JOURNAL OF VIROLOGY, Sept. 2010, p. 8811–8820
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 17
Delaying the Expression of Herpes Simplex Virus Type 1 Glycoprotein
B (gB) to a True Late Gene Alters Neurovirulence and Inhibits the
gB-CD8?T-Cell Response in the Trigeminal Ganglion?
Srividya Ramachandran,1,2Katherine A. Davoli,2Michael B. Yee,2
Robert L. Hendricks,2,3,4and Paul R. Kinchington2,3*
Molecular Virology and Microbiology Graduate Program,1and Departments of Ophthalmology,2Molecular Microbiology and
Genetics,3and Immunology,4University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
Received 5 March 2010/Accepted 15 June 2010
Following herpes simplex virus type 1 (HSV-1) ocular infection of C57BL/6 mice, activated CD8?T cells
specific for an immunodominant epitope on HSV-1 glycoprotein B (gB-CD8 cells) establish a stable memory
population in HSV-1 latently infected trigeminal ganglia (TG), whereas non-HSV-specific CD8?T cells are lost
over time. The retention and activation of gB-CD8 cells appear to be influenced by persistent viral antigenic
exposure within the latently infected TG. We hypothesized that the low-level expression of gB from its native
promoter before viral DNA synthesis is critical for the retention and activation of gB-CD8 cells in the TG
during HSV-1 latency and for their ability to block HSV-1 reactivation from latency. To test this, we created
a recombinant HSV-1 in which gB is expressed only after viral DNA synthesis from the true late gC promoter
(gCp-gB). Despite minor growth differences compared to its rescuant in infected corneas, gCp-gB was signif-
icantly growth impaired in the TG and produced a reduced latent genome load. The gCp-gB- and rescuant-
infected mice mounted similar gB-CD8 effector responses, but the size and activation phenotypes of the
memory gB-CD8 cells were diminished in gCp-gB latently infected TG, suggesting that the stimulation of
gB-CD8 cells requires gB expression before viral DNA synthesis. Surprisingly, late gB expression did not
compromise the capacity of gB-CD8 cells to inhibit HSV-1 reactivation from latency in ex vivo TG cultures,
suggesting that gB-CD8 cells can block HSV-1 reactivation at a very late stage in the viral life cycle. These data
have implications for designing better immunogens for vaccines to prevent HSV-1 reactivation.
Herpes simplex virus type 1 (HSV-1) is a ubiquitous human
pathogen that is responsible for repeated corneal infections
that can induce blinding keratitis. The murine model of ocular
HSV-1 infection has elucidated the role of host immunity in
the establishment and maintenance of viral latency in trigem-
inal ganglia (TG). HSV-1 infection of a scarified mouse cornea
leads to a short-lived epithelial lesion caused by acute viral
replication in and destruction of corneal epithelial cells. Dur-
ing replication in culture, viral genes are expressed in a tightly
regulated temporal cascade characterized by the sequential
expression of immediate-early (IE) (?) genes and early (?)
genes before viral DNA synthesis. The late ? genes are maxi-
mally expressed after viral DNA replication and can be subdi-
vided into ?1 genes, which are expressed in small amounts
before viral DNA replication, and ?2 genes, which are abso-
lutely dependent on DNA replication for expression (7). Con-
firmation of the expression kinetics of HSV-1 genes in vivo has
proven to be difficult, as uniform infections cannot be estab-
lished. Previous studies seeking to address viral replication
kinetics in neurons have given rise to controversial conclusions
(31). However, our group has previously demonstrated that
during reactivation, the ?1 gene promoter of glycoprotein B
(gB) is active before the ?2 gene promoter of glycoprotein C
(gC), suggesting that the expression kinetics of the ?1 and ?2
genes are similar during both lytic replication and reactivation
(24). gB is a multifunctional structural glycoprotein that con-
tains an immunodominant epitope spanning amino acids 498
to 505 (gB498-505) recognized by a majority of CD8?T cells
(gB-CD8 cells) in C57BL/6 mice within 2 h of target cell in-
fection (9, 19, 35).
Replicating HSV-1 in the corneal epithelium accesses the
termini of interdigitating sensory neurons and travels via ret-
rograde axonal transport to the neuronal soma in the TG. The
viral genome is maintained in sensory neurons in a latent state
in which no infectious virus is produced. Latency is character-
ized by the repression of most viral lytic cycle genes and the
abundant expression of viral RNAs known as latency-associ-
ated transcripts (LATs) with no known protein products (11,
13). The repression of viral protein synthesis during latency has
led to the prevalent view of latency as being a quiescent and
antigenically silent infection that is ignored by host immunity.
However, very low levels of gene transcripts and proteins from
all kinetic classes have been detected in latently infected mu-
rine TG (4, 12). Furthermore, the findings of recent immuno-
logical studies of HSV-1 latency in mice are inconsistent with
the notion that latent virus is ignored by the host immune
system. CD8?T cells infiltrate the TG during acute HSV-1
infection, with peak accumulation occurring coincident with
the elimination of replicating virus and the establishment of
latency (9, 35). In C57BL/6 mice, gB-CD8 cells represent about
half of the CD8?T-cell infiltrate in the TG (9). Most if not all
* Corresponding author. Mailing address: Department of Ophthal-
mology, University of Pittsburgh School of Medicine, 1020 Eye and
Ear Building, 203 Lothrop Street, Pittsburgh, PA 15213. Phone: (412)
647-6319. Fax: (412) 647-5880. E-mail: email@example.com.
?Published ahead of print on 23 June 2010.
of the remaining CD8?T cells in infected TG appear to rec-
ognize as-yet-undefined HSV-1 proteins (27). The effector
CD8?T-cell population in the acutely infected TG undergoes
contraction as latency is established, giving rise to a small but
stable memory population with the same 50:50 ratio of gB-
specific to non-gB-specific cells (9).
In both mice and humans, CD8?T cells are found in the
HSV-1 latently infected TG in direct apposition to neurons (9,
10, 32). In humans, HSV-1-specific CD8?T cells surrounding
HSV-1 latently infected neurons demonstrate an activated and
effector memory phenotype (34). Using tetramers that bind to
the T-cell receptor of gB-CD8 cells in C57BL/6 mice, we have
demonstrated that these cells form an immunological synapse
with neurons in latently infected TG and even release lytic
granules into the synapse. Thus, CD8?T cells can detect and
respond to latent virus during immunosurveillance (9, 10). The
TG-resident gB-CD8 cell population exhibits a more activated
phenotype (CD69 and granzyme B expression) and is less
dependent on homeostatic proliferation signals than its coun-
terparts in noninfected tissue, such as the lungs and spleen
(27). gB-CD8 cells can employ the cytokine gamma interferon
(IFN-?) and lytic granules to prevent HSV-1 reactivation from
latency in vivo and in ex vivo TG cultures without inducing
neuronal apoptosis (3, 10).
The HSV-1 protein ICP47 inhibits CD8?T-cell recognition
of infected targets by impairing the transport of peptides into
the endoplasmic reticulum for loading onto major histocom-
patibility complex (MHC) class I molecules and transport to
the cell surface (1). ICP47 more efficiently inhibits human
transporter associated with antigen processing (TAP) trans-
porters than their mouse counterparts. This might lead to a less
efficient recognition of viral epitopes by CD8?T cells in hu-
man TG and contribute to the frequent HSV-1 reactivation
events that are observed for some humans but are absent or
very infrequent in mice. Indeed, HSV-1 strains that incorpo-
rate murine cytomegalovirus (CMV) immune evasion mole-
cules do show spontaneous reactivation in mice (21). Despite
more-efficient immune evasion in humans, latently infected
neurons in human TG are surrounded by contiguous CD8?T
cells that exhibit an activation phenotype similar to that seen in
mice (9, 34). In both humans and mice, stimuli known to
impair CD8?T-cell function (stress, immunosuppressive
drugs, and exposure to UV irradiation) lead to HSV-1 reacti-
vation from latency (5, 14, 25, 26, 28). These findings are
consistent with the notion that in both mouse and human TG,
CD8?T cells provide immunosurveillance of latently infected
neurons with low-level and likely intermittent T-cell receptor
(TCR) recognition of viral epitopes during partial reactivation
The importance of appropriate kinetic expression of HSV-1
genes for virulence and the generation of host immunity has
not been explored. Here we constructed a recombinant HSV-1
that expresses the ?1 gB gene as a ?2 gene, eliminating low-
level expression prior to viral DNA synthesis. We investigated
whether this kinetic change of gB expression would influence
HSV-1 virulence, the generation and homing of gB-CD8 cells
to the TG, and the ability of gB-CD8 cells to inhibit HSV-1
reactivation from latency.
MATERIALS AND METHODS
Cells and viruses. Vero cells (ATCC, Manassas, VA) were grown in Dulbec-
co’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine
serum (FBS), 5% Serum Plus (SAFC Biosciences, Lenexa, KS), penicillin G (100
units/ml), streptomycin (100 mg/ml), and amphotericin B (Fungizone) (250 mg/
ml). The RE strain of HSV-1 (18) was used as the basis for all studies.
Construction and analysis of recombinant HSV-1. The recombinant virus
containing gB under the late glycoprotein C (gC) promoter (HSV-1 gCp-gB) in
the HSV-1 RE background and its corresponding rescuant (HSV-rescue) were
constructed as shown in Fig. 1. The sequences of gB and its promoter were PCR
amplified from bp 54817 to 56640 in two sections: gB was amplified by using
primers gB-E (5?-AGCAAGCTTGTAGAAGCCGTCGACCTGCTTGAA [the
HindIII site is underlined]) and gB-S (5?-GAGGGATCCCCGCCATGCGCCA
GGGCGCC [the BamHI site is underlined]), and the promoter was amplified by
using primers 28-E (5?-CGGGATCCGCACGCTAGCTGGCGCATGGCGGG
ACTACGG [the NheI/BamHI sites are underlined]) and 28-S (5?-GAGAATT
CTGACGAAGCGGTCGTTGGCCAGCC [the EcoRI site is underlined]).
All DNAs were amplified from the RE genomic template by using Expand
proofreading polymerase (Roche) under hot-start conditions and in reaction
mixtures containing 5% dimethyl sulfoxide (DMSO). Each PCR fragment was
cut with the terminal engineered restriction sites and triple ligated into vector
pUC19 as a HindIII-BamHI-EcoRI fragment to generate plasmid pK1968, which
contained a unique BamHI and NheI site just upstream of the gB ATG start
codon. A portion (508 bp) of the region immediately upstream of the gC initi-
ating ATG (bp 96227 to 95820) containing the gC promoter (17) was similarly
PCR amplified by using primers gCp-S (5?-CGGGATCCGCCCGACGCCTCC
CCCTCGCGA [the BamHI site is underlined]) and gCp-B (5?-GCGCGCTAG
CAGATCTCTTAAGGCAGGTCATCAACCTCGGGTT [the Nhe-BglII-AflII
site is underlined]). The PCR product was then cut with BamHI and NheI
inserted into the corresponding sites engineered upstream of gB in pK1968. The
resulting construct was digested with NheI and AflII, and an NheI-AflII fragment
of pEGFP-C1 containing enhanced green fluorescent protein (EGFP) and its
polyadenylation signal were inserted to be upstream of the gC promoter and
downstream of and driven by the gB promoter. A plasmid derivative for the
rescuant was developed by PCR amplifying a portion of the gB gene and the
promoter/UL28 region using primers 54810F-EcoRI (5?-GGCCGAATTCGCG
CGCGTAGAAGCCGTCGACC [the EcoRI site is underlined]) and 55812R
AvrII (5?-GTCCTCCAGCACCTCGCCCCTAGGCTACCTGACG) and primer
55812F AvrII (5?-GTCAGGTAGCCTAGGGGCGAGGTGGAGGAC) with
56801R HindIII (GCCCAAGCTTACGACGGGGACCGTGTCGCCGT) (mu-
tations are in boldface type). Each PCR fragment was digested with the terminal-
end restriction sites, combined, and triple ligated into EcoRI- and HindIII-
digested pUC19. The single mutation resulted in the insertion of a silent
noncoding unique AvrII restriction site. All DNA inserts were sequenced for
HSV gCp-gB was derived by the cotransfection of infectious wild-type (WT)
HSV-1 RE viral DNA with plasmids linearized with SspI into Vero cells by using
Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). GFP-positive virus
plaques were picked and plaque purified to homogeneity, as detailed previously
(24). The rescuant (HSV-rescue virus) was derived by cotransfecting viral DNA
from the EGFP-positive gCp-gB virus with the promoter-gB plasmid containing
the novel AvrII site. Rescuant plaques were selected and picked based on the loss
of fluorescence followed by the subsequent titration of the mixed progeny from
the transfection (Fig. 1i to iii). The recombinants were positively identified for
inserted DNA sequences (gCp-gB) or for the inserted AvrII site (rescue virus) by
Southern blot analysis of restricted viral DNA. Viral DNAs from both viruses
were cut with NcoI, AvrII, and NcoI-AvrII double digests and probed with 5?
32P-radiolabeled oligonucleotides derived from the gB coding sequence or the
UL28 coding sequence (data not shown).
A recombinant with US3 deleted in the HSV-1 RE background was also
derived for this study. Sequences of part of the US3 protein were PCR amplified
from the HSV-1 RE genomic template by using the GC-Rich PCR system
(Roche) under hot-start conditions and primers US3.5F (5?-GGG AAT TCA
TGT ACG GAA ACC AGG ACT AC-3?) and US3.5R (5?-GGA AGA TCT
TCA TTT CTG TTG AAA CAG CGG CAA-3?). The PCR product was digested
with BglII and EcoRI and ligated into pEGFP-C2 digested with BamHI and
EcoRI. The collapse of this construct with XhoI resulted in the removal of the
N-terminal part of the US3 coding sequence encoding residues 1 to 169. The
remaining part lacks ATG, is out of frame with respect to EGFP, and lacks
domains required for kinase activity. DNA containing the promoter sequence
upstream of the US3 gene was PCR amplified by using primers US3PF (5?-GCG
CCC TAG GGC TAG CTC GCC GCA CCG TGA GTG CCA-3?) and US3PR
8812 RAMACHANDRAN ET AL.J. VIROL.
(5?-GCC ATT AAT ATT AAT GCC GCG AAC GGC GAT CAG AGG GTC
AGT-3?). This PCR product was digested with AvrII and AseI and used to
replace the CMV IE promoter. The resulting construct contained EGFP flanked
by the US3 promoter and the distal end of US3 (Fig. 1v). Viruses were selected
from cotransfections of HSV-1 RE viral DNA with the linearized plasmid and
were identified and plaque purified based on the gain of EGFP fluorescence. The
insertion of GFP and the deletion of the N termini of the US3 coding sequences
were confirmed by Southern blot analysis of viral DNA (data not shown).
A recombinant HSV-1 in which the gB protein is fused to EGFP was also used
for this study. HSV gB-EGFP (gB-EGFP) was generated on the RE backbone
(Fig 1iv). Briefly, a plasmid was developed in which the initiating methionine of
EGFP was placed in frame with the last residue of the gB coding sequence,
followed by the sequences immediately downstream of gB. A portion of the gB
sequence was PCR amplified with primers 5?-GACCGAATTCGACAACGTG
ATCGTCCAAAACTC and 5?-GCCGAGATCTTCACCCTAGGAGGTCGTC
CTCGGTCGG and cloned into pGEM-T Easy using TA cloning. The construct
was sequenced and verified for integrity. A portion of the sequences immediately
downstream of the gB stop codon containing the ORF26 coding sequences was
then generated by PCR using primers 5?-GCCG AGATCT GAC GCG GAC
GAC CTG TGA TG and ACGCAAGCTTGGACTACCCGTACTA digested
with HindIII and BglII and cloned downstream of the first gB fragment. The
resulting construct was then digested with BglII and AvrII, and an NheI-BglII
fragment derived from pEGFP C1 was inserted into EGFP in frame with the last
residue of the gB coding sequence. This plasmid was linearized and cotransfected
with infectious HSV-1 RE DNA, and recombinant viruses were picked and
purified based on the gain of EGFP fluorescence when visualized by blue UV
light. All viruses were carefully picked to reduce UV exposure to minimal levels.
DNAs were verified by Southern blotting, and the fusion of EGFP to gB was
determined by Western blot analyses (data not shown). Growth curves for Vero
cells established that this virus grew to levels deemed not significantly different
from those of wild-type RE (data not shown).
Analysis of HSV-1 protein expression. Subconfluent Vero cell monolayers
were infected with either HSV-1 RE, gCp-gB, or rescue virus at a multiplicity of
infection (MOI) of 10 PFU/cell for 1 h at room temperature (25°C) in medium
lacking or containing 350 ?g/ml phosphonoacetic acid (PAA; Lancaster Synthe-
sis, Pelham, NH). Following medium replacement under the same conditions,
the monolayers were incubated at 37°C, and cells were harvested at the indicated
time points. Cells were washed in ice-cold phosphate-buffered saline (PBS) and
then directly solubilized in sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE) buffer, boiled, and separated by SDS-PAGE. Proteins
were transferred onto Immobilon-P membranes (Millipore, Billerica, MA) for
immunoblot analyses. Antibodies to gB and gC (pooled monoclonal antibodies,
a kind gift of W. Goins, University of Pittsburgh) were detected by using horse-
radish peroxidase and West Dura chemiluminescent reagents (Pierce Biotech-
nology Inc., Rockford, IL).
Multistep in vitro growth kinetics. Vero cells were infected at an MOI of 0.01
PFU/cell and incubated for 4, 24, and 48 h. Cells and supernatants were har-
vested, and infectious virus that was released following three freeze-thaw cycles
was detected by a plaque assay on confluent Vero cells.
Mice and ocular infections. Three- to five-week-old female wild-type C57BL/6
mice (Jackson Laboratories, Bar Harbor, ME) were anesthetized by the intra-
peritoneal injection of 2.0 mg of ketamine hydrochloride and 0.04 mg of xylazine
(Phoenix Scientific, St. Joseph, MO) in 0.2 ml of Hanks buffered salt solution
(BioWhittaker, Walkersville, MD). Both corneas were scarified and infected
topically with HSV-1 viruses at the indicated PFU/eye in 3 ?l of RPMI medium
(BioWhittaker). All viruses were Percoll gradient purified as detailed previously
(24). All animal studies were carried out under University of Pittsburgh IACUC-
approved protocols and in accordance with the ethical treatment of animals, as
defined by the Association for Research in Vision and Ophthalmology for the
use of animals in ophthalmic and vision research.
Viral replication and spread. Viral replication in the cornea was determined
with tear film samples obtained by using a foam-tipped applicator (Kettenbach,
FIG. 1. Construction of HSV gCp-gB, HSV-rescue, and HSV-1 US3KO viruses. (i) Representation of the HSV-1 genome showing the
UL27/UL28 locus, which contains the gB promoter (arrow) in the UL28 coding sequences upstream of the gB ORF. The restriction sites used to
identify the insertion, as detailed in the text, are indicated on the gene locus at their approximate positions. (ii) Expansion of the corresponding
region in the recombinant HSV-1 gCp-gB virus showing that in place of its native promoter, gB is driven by the well-characterized ?2 gC promoter
in gCp-gB so that the protein is expressed only following DNA replication. The strategy required the maintenance of the gB promoter in the
genome, because it is concurrent with the upstream UL28 ORF encoding an essential terminase subunit. As such, HSV-1 gCp-gB had the gB
promoter driving EGFP followed by a polyadenylation motif to terminate RNA made from the gB promoter. (iii) Representative structure of the
HSV-rescue virus showing that gB expression is restored to its native promoter but that a unique noncoding AvrII site distinguishes it from the
parental strain. (iv) Representative structure of the HSV gB-EGFP virus showing that the gB protein is expressed as a fusion with EGFP at its
C terminus. (v) Representation of the genome showing the position of the US3 locus, which was modified such that the US3 promoter drives the
expression of EGFP, followed by an untranslated portion (amino acids 170 to 481) of the US3 ORF. If spurious transcription/translation allowed
the expression of the remaining part of the US3 reading frame, that part would not be kinase functional, since it lacks the critical ATP binding
domain and the first part of the catalytic domains.
VOL. 84, 2010 DELAYING EXPRESSION OF HSV-1 GLYCOPROTEIN B8813
Germany). The foam tips were transferred into DMEM and vortexed, and virus
was titrated on Vero cells by a standard plaque assay. To determine viral titers
in the ganglia, TG were surgically excised at 4, 5, and 8 days postinfection (p.i.)
from euthanized animals, homogenized, and subjected to three freeze-thaw
cycles prior to titration on Vero cells.
Flow cytometry. At the indicated times after infection, mice were euthanized
by exsanguination. TG were excised and resuspended in DMEM containing 10%
FBS and 400 U/ml collagenase type 1 (Sigma-Aldrich) per TG for 1 h at 37°C.
Cells were then dissociated into single-cell suspensions by trituration. Draining
lymph nodes (DLN) were excised, mechanically dispersed with the use of a nylon
filter (BD Pharmingen, San Diego, CA), and treated with red blood cell lysis
buffer (0.16 M NH4Cl, 0.17 M Tris in distilled water [dH2O] [pH 7.2]) prior to
staining. Single-cell TG or DLN suspensions were stained for flow cytometric
analysis as previously described (5). Fluorochrome-conjugated antibodies against
CD8? (clone 53-6.7) and CD45 (clone 30-F11) and the proper isotype control
antibodies were purchased from BD Pharmingen. Antibodies against granzyme
B (clone GB11) and the respective isotype controls were purchased from Caltag.
Phycoerythrin (PE)-conjugated H-2Kbdimers (BD Pharmingen) were incubated
with the SSIEFARL peptide at 37°C overnight prior to use to identify the
H-2Kb-restricted HSV-1 gB498-505-specific CD8?T-cell population.
Quantitative real-time PCR. The HSV-1 genome copy number in HSV-1-
infected TG was determined by quantitative real-time PCR as previously de-
scribed by using primers that recognize the sequences of the gH gene (5).
Ex vivo TG cultures. Latently infected TG suspensions (34 days p.i.) were
depleted of endogenous CD8? T cells by antibody/complement-mediated lysis
using Low-Tox M rabbit complement (Cedarlane). The efficiency of depletion
was assessed by flow cytometry. Single-cell TG suspensions were plated at one-
fifth TG equivalents per well in 48-well culture plates in 400 ?l of DMEM
containing 10% FBS, 10 mM HEPES buffer (Gibco), 10 U/ml recombinant
murine interleukin-2 (IL-2) (R&D Systems), and 50 ?M 2-mercaptoethanol.
Where indicated, cultures were supplemented with exogenous gB-CD8?T cells
at 2 ? 104CD8?T cells/well as determined previously (10). TG cultures were
monitored for reactivation by testing culture supernatant fluid for live virus by
standard viral plaque assays as previously described (16). Supernatants were
tested every 2 days for a total of 8 days in culture. Data are represented as the
percentage of wells that were positive for viral reactivation.
Construction and characterization of recombinant HSV-1.
We created HSV-1 gCp-gB, which exhibits gB expression with
true late kinetics and complete dependence on viral DNA
replication. The construction of the virus and its rescuant (res-
cue virus) is depicted in Fig. 1. In place of its native promoter,
gB is driven by a ?2 gC promoter in gCp-gB so that the protein
is expressed only following DNA replication. The strategy re-
quired the maintenance of the gB promoter in the genome,
because it is contiguous with the upstream UL28 essential open
reading frame (ORF) encoding a terminase subunit. As such,
HSV-1 gCp-gB was engineered so that the gB promoter drove
the expression of EGFP followed by a polyadenylation motif to
terminate RNA made from the gB promoter (Fig. 1ii). The
rescuant (rescue virus) engineered on the gCp-gB background
restored gB expression back to its native promoter and could
be differentiated from the parental virus (HSV-1 RE) by the
insertion of a silent and novel AvrII restriction site (Fig. 1iii).
An immunoblot analysis performed at 8 h p.i. revealed an
abundant expression of gB in cells infected with wild-type
HSV-1 RE (WT RE) and rescue virus but only trace expres-
sion in cells infected with gCp-gB virus (Fig. 2A). This was
similar to the low levels of the gC protein expressed by all the
viruses (Fig. 2B). By 12 h p.i., similar levels of gB expression
were detected in WT RE, rescue, and gCp-gB viruses. As
expected, the presence of the HSV-1 DNA replication inhibi-
tor phosphonoacetic acid (PAA) blocked the expression of gC
in all viruses and of the gB protein in gCp-gB (Fig. 2A and B,
lane 6) while permitting the low-level expression of gB from its
native promoter in WT RE and the rescue virus (Fig. 2A, lane
6). These data confirm that gB expression kinetics in gCp-gB
are regulated as a ?2 late gene, eliminating the low-level gB
protein expression prior to DNA replication observed when
expression is regulated by its native promoter.
Viral replication of gCp-gB is impaired in vivo but not in
culture. Following the infection of Vero cell monolayers at a
low multiplicity of infection (MOI of 0.01 PFU/cell), gCp-gB
exhibited a growth curve that was nearly identical to those of
the rescuant (rescue virus), demonstrating normal replication
efficiency for gCp-gB in vitro (Fig. 3A). However, the gCp-gB
virus showed some growth impairment in vivo in mice. Follow-
ing corneal infection of C57BL/6 mice with wild-type HSV-1,
viral titers peaked at 1 day p.i., followed by clearance by 7 days
p.i. (24). Here we observed a significant (approximately 2-fold)
decrease in tear film titers for gCP-gB compared to those for
its rescuant at 1 day p.i. (Fig. 3B), but titers equalized there-
after. By 7 days p.i., infectious virus could not be detected in
the tear film of mice infected with either virus, demonstrating
that the gCp-gB virus is cleared with normal kinetics in the
cornea. Virus replication was assessed in the TG at 4, 5, and 8
days p.i. based on previous findings of peak replication at 4
days p.i. and viral clearance and the uniform establishment of
latency by 8 days p.i. (24). The replication of the rescue virus
was comparable to that of our parent wild-type strain RE at 4
and 5 days p.i., and replicating virus of both strains was cleared
by 8 days p.i. (Fig. 3C). Having established similar replication
kinetics of wild-type and rescue viruses in the cornea and TG,
only rescue virus was used in subsequent experiments as the
appropriate control. The replication of gCp-gB was detectable
FIG. 2. HSV gCp-gB expresses gB with true late kinetics. Conflu-
ent monolayers of Vero cells were infected with HSV-1 RE, gCp-gB,
or rescuant at an MOI of 10 with or without the addition of 350 ?g/ml
of phosphonoacetic acid (PAA). Total SDS-PAGE-separated proteins
were analyzed by immunoblotting for gB (A) or gC (B) using pools of
monoclonal antibodies. The times of harvest are shown above each
figure and lane designation in hours. The top of each figure represents
infection with HSV-1 RE, the middle represents infection with HSV-
rescue virus, and the bottom represents infection with HSV gCp-gB.
Only the regions corresponding to the main glycoprotein products are
8814 RAMACHANDRAN ET AL.J. VIROL.
but severely impaired at 4 and 5 days p.i. compared to those of
the rescue and wild-type viruses and was also completely
cleared by 8 days p.i. (Fig. 3C). Thus, HSV-1 virulence is
compromised in the cornea and more profoundly in the TG
when gB expression prior to viral DNA replication is pre-
HSV-1 gCp-gB establishes a reduced latent viral load in the
TG. We next determined if gCp-gB induced a reduced latent
viral load in the TG, as shown previously for other HSV-1
strains that exhibited impaired replication in the TG (9). Cor-
neal infection of mice with 1 ? 105PFU/eye of gCp-gB indeed
resulted in a reduced latent viral load in the TG compared to
its rescuant when assessed at 14, 34, and 64 days p.i. (Fig. 4).
Unexpectedly, we observed an increase in viral genome copy
numbers in rescue virus-infected mice from 14 to 34 days p.i.
The cause of this increase is currently unknown and has not
previously been observed for wild-type HSV-1 RE. However,
we were able to equalize the latent loads of gCp-gB and its
rescuant by reducing the infectious dose of the rescue virus by
3-fold (Fig. 4).
Expansion of gB498-505-specific CD8?T cells in the lymph
nodes and their accumulation and retention in infected TG.
We determined if the altered kinetics of expression of gB
would influence the initial expansion of gB-CD8 cells in the
draining lymph nodes following corneal infection. At 8 days
p.i., a similar total number (not shown) and a similar frequency
of gB-CD8 cells (Fig. 5A) were observed for lymph nodes of
mice infected with gCp-gB and the rescuant, indicating that
delayed gB expression did not impair the priming of naïve
CD8?T cells. The total CD8?T-cell population in TG in-
fected with both gCp-gB and rescue virus exhibited the ex-
pected expansion (8 days p.i.), contraction (8 to 30 days p.i.),
and stable memory (?30 days p.i.), but the size of the CD8?
T-cell population in gCp-gB-infected TG was significantly re-
duced at all times tested (Fig. 5B). The frequency of gB-CD8
FIG. 3. Viral replication titers are reduced in corneal tear films and
TG of gCp-gB-infected mice but not in culture. (A) Vero cell mono-
layers were infected at an MOI of 0.01 with gCp-gB or rescue viruses.
Cells and supernatants were collected at the designated hours p.i. and
subjected to three freeze-thaw cycles, and PFU/ml of HSV-1 were
measured on Vero cells. The difference between the viral titers of
gCp-gB and rescue was not significantly different at any time tested
(P ? 0.01). The experiment was repeated two independent times, with
similar results. (B) Mice were infected with rescue or gCp-gB virus at
1 ? 105PFU/eye. Eye swabs were performed at the indicated days p.i.,
and the titers of HSV-1 were determined on Vero cells. The viral titers
are shown as means ? standard errors of the means (SEM). An
asterisk indicates that titers were significantly different as assessed by
a Student’s t test (P ? 0.05). (C) Mice were infected with RE, rescue,
or gCp-gB virus at 1 ? 105PFU/eye. Infected TG were excised, ho-
mogenized, and subjected to three freeze-thaw cycles, and HSV-1
titers were determined on Vero cells. Each data point represents the
mean viral titer from a single TG as determined by plaque assay. The
data shown are combined data from two independent experiments.
The significance of differences in TG titers was assessed by a Student’s
t test (??, P ? 0.0008; ?, P ? 0.0573). At 8 days p.i., no infectious virus
could be detected from TG infected with either virus. ns, not signifi-
FIG. 4. HSV gCp-gB establishes latency with fewer genome copies
than the rescue virus. Corneas of mice were infected at an infectious
dose of 1 ? 105or 3 ? 104PFU/eye. TG were excised at 14, 34, and 64
days p.i., and genome copy numbers were determined by real-time
PCR. Each data point represents the viral genome copy numbers from
a single TG. The data shown are combined data from two independent
experiments. At a similar infectious dose (1 ? 105PFU), the rescue
virus induced a significantly higher (P ? 0.05) latent viral load than the
gCp-gB virus at all times tested. Reducing the infectious dose of rescue
virus 3-fold relative to that of gCp-gB virus resulted in latent viral loads
that were not significantly different (P ? 0.05). Data were analyzed by
a Student’s t test.
VOL. 84, 2010DELAYING EXPRESSION OF HSV-1 GLYCOPROTEIN B 8815
cells in TG latently infected (34 and 64 days p.i.) with rescue
virus was approximately 50% (Fig. 5B), as previously reported
for TG latently infected with wild-type RE (9). Interestingly,
the delayed expression of gB by gCp-gB was associated with a
dramatically reduced frequency of gB-CD8 cells in both acutely
and latently infected TG (Fig. 5B). The reduced frequency of
gB-CD8 cells in gCp-gB-infected TG was not related to the latent
genome copy number, since infection with a reduced infectious
dose of rescue virus dramatically reduced the latent genome copy
number at 34 days p.i. (Fig. 4) but did not alter the frequency of
gB-CD8 cells in the TG (data not shown).
Late expression of gB contributes to the diminished gB-CD8
cell population in gCp-gB latently infected TG. To determine
if the altered gB-CD8 cell frequency in gCp-gB-infected TG
during latency resulted from impaired virus replication during
acute infection, we compared the absolute numbers and fre-
quencies of gB-CD8 in TG that were latently infected with
gCp-gB or two other recombinant HSV-1 strains with similarly
impaired TG replication. The first virus contains a disruption
of the US3 kinase gene (US3KO) (Fig. 1v), and the second
virus expresses a gB protein fused to EGFP (gB-EGFP) (Fig.
1iv). Both US3KO and gB-EGFP viruses express gB under the
control of its native promoter, and kinetic studies revealed that
both viruses expressed gB at low levels prior to viral DNA
synthesis and in the presence of DNA replication inhibitors, as
seen for RE (data not shown). The gCp-gB, US3KO, and
gB-EGFP viruses induced similar loads of latent virus in the
TG at 34 days p.i. (Fig. 6A) and similar overall CD8?T-cell
infiltrates into the TG at 34 days p.i. (Fig. 6B). Thus, the
reduced sizes of the CD8?T-cell populations in TG latently
infected with gCp-gB, US3KO, and gB-EGFP relative to that
observed for TG latently infected with rescue virus (Fig. 5B)
appear to reflect the reduced load of latent virus. Importantly,
TG harboring the latent US3KO or gB-EGFP virus exhibited a
significantly higher frequency of gB-CD8 cells (33% and 37%,
respectively) than did those harboring gCp-gB (?20%) (Fig.
6), suggesting that delayed gB expression and not the latent
viral load resulted in the reduced frequency of gB-CD8 cells in
gCp-gB latently infected TG.
gCp-gB fails to activate gB-CD8 cells during latency. We
previously demonstrated that non-HSV-specific CD8?T cells
are lost from HSV-1 latently infected TG during latency and
that gB-CD8 cells form an immunological synapse with neu-
rons within latently infected TG and maintain an activation
phenotype, including granzyme B expression (9,10, 27). These
observations suggest that antigenic exposure maintains gB-
CD8 cells in an activated state during latency. The reduced
frequency of gB-CD8 cells in TG harboring latent gCp-gB
suggested reduced antigenic exposure, and we hypothesized
that this would also result in reduced activation, as assessed by
intracellular granzyme B expression. Indeed, the absolute
number (Fig. 5C) and frequency (not shown) of granzyme
B-positive gB-CD8 cells were significantly reduced in gCp-gB-
infected TG at 34 days p.i.
gB-CD8 cells are capable of blocking reactivation of gCp-gB.
The early expression of gB before viral DNA synthesis might
provide a requisite window of opportunity for gB-CD8 cells to
prevent full reactivation and virion formation. However, it was
of interest to ask if gB-CD8 cells are still able to block reacti-
vation if they encounter a neuron late in reactivation after
DNA synthesis is initiated. To address this, cultures of dis-
persed TG harboring similar loads of latent gCp-gB or rescu-
ant virus were depleted of endogenous CD8?T cells, recon-
stituted or not with purified memory gB-CD8 cells that were
previously expanded from latently infected TG (10), and mon-
itored for HSV-1 reactivation based on the recovery of infec-
tious virus from culture supernatants. Figure 7A verifies that
the TG cell suspensions were effectively depleted of endoge-
nous CD8?T cells. In the absence of added gB-CD8?T cells,
reactivation was observed in neurons harboring both gCp-gB
and the rescuant, although the reactivation frequency was
slightly lower in neurons harboring gCp-gB (Fig. 6B). Notably,
late gB expression did not impair the ability of the gB-CD8?T
cells to block reactivation. Thus, gB-CD8 cells can act very late
FIG. 5. HSV gCp-gB-infected mice contain fewer gB498-505-specific
CD8?T cells in their TG but not lymph nodes, and fewer gB498-505-
specific CD8?T cells in the TG of gCp-gB-infected mice are activated.
TG or lymph nodes were excised; dispersed into single-cell suspen-
sions; stained for CD45, CD8, gB498-505T-cell receptor or intracellular
granzyme B expression; and analyzed by flow cytometry. The data are
represented as the means ? SEM. (A) Graph representing the per-
centage of gB-CD8?T cells in draining lymph nodes (DLN) of mice
infected with 1 ? 105PFU/eye of either rescue or gCp-gB virus at 8
days p.i. The data shown are combined data from two independent
experiments. The mean percentages of gB-CD8?T cells are not sig-
nificantly different between rescue and gCp-gB viruses as assessed by a
Student’s t test (P ? 0.05). (B) Total number of CD8?T cells (left) and
percentage of gB-CD8?T cells (right) per TG from mice infected with
1 ? 105PFU/eye HSV. The mean for the percentage of gB-CD8?T
cells is shown within each bar graph. The data are combined data from
three independent experiments. The mean percentages of total and
gB498-505-specific CD8?T cells between rescue and gCp-gB viruses are
significantly different at all time points tested as assessed by a Student’s
t test (P ? 0.05). (C) Graph representing the absolute number of
gB-CD8?T cells in infected TG expressing intracellular granzyme B at
34 days p.i. from mice infected with 3 ? 104PFU/eye rescue virus and
1 ? 105PFU/eye gCp-gB virus to yield equal genome loads. The data
are combined data from two independent experiments. The differences
between the absolute numbers are significantly different as assessed by
a Student’s t test (P ? 0.0016).
8816 RAMACHANDRAN ET AL.J. VIROL.
in the viral life cycle to block the full reactivation and forma-
tion of infectious virus.
We investigated the influence of gB gene expression kinetics
on HSV-1 virulence and targeting by the host immune system.
To test this, we created a recombinant virus (gCp-gB) that
expresses gB as a late protein from the ?2-regulated gC pro-
moter, thus abrogating the expression of the gB protein prior
to DNA synthesis. This should not influence the level of gB
expression since gB and gC are expressed at similar levels at
late times postinfection (29). We observed that gCp-gB exhib-
ited reduced virulence in the mouse cornea and particularly in
the TG. HSV-1 gB is required for viral entry into cells by
mediating the fusion of the viral envelope to the host cell
membrane (6, 33). However, several recent studies have dem-
FIG. 6. Delaying gB contributes to the diminished gB-CD8?T-cell response in the TG. Mice were infected at 1 ? 105PFU/eye with gCp-gB
or two other TG replication-impaired HSV-1 strains, one lacking the US3 kinase (US3KO) and the other expressing gB as a fusion protein with
EGFP (gB-EGFP). The data shown are combined data from two independent experiments. (A) Viral genome copy numbers were determined by
real-time PCR at 34 days p.i. The data are represented as the means ? SEM. The differences between gCp-gB and US3KO genome copy numbers
and gCp-gB and gB-EGFP genome copy numbers are not significantly different as assessed by a Student’s t test (P ? 0.05). (B and C) Single-cell
suspensions of TG infected for 34 days were stained for CD45, CD8, and gB498-505TCR expression and analyzed by flow cytometry. The data are
presented as the means ? SEM. (B) The total number of CD8?T cells retained in TG of mice infected with gCp-gB or US3KO and gCp-gB or
gB-EGFP is not significantly different as assessed by a Student’s t test (P ? 0/05). (C) The percentage of gB498-505-specific CD8?T cells is
significantly different between gCp-gB and US3KO (P ? 0.0013) and gCp-gB and gB-EGFP (P ? 0.0003) viruses at all time points tested as assessed
by a Student’s t test.
FIG. 7. gB498-505-specific CD8 T cells can block gCp-gB reactivation. Mice were infected with 3 ? 104PFU/eye rescue virus and 1 ? 105
PFU/eye gCp-gB virus to establish equal numbers of genome copies during latency. TG were excised at 34 days p.i., and single-cell suspensions
were depleted of CD8 as described in Materials and Methods. Depleted TG were plated as one-fifth TG cultures, half of the cultures received an
add-back of gB498-505-specific CD8 T cells (gB-CD8 T cells), and the other half did not. Reactivation was monitored by sampling supernatants for
infectious virus via plaque assay. (A) Representative dot plots before and after CD8 depletion, showing depletion efficacy. (B) Representative
graph showing reactivation frequencies of rescue and gCp-gB viruses with or without gB-CD8 cells added back. For both viruses, the difference
in reactivation frequencies between cultures with gB-CD8 cells added back and without CD8?T cells is statistically significant (*, P ? 0.0114;**,
P ? 0.0017), as assessed by a survival curve analysis (log rank test). The experiment was repeated three independent times, with similar results.
VOL. 84, 2010 DELAYING EXPRESSION OF HSV-1 GLYCOPROTEIN B8817
onstrated other functions for gB during the viral life cycle, and
it is possible that delaying the expression of gB might impair
replication by modulating one or more of these functions. For
instance, a delay of gB expression could delay gB binding to the
cellular stress sensor PKR-like endoplasmic reticulum kinase
(PERK) and the resulting augmentation of viral protein trans-
lation (20). In our studies we observed some growth impair-
ment of the gCp-gB virus in the cornea, consistent with our
recent observation of gCp-gB virus growth impairment and
increased phosphorylation of the alpha subunit of eukaryotic
initiation factor 2 (eIF2?) in primary corneal fibroblasts (our
unpublished observations) but not in Vero cells in vitro. More
severe gCp-gB growth impairment was observed in the TG,
suggesting that the activity on this pathway might be particu-
larly important for TG neurons. The transport of gCp-gB cap-
sids from the cornea to the nerve body might also be hindered,
since the phosphorylation of gB is needed for the efficient
egress of nucleocapsids from the inner nuclear membrane (36).
HSV-1 gB is also a highly glycosylated protein, and delayed
expression kinetics could also contribute to altered glycosyla-
tion patterns, which could negatively affect protein function.
Our findings emphasize the selective importance of the kinet-
ics of gB expression for viral growth in certain cell types. The
mechanisms underlying the growth impairment of gCp-gB are
currently under investigation.
To our knowledge this is the first study to assess the influ-
ence of viral gene expression kinetics on host immunity. The
expansion of naïve HSV-specific CD8?T cells in the lymphoid
organs appears to result from a cross-presentation of viral
antigens rather than by direct presentation by infected antigen-
presenting cells (2). Accordingly, any of the kinetic classes of
HSV-1 proteins could theoretically be presented, and a de-
layed expression of gB should not greatly influence the initial
expansion of gB-CD8?T cells in mice infected with gCp-gB.
This was in fact observed, as quantitatively similar expansions
of gB-CD8 cells were observed for draining lymph nodes of
mice infected with gCp-gB and rescuant viruses. Thus, the
immunogenic properties of gB are not influenced by altering
the kinetics of expression. In contrast, a delay in gB expression
kinetics could impact gB-CD8 cell cognate recognition of gB
epitopes in latently infected neurons and their capacity to
prevent full reactivation and virion formation. The sequential
expression of HSV-1 genes during lytic infection has been
appreciated for some time, but little is known about the im-
portance of expression kinetics for the targeting of viral pro-
teins by host immunity. By use of classical molecular ap-
proaches, gB was shown previously to be produced early in the
viral life cycle before viral DNA replication, leading to its
classification as a leaky late ?1 gene (22, 23). Very low levels of
protein synthesis are required to sensitize cells for recognition
by CD8?T cells (30). Accordingly, while gB is hardly detect-
able by immunoblotting at very early stages of infection, Muel-
ler and colleagues showed previously that de novo gB synthesis
can be detected by gB-CD8 cells as early as 2 h postinfection
HSV-1 latency differs from chronic infections in that viral
DNA replication does not take place and viral protein expres-
sion is largely silenced. However, HSV-1 latency is no longer
considered an entirely antigenically silent state. The recogni-
tion of latent virus by CD8?T cells was demonstrated by the
observations that HSV-specific CD8?T cells surround neu-
rons in latently infected murine TG, form immunological syn-
apses with the neurons, and release lytic granules into the
synapse in situ and that HSV-specific CD8?T cells can block
HSV-1 reactivation from latency in dispersed TG cultures
(9,10). Moreover, non-HSV-specific CD8?T cells that enter
the TG during acute infection are lost during latency, suggest-
ing that an antigenic encounter is required to maintain the
CD8?T-cell population in the TG (27). Presumably, these
antigenic encounters occur during partial reactivation events
that are terminated by CD8?T-cell effector functions, includ-
ing IFN-? and lytic granule release (3, 10, 15). The fact that the
HSV-1 latent genome copy number remains constant during
latency and that CD8?T cells can terminate reactivation prior
to the expression of the true late gC gene in TG cultures (16)
suggests that gB-specific CD8?T cells recognize their epitope
on neurons early in the viral life cycle prior to viral DNA
synthesis. This raises the question of whether gB expression
prior to HSV-1 DNA synthesis is required for the retention of
gB-CD8 cells in latently infected TG and for their ability to
block HSV-1 reactivation from latency.
One of the most striking observations of our study was that
the frequency of gB-CD8 cells was diminished over time in TG
harboring latent gCp-gB. These findings suggest a lack of an-
tigenic exposure to gB-CD8 when gB is expressed as a true late
gene in gCp-gB. We conclude that reduced antigenic exposure
due to the altered kinetics of gB expression and not the re-
duced latent genome copy number in gCp-gB-infected TG is
responsible for the loss of gB-CD8 based on four observations.
First, reducing the infectious dose of the rescue virus 3-fold
caused a corresponding approximately 3-fold reduction in the
latent genome copy number in the TG but did not alter the
frequency of gB-CD8 cells (data not shown). Second, although
the gCp-gB, US3KO, and gB-EGFP viruses all induced com-
parable latent genome copy numbers in the TG, the gB-CD8
cell frequency was significantly lower in the TG harboring
gCp-gB. Third, the fact that gB-specific CD8?T cells are
selectively lost from the TG argues against the possibility that
delaying gB expression simply reduces the levels of inflamma-
tory mediators such as chemokines and cytokines, which would
not be expected to result in the selective loss of CD8?T cells
of a particular specificity. Fourth, the activation of gB-CD8
cells, as indicated by intracellular granzyme B levels, was dra-
matically reduced in TG harboring latent gCp-gB compared to
those harboring rescuant virus, even when the load of latent
virus was equalized. We are currently investigating whether the
loss of gB-CD8 cells from the TG and the concomitant loss of
granzyme B expression within gB-CD8 cells in TG harboring
gCp-gB correspond to lower levels or an absence of gB tran-
scripts within latently infected TG.
We have previously shown that gene expression kinetics are
similar during reactivation and lytic replication, at least with
respect to the ?1 (gB) and ?2 (gC) genes (24). In a wild-type
virus, gB is expressed relatively early, so it seems obvious to
surmise that gB-CD8 cells can recognize their viral antigen
early and shut down reactivation before DNA replication. We
hypothesized that once DNA replication takes place, it is too
late in the viral life cycle for CD8?T cells to block reactivation,
as the virus has already committed to assembly and egress.
However, our findings establish that gB-CD8 cells can indeed
8818 RAMACHANDRAN ET AL. J. VIROL.
block reactivation even after viral DNA replication. This find-
ing suggests that even if a gB-CD8 cell encounters a neuron
late in the reactivation process, it will still be able to block full
reactivation and virion formation. This is consistent with the
ability of the CD8?T-cell effector molecule IFN-? to block
reactivation even after late gene expression (3). CD8?T cells
can employ lytic granules and IFN-? to block the reactivation
of wild-type HSV-1 from latency without neuronal destruction
(10). It remains to be determined if the effector mechanism(s)
employed by gB-CD8 cells to block reactivation when gB is ex-
pressed only after viral DNA synthesis is compatible with neuro-
nal preservation. The fact that the CD8?T cells in infected TG
that are not specific for the immunodominant gB498-505epitope
appear to be HSV specific implies that the reduced frequency
of gB-CD8 cells in TG harboring gCp-gB is associated with an
increased frequency of HSV-specific CD8?T cells reactive to
subdominant epitopes or to new epitopes that arise in these
mice. Clarification of this issue will await the identification of
the subdominant epitopes recognized by these cells.
The data from this study can be used to design better ther-
apeutic vaccines for HSV-1. While CD8?T cells responding to
late antigens are still able to block reactivation, they are not
retained in high numbers in the TG during latency. This makes
late viral antigens a poor choice as the sole immunogen. There
is an obvious utility to targeting viral proteins expressed before
DNA replication. A block in reactivation after viral DNA syn-
thesis would permit an accumulation of viral genomes in neu-
rons, which is associated with a greater likelihood of reactiva-
tion (8). A study reported previously by Hoshino and
colleagues demonstrated that the rate of HSV-1 reactivation is
proportional to the number of latent genomes and inversely
proportional to the number of CD8?T cells retained in the
TG. Those researchers therefore suggested that vaccines
should be evaluated for their ability to induce and maintain a
virus-specific CD8?T-cell response as well as a low genome
load in the ganglia (8). Our findings suggest that immunization
against viral ?, ?, and ?1 proteins that are expressed before
DNA replication would optimize the capacity of the immune
system to maintain HSV-1 in a latent state and prevent recur-
This work was supported by Public Health Service grants EY015291
(P.R.K.) and EY05945 (R.L.H.), a core grant from the NEI
(EY08098), and unrestricted funds from Research To Prevent Blind-
ness, Inc., and the Eye & Ear Foundation of Pittsburgh.
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