent upper cell surface, al-
though this is likely an underestimation, since some virions along
the upper cell surfaces are expected to be noninfectious parental
virus particles that did not enter the cell. At both the substrate-
adherent surface and at cell-cell contact site egress locations, ad-
ditional membrane was present allowing a curvature in the mem-
to C). This was not the case in uninfected cell samples; the adher-
ent cell membrane of mock-infected Vero cells was tightly ap-
posed to the coverslip surface (Fig. 1E and F). Although many
virions were observed exterior to the plasma membrane in in-
brane (Fig. 1A to D). The few virions seen on other membrane
surfaces were often in areas adjacent to cell-cell contacts (Fig. 1D,
bracket). Similar results were obtained whether the cells were in a
by a lack of available cell-cell-adherent surfaces.
Confocal microscopy was utilized to observe the release of vi-
rions in larger numbers of infected Vero cells. Cells grown on
glass coverslips were infected with a GFP-tagged VP26 mutant
(K26GFP) and fixed at 12 hpi. Confocal Z-stack images showed
that GFP-labeled virions were concentrated along the adherent
surfaces of cells. A representative series is shown in Fig. 1G. Most
to the coverslip (those marked by an asterisk). The majority of
virions that were visible above the two planes were located at cell-
cell contact points (Fig. 1G, arrows in slices 4 to 6). The large
GFP-containing areas located in the nucleus are capsid assembly
compartments. In the following sections we further characterize
cell surface were best visualized using total internal reflection flu-
orescence (TIRF) microscopy. This method allowed the area of
excitation to be restricted to a small plane above the coverslip. A
of the laser can be adjusted to obtain the desired excitation field
the maximum angle to allow the excitation of the largest area
possible, which is 300 nm above the coverslip.
Using TIRF microscopy, we found that GFP-labeled virions
clustered at specific sites along the adherent cell surface in a man-
ner similar to that observed with EM (Fig. 2). In addition, infec-
tion induced the recruitment of glycoproteins to egress locations.
N-acetylneuraminic acid (sialic acid) residues on glycoproteins,
whether viral or cellular in origin (45). When infected Vero cells
were fixed, permeabilized, and stained with rhodamine-labeled
WGA, it was observed that viral release sites were stained much
more strongly than the surrounding cell membrane (Fig. 2A and
E), indicating that the regions where cell-associated viral particles
Such focal concentrations were absent from uninfected cells,
staining outside the patches was much brighter in infected cells
than in uninfected cells, suggesting a greater amount of surface
glycoprotein expression overall. There were often several viral
egress patches per infected cell, yet cells with a single large patch
were also seen. Patches could be expansive and were generally
peripheral, as shown in Fig. 2B.
Viral glycoproteins were found to be a component of WGA-
stained patches. Infected Vero cells were fixed at 12 hpi and
stained with ?-gB, ?-gD, ?-gH, or ?-gE monoclonal antibody.
Staining for gB and gD are shown in Fig. 2B and C, while gH and
gE staining is not shown. All viral glycoproteins were found in
greater amounts in the regions where cell-associated virus was
concentrated compared to the surrounding membrane. Viral gly-
staining (Fig. 2F). Viral proteins appear to compose a large por-
tion of the glycoprotein patches, but cellular proteins also may be
recruited. Since both viral glycoprotein staining and WGA stain-
ing similarly define the size and shape of egress locations, patches
were labeled using rhodamine-WGA in the following figures.
Patches were observed in all cells at 12 hpi that were labeled with
viral glycoprotein antibodies, suggesting that the majority of in-
fected cells form these structures (data not shown). Many glyco-
protein-stained patches resembled a donut in shape (Fig. 2C, ar-
rowheads). This phenomenon was due to the pocket-like
structure of these sites. The holes in the donuts were the result of
Mingo et al.
jvi.asm.org Journal of Virology
the membrane rising above the 300-nm excitation limit in the
center of the patches.
Concentrations of virions and glycoproteins along the adherent
surface were not specific to Vero cells but seemed to be associated
with a nonpolarized state. Similar adherent surface glycoprotein
patches were observed in infected HeLa cells (a nonpolarized cell
FIG 1 Location of progeny virions in HSV-1-infected Vero cells. (A to D) Thin-section electron micrographs of infected Vero cells fixed and processed on
with some virus above cell-cell contacts (bracket). Panel B is an enlargement of the left arrowhead in panel A; panel C is an enlargement of the right arrowhead
sections above the coverslip where the majority of virions can be seen. Other sections have far fewer virions. The nucleus (containing VP26-GFP-labeled capsid
assembly areas) is outlined. Arrows indicate virus released along a cell-cell contact. Images were obtained with a Philips 400T transmission electron microscope
(A to F) and a Zeiss LSM 510 confocal microscope with an inverted 100? lens (G).
HSV Release Sites
July 2012 Volume 86 Number 13 jvi.asm.org 7087
Glycoprotein patches form independently from trafficking
virions. Vero cells infected at an MOI of 10 showed neither gly-
coprotein staining nor VP26-GFP virus signal at 4 hpi (Fig. 3A,
spread and low levels of virus were consistently detected associat-
arrived first, modifying the composition of the egress site mem-
To further explore the differential trafficking between virions
and glycoproteins, Vero cells were infected with a ?UL25 mutant
(KUL25NS). The gene product of UL25 is needed for the packag-
and capsids do not exit the nucleus. However, protein expression
continues normally (31). If glycoprotein movement to the cell
would expect to see a drop in the size of glycoprotein patches in
FIG 2 Glycoprotein enrichment at coverslip-adherent surface egress sites. (A) TIRF micrographs of a VP26-GFP HSV-1-infected Vero cell at 12 hpi fixed,
permeabilized, and stained with rhodamine-conjugated WGA (Rh-WGA) to mark glycosylated proteins. As in EM pictures, GFP-labeled virions were found to
micrographs of a VP26-GFP HSV-infected Vero cell at 12 hpi that had been treated with ?-gB (DL16) antibody. (C) TIRF micrographs of a VP26-GFP
HSV-infected Vero cell treated with ?-gD antibody (DL11). Staining indicates that viral glycoproteins accumulate at egress sites. Arrowheads mark two egress
sites where the pocket-like structure of the sites is apparent. The holes in the donuts are areas where the membrane has extended beyond the 300-nm laser
image until the edge was visible. The size bar in panel C is also relevant for panels A to D. (F) HSV-1-infected Vero cell stained with both rhodamine-WGA and
?-gD antibody. Note that there is complete colocalization between the two. Alexa 594-conjugated secondary antibodies were used in panels B, C, and F.
Mingo et al.
jvi.asm.org Journal of Virology
cells infected with the ?UL25 virus compared to the WT. If the
on the surface with the ?UL25 mutant.
HSV-1 or the ?UL25 mutant for 12 h. Cells were then fixed, per-
meabilized, and stained with WGA. Capsids were labeled with
tibody. In the KOS-infected cells, virus and glycoproteins were
found to colocalize at release site patches as expected (Fig. 3B,
top). In ?UL25 mutant-infected cells, glycoproteins formed nor-
mal patches even though capsids were not present (Fig. 3B, bot-
those formed in the infection with wild-type UL25 gene product
(data not shown). These results provide evidence that patch gly-
coproteins are in the cell membrane of infected Vero cells, and
glycoproteins traffic to and accumulate at viral egress sites inde-
pendently of virions.
previously shown that the cellular cytoskeleton is altered during
HSV-1 infection. The microtubule organizing center (MTOC) is
disrupted and microtubules become sparse and disorganized (23,
bules could be seen in our samples, results showed that these mi-
infection (16, 41, 59, 63). When infected Vero cells were stained
for actin with Texas Red-labeled phalloidin, few stress fibers were
lining the cell membrane) could still be seen in most cases (Fig.
the viral egress sites. It is unlikely that the actin holes observed
were due to limitations of the TIRF laser; depleted areas were still
In addition, the majority of areas where actin was cleared con-
tained a glycoprotein patch whose edges closely followed the line
of actin depletion even if few virions were visible (data not
shown). A similar result was observed with focal adhesions (Fig.
4C). Although patches often formed shapes that mimicked large
sites; they were depleted in areas where the patches formed. Areas
of microtubule, actin, and focal adhesion depletion were rarely
after HSV infection.
Both actin and microtubules contribute to glycoprotein
viral egress sites were depleted of cytoskeletal elements, it was
ficking. The expected size and density of virus-containing vesicles
along with the nonrandom release of virions suggested that a
structural element was assisting their movement and delivery to
their destination. The possible role of microtubules and actin was
vals after infection. VP26-GFP HSV-1-infected cells were treated
at 4 hpi (before patches were visible and infectious virus detect-
FIG 3 TIRF micrographs showing formation of glycoprotein patch sites un-
fixed at 4, 6, and 8 hpi. Patch glycoproteins were labeled with rhodamine-
of the adherent surface of the cell (as determined by increasing the brightness
of the image) is outlined. Note that neither glycoprotein patches nor virus are
visible at 4 hpi. By 6 hpi, glycoprotein patches are beginning to form (arrow)
on the adherent surface, but virus still is not evident. At 8 hpi, there is pro-
accumulate in these areas. Images indicate that patches form before viral
egress. (B) Vero cells infected with wild-type KOS HSV-1 (top) or UL25?
mutant HSV (bottom) were fixed at 12 hpi. In a UL25? infection, capsids are
retained in the nucleus. Egress site glycoproteins were labeled with WGA.
Virus was labeled with ?-VP5 major capsid protein antibody and Alexa 488-
tein patches form independently of viral release.
HSV Release Sites
July 2012 Volume 86 Number 13jvi.asm.org 7089
or 11.5 hpi (patches were large and contained many virus parti-
cles). Treatment included either 10 ?g/ml nocodazole to depoly-
merize microtubules, 1.7 ?g/ml cytochalasin B to depolymerize
actin, or 3.3 ?l/ml DMSO as a control. Each drug concentration
the cytoskeleton. Treated cells were fixed at 12 hpi and stained
with rhodamine-WGA to define the patch outlines. Resulting
TIRF images (Fig. 5A to C) were then analyzed as described in
Materials and Methods.
It was found that the depolymerization of the microtubules
during infection caused a significant reduction in the percentage
addition, depolymerization at early time points caused a reduc-
the adherent surface (Fig. 5B and E). No significant effect was
observed when nocodazole was added later in infection after
nation of virus titers showed that there was no statistically signif-
icant effect on titer throughout several experimental repetitions
(data not shown), although small reductions in infectious virus
the lack of microtubules impedes the trafficking of glycoproteins
to the cell surface. The decrease in virus at the cell membrane is
also likely due to inhibited transport to the surface, although the
velopment of virus.
eton decreased the number and size of egress patches at all time
points tested (Fig. 5C and D). This effect was more pronounced
was also seen when actin was depolymerized after patches had
already formed. As actin was disrupted, the patches that had
FIG 4 Location of cytoskeletal elements in relation to viral egress sites on the coverslip-adherent surface of HSV-1-infected Vero cells as shown by TIRF
microscopy. (A to C) VP26-GFP HSV-infected Vero cells stained with ?-tubulin antibody to label microtubules (A), Texas Red-phalloidin (TR-Phalloidin) to
lined areas mark representative egress sites where the depletion has occurred. The starred area in panel B is an adjacent uninfected cell. (D to F) Mock-infected
Mingo et al.
jvi.asm.org Journal of Virology
FIG5 Role of microtubules and actin in virus/glycoprotein trafficking and patch formation. Cells were infected with VP26-GFP HSV-1 and fixed at 12 hpi. (A)
TIRF micrograph of DMSO-treated sample stained with rhodamine-WGA to mark glycoprotein patches. (B) TIRF micrographs of infected cells treated for the
noted intervals during infection with 10 ?g/ml nocodazole (Noc). Cells were stained with WGA. Note that patches in the 4- to 12-hpi treatment sample are
smaller, with fewer virions than the control. (C) TIRF micrographs of infected cells treated for the noted periods with 1.7 ?g/ml cytochalasin B (CyB). Patches
were labeled with WGA. Note that treatment has disrupted glycoprotein patch structures. (D) Graph depicts the percentage of cell membrane that is covered in
glycoprotein-rich patches. Numbers were obtained by dividing the sum of the patch areas (that were above the 300-pixel cutoff) by the total area of the cellular
adherent membrane visible in TIRF micrographs. (E) Graph depicts the amount of virus on the cellular adherent membrane visible by TIRF microscopy.
Twenty-five cells per sample set were analyzed. Error bars are for the standard errors of the means. P values of treated samples compared to DMSO control
samples are labeled the following: *, ?0.05; **, ?0.005; and ***, ?0.0005.
July 2012 Volume 86 Number 13jvi.asm.org 7091
formed were dispersed into very small glycoprotein aggregations
with the occasional associated virion. The result that previously
interpreted to indicate that actin filaments are maintaining the
structure of egress sites.
Treatment with cytochalasin B early in infection decreased the
amount of virus on the cell surface (Fig. 5E). This suggested that
maintaining patch integrity. There was no statistically significant
difference in titers of cell-associated or supernatant virus for all
not shown). The observed decrease in detectable virus on the
membrane when cells were treated from 11.5 to 12 hpi was prob-
assisting the trafficking of virions from the cytoplasm to the cell
surface, or it could be assisting newly secreted virus to move to
tributes to both virus trafficking to egress sites and to the mainte-
nance of viral and possibly cellular components at these sites.
The structure of egress sites can reform after disruption.
During the process of secretion in secretory cell types, actin is
locally depolymerized in the cortex, allowing the passage of a ves-
icle. Existing studies have found that the depolymerized areas are
explanation that we considered for the existence of actin holes at
viral egress sites was that actin depolymerized to allow the exocy-
tosis of the virus, and the holes remained due to a global block in
actin cortex as a whole is lost during infection (likely through a
where this has occurred.
were treated with 1.7 ?g/ml cytochalasin B for 30 min. Actin was
rinsed out and infection continued for another 45 min. Samples
were stained with either WGA or phalloidin. The results showed
that the actin cortex was able to reform after depolymerization;
there was no apparent block in actin filament polymerization or
and GFP-labeled virus were visible after the 45-min recovery pe-
riod (Fig. 6C). It is unclear whether the dispersed virus/glycopro-
virus released since the restoration of the actin cortex. In either
case, we conclude that actin was able to polymerize normally and
infected cells were able to create new egress sites late in infection.
The clearance of actin at these sites appears to be locally rather
than globally induced.
Glycoprotein E is necessary for glycoprotein patch forma-
polarized cells such as keratinocytes and neurons is dependent
upon viral glycoprotein E (gE). gE null mutants produce normal
of this protein in HSV-1-infected nonpolarized cells. To test the
function of gE in the trafficking of viral components to the cover-
slip-adherent cell surface, we ultilized ?gE, gE?CT, and gE?CT
rescue mutants. Using a bacterial artificial chromosome system,
the start codon was replaced with a stop codon in the gE gene
sequence. To create the gE cytoplasmic tail deletion mutant, two
stop codons were added at residue 446 directly after the trans-
membrane region (14). The expression of gE was rescued by re-
moving these stop codons.
The mutants were verified by harvesting the supernatant and
cell lysates of infected Vero cells and staining for the appropriate
the WT and rescue infection but was absent from the cell lysates
and virions of the gE deletion mutants. The gE cytoplasmic tail
FIG 6 TIRF micrographs showing egress site reformation after actin depo-
lymerization-induced disruption. VP26-GFP-infected Vero cells at 11.5 hpi
(A) were treated with 1.7 ?g/ml cytochalasin B for 30 min (B). (C) Toxin was
then rinsed out and the infection continued for another 45 min. Sample cells
were fixed at each step and stained with Texas Red-phalloidin or rhodamine-
WGA. (D) Untreated control. Note that both the actin cortex and viral egress
sites were able to reorganize after disruption. There is no global block in actin
Mingo et al.
jvi.asm.orgJournal of Virology
level and was less stable than its full-length counterpart. In cell
due to the presence of large amounts of immature gE (Fig. 7).
To test the role of gE in the creation of release sites and the
were infected with ?gE, gE?CT, and gE?CT rescue mutants. A
low MOI of 0.3 was used to ensure infections arose from a single
at 10 hpi, a time at which each infected cell was producing virus
but second-generation infected cells were not yet doing so. In this
way, the effect of the gE deletions on the formation of infectious
virus could be determined separately from effects on cell-to-cell
spread. At 12 hpi, infected Vero cells were fixed with 4% PFA and
treated with ?-VP5 antibody (labeling major capsid protein) and
Alexa 488-conjugated secondary antibody. Samples were then
stained with rhodamine-WGA to label glycoprotein patches and
were measured as described in Materials and Methods for both
patch coverage and density of virions on the cell surface.
caused a reduction in the percentage of cell surface covered in
and B). However, while there was an inhibition in glycoprotein
patch formation, the number of virions on the cell surface of the
?gE mutants increased slightly (Fig. 8A and C) for unknown rea-
sons. There was no statistical difference in titers, indicating that
the expansion of glycoprotein patches at egress locations, but un-
infection in polarized and nonpolarized epithelial cells.
both highly differentiated and less differentiated epithelial cells.
While there has been some study of directed egress in polarized
membranes, and high TER), egress in nonpolarized cell types has
not been explored as extensively. Here, we report the results of a
study in which the release of HSV virions in nonpolarized Vero
cells was investigated. The major findings are summarized as fol-
lows. (i) HSV-1 egress in Vero cells is a directed process that oc-
curs at adherent surfaces. (ii) Both the membrane composition
and cytoskeletal structure of egress sites are modified during in-
fection. These modifications begin before virus is released, and
they proceed even when capsid trafficking is blocked. (iii) Traf-
microtubules and, to a lesser extent, actin. (iv) Actin determines
the structure of egress locations; when actin is depolymerized the
sites disperse. (v) Viral glycoprotein E aides in the formation of
egress sites. These findings are discussed in greater detail below.
Virus is directed to adherent surfaces rather than cell-cell
junctional proteins in Vero cells. The observation that the ma-
specific sites along cell-cell and cell-coverslip contact areas indi-
cates that the sorting of virus in nonpolarized cells is a directed
rather than random process. Previous reports have shown a ran-
dom, dispersed association of virions with the surface of HEp-2
nonpolarized cells at 17 hpi (21). We observed directed egress in
Vero cells between 8 and 15 hpi. By 17 hpi, Vero cells were
is possible that HEp-2 cells exhibit the directed sorting of virions
at earlier time points as well. Although virus titers in Vero cells
was observed to transfer to adjacent noninfected cells before 12
12-h time period thus gives the most information about viral
egress during cell-to-cell spread. Although it has been hypothe-
polarized epithelial cells is determined by the presence of cell-cell
junctional complexes (9), our results suggest that other elements
E-cadherin, and exogenously expressed nectin-1 does not traffic
to egress sites (our unpublished observations). The state of being
adherent to a substrate or cell may, therefore, be sufficient to di-
rect egress in some cell types, including Vero and HeLa cells. This
may be due to the directionality of the cytoskeleton or to cellular
proteins expressed along adherent surfaces.
Infection induces the creation of specialized egress sites. In
the course of infection, the adherent surfaces of Vero cells were
modified to create discernible viral egress sites. First, pocket-like
ible by both EM and TIRF microscopy. In contrast, the plasma
Second, the cell membrane at egress sites was extremely rich in
glycoproteins. Comparable concentrations were never observed
release sites with the depletion of microtubules, actin, and focal
Virological synapses form at areas of adhesion between infected
and noninfected cells. Viral and cellular proteins are recruited to
synapse sites, exocytosis is polarized toward these areas, and viri-
ons are released between the two cells (22). The observation that
HSV-1 egress sites formed on adherent surfaces of Vero cells is a
reasonable finding; in vivo the majority of adherent surfaces are
those in contact with adjacent cells. In addition, viral glycopro-
teins concentrated at egress sites, and the lack of patches on non-
FIG 7 Western blot analysis of gE expression in deletion and rescue mutants.
Cell lysates and supernatants of wild-type HSV- and gE mutant-infected Vero
transferred, and blotted with ?-gE antibody. The gE antibody used is against
that gE is expressed normally in the WT and rescue samples (gE?CT.R) but
not in the deletion mutant-infected samples. The arrowhead points to a non-
specific band. Truncated gE is indicated by a star.
HSV Release Sites
July 2012 Volume 86 Number 13 jvi.asm.org 7093
Mingo et al.
jvi.asm.org Journal of Virology
adherent surfaces, suggests that secretion was polarized toward
these structures. Future work will determine whether HSV-1
egress patches function as virological synapses and assist the cell-
to-cell spread of infection.
The cellular cytoskeleton assists egress in several possible
ways. Although viral release sites were depleted of cytoskeletal
elements, both microtubules and, to a lesser extent, actin were
found to be important for the movement of viral components to
egress locations. Microtubules are known to function in the long-
distance movement of secretory vesicles, while actin has been re-
ported to serve as both a barrier and an aide to secretion.
It is possible that actin surfing is playing a role in virus/glyco-
protein patch formation. Virus may be released elsewhere on the
to the sites where patches form. We observed that when actin
motility was inhibited with blebbistatin, patch size was decreased
In addition to assisting virus/glycoprotein trafficking, actin
was found to play a structural role in the maintenance of HSV
egress sites; when the actin cortex was depolymerized with cy-
tochalasin B, glycoprotein patches and associated virions dis-
persed across the cell surface (Fig. 5 and 6). The maintenance of
virions at the observed sites suggests an interaction with a protein
or lipid within the glycoprotein-rich areas. The putative protein
could be maintained at egress sites by actin in two ways. The viri-
on-interacting protein could be bounded by an actin fence (24,
36); that is, the protein or lipid could be unable to move past the
peripheral cortical actin and its membrane-associated proteins. It
is also possible that the protein is directly/indirectly bound to the
actin cytoskeleton. The depletion of actin we observed at release
sites makes the former a likely possibility, although there may be
low levels of actin at these sites that are not detectable by phalloi-
din. Future work should be able to characterize the proposed as-
sociation between virus and cell surface protein and what role it
plays in cell-to-cell spread.
Glycoprotein E is important for the formation of egress site
glycoprotein patches. gE has been reported to direct the traffick-
ing of progeny virions to cell junctions in epithelial cells and syn-
apses in neurons (4, 6, 21). Surprisingly, the deletion of gE in a
Vero cell infection did not decrease the amount of virus associat-
ing with bottom surface egress locations; in fact, the number was
increased. At this time, we do not have a theory for why this oc-
curs. The absence of gE did decrease the amount of glycoproteins
at these sites, resulting in much smaller egress patches. Therefore,
ized cell infections.
size was due to the loss of gE stabilization of the surface glycopro-
tein aggregations. Previous reports have indicated that gE inter-
acts with multiple glycoproteins and can stabilize these associa-
and gE?CT-infected cells (Fig. 8), it is not definitively more dis-
persed than it is in the rescue virus images. If the lack of gE did
result in the disruption of glycoprotein associations but this dis-
persal was not visible, then those dispersed glycoproteins would
think it unlikely that slightly disrupted (complete disruption
would lead to no patches) glycoprotein aggregations would be
endocytosed at the egress sites, and minimally disrupted aggrega-
tions would be unlikely to diffuse past the actin fence to areas
previous studies have not been able to observe a link between gE
and the cytoskeleton; when the cytoskeleton is isolated from de-
tergent-disrupted cells, gE remains in the soluble portion (9).
A small drop in patch size would be expected when a viral
glycoprotein is deleted. The absence of gE as the cause of the ob-
served decrease suggests that there is more gE in patches than the
other cellular and viral glycoproteins combined. There does not
appear to be more gE in egress patches than gD as shown by im-
munofluorescence (not shown); however, the difference may be
hidden by different antibody affinities.
containing vesicles to the cell surface. Previous works suggested
that viral glycoprotein-containing vesicles can traffic indepen-
dently to egress locations (9, 51, 52, 55, 65). In some HSV-1-
infected neurons, capsids are shipped down the axon in unenvel-
oped form (33, 52, 66), and glycoproteins are transported on
(28, 51, 60). This indicates that gE can direct the movement of
viral proteins by direct or indirect means. gE’s targeting of vesicle
transport may be limited to glycoproteins in nonpolarized cell
Hypothesized function of glycoprotein patches. There are
several roles that glycoprotein patches could play in an epithelial
cell infection. Viral glycoproteins concentrated in the cell mem-
brane may be able to recruit entry receptors on adjacent unin-
a target cell. gE in concert with gI is known to function as an Fc
receptor (12, 15, 19, 38), and the observed reduction in patch size
with the ?gE mutants suggests that there are large amounts of gE
protein in the patches. It is possible that gE at the egress sites can
function to protect progeny virus from attack from the humoral
patch membrane are able to assist with the fusion of the outgoing
virus-containing vesicle with the cell surface. Previous reports
have concluded that Vero cells infected with a ?gE mutant are
FIG 8 Effect of gE and gE cytoplasmic tail deletions on patch formation and progeny virion trafficking. (A) TIRF images of Vero cells infected with the ?gE
mutant, the gE?CT mutant, or the gE?CT rescue virus at an MOI of 0.3 for 12 h. Cells were fixed and treated with rhodamine-WGA and ?-VP5 antibody with
Alexa 488-conjugated secondary antibody. Note that patches are smaller in size in the gE deletion mutant-infected cells, but the virion number in those patches
on the total adherent membrane. Quantifications support the visual data. (D) Infected cells were harvested at 10 hpi, and the titer was determined by
on the adherent surface was increased. P values of deletion mutants compared to rescue virus are labeled the following: *, ?0.05; **, ?0.005; and ***, ?0.0005.
Bars represent standard errors of the means.
HSV Release Sites
July 2012 Volume 86 Number 13jvi.asm.org 7095
study it was found that the transfer of virus from infected cells to
Assuming the viral egress sites that form along cell-glass contact
surfaces function similarly to those that form between cells, the
adjacent cell; the buildup of cellular and/or viral proteins at these
locations may be necessary for cell-cell spread. Since free virus is
is likely not obligatory for glycoproteins on an infected cell to
induce the creation of a specialized entry site on an adjacent non-
infected cell. However, since the majority of progeny virions re-
for viral detachment. It is our hope that future studies in this area
will clarify the role that the recruited glycoproteins play in the
cell-to-cell spread of HSV-1 infection.
We are extremely grateful to Alan (Rick) Horwitz for the use of his TIRF
nical support. In addition, we acknowledge the procedural support pro-
vided by Stacey Guillot of the Advanced Microscopy Facility at the Uni-
versity of Virginia. We are indebted to Gary Cohen, Roselyn Eisenberg,
and Harvey Friedman for the kind gifts of their anti-HSV glycoprotein
antibodies. Likewise, we thank Fred Homa and Prashant Desai for pro-
viding HSV-1 mutants. Furthermore, we are grateful to Amy Bouton and
Keena Thomas for their generosity in advice and reagents. Lastly, we
thank Jesse Seamon for computer access and technical support.
Ulrike Lorenz, Tom Parsons, Dorothy Schafer, Judy White, and John
Wills for helpful advice on experimental design.
This work was supported by NIAID training grant AI007046, NIH
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