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, firstname.lastname@example.org.
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
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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