JOURNAL OF VIROLOGY, Apr. 2007, p. 3731–3739
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 8
Sialic Acid on Herpes Simplex Virus Type 1 Envelope Glycoproteins
Is Required for Efficient Infection of Cells?
Jeremy R. Teuton1and Curtis R. Brandt1,2,3*
Program in Cell and Molecular Biology,1Department of Medical Microbiology and Immunology,2and Department of
Ophthalmology and Visual Sciences,3University of Wisconsin–Madison, Madison, Wisconsin 53706
Received 13 October 2006/Accepted 8 January 2007
Herpes simplex virus type 1 (HSV-1) envelope proteins are posttranslationally modified by the addition of
sialic acids to the termini of the glycan side chains. Although gC, gD, and gH are sialylated, it is not known
whether sialic acids on these envelope proteins are functionally important. Digestion of sucrose gradient
purified virions for 4 h with neuraminidases that remove both ?2,3 and ?2,6 linked sialic acids reduced titers
by 1,000-fold. Digestion with a ?2,3-specific neuraminidase had no effect, suggesting that ?2,6-linked sialic
acids are required for infection. Lectins specific for either ?2,3 or ?2,6 linkages blocked attachment and
infection to the same extent. In addition, the mobility of gH, gB, and gD in sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis gels was altered by digestion with either ?2,3 specific neuraminidase or nonspecific
neuraminidases, indicating the presence of both linkages on these proteins. The infectivity of a gC-1-null virus,
?gC2-3, was reduced to the same extent as wild-type virus after neuraminidase digestion, and attachment was
not altered. Neuraminidase digestion of virions resulted in reduced VP16 translocation to the nucleus,
suggesting that the block occurred between attachment and entry. These results show for the first time that
sialic acids on HSV-1 virions play an important role in infection and suggest that targeting virion sialic acids
may be a valid antiviral drug development strategy.
Herpes simplex virus type 1 (HSV-1) is a widespread human
pathogen with 70 to 90% of the adults in the United States
testing seropositive for the virus (92). The most common man-
ifestation is mucous membrane infection resulting in ulcerative
lesions that are usually self-limiting in immunocompetent in-
dividuals. However, serious illnesses, including lethal neonatal
HSV, encephalitis, and blinding keratitis, can occur (43, 49, 83,
92). Primary and recurrent infections in the immunocompro-
mised, such as transplant recipients, those on chemotherapy,
or those infected with human immunodeficiency virus (HIV)
can be life-threatening (64, 80, 94). A number of antivirals are
approved for HSV-1 treatment (21, 39, 60), but they are not
completely effective. One significant problem in dealing with
HSV infections is the ability of the virus to persist in the host
as a latent infection (65). None of the currently available an-
tivirals can eliminate a latent infection. Preventing the estab-
lishment of a persistent infection, which could be accomplished
either by blocking infection or the establishment of latency,
would be an ideal strategy for dealing with this virus.
The development of agents to block HSV infection requires
a greater understanding of HSV entry. Infection is initiated by
the binding of viral glycoprotein C (gC) or gB to cell surface
heparan sulfate proteoglycans (37, 73, 74). After attachment,
gD can bind to any of several cellular receptors including
herpes virus entry mediator, nectin-1, nectin-2, or 3-O-sulfated
heparan sulfate (17, 30, 54, 70, 84), triggering a conformational
change in gD (18, 28, 48). The conformational change in gD is
thought to be required for the assembly of the entry-fusion
complex which consists of gD, gB, and the gH-gL heterodimer.
Recent evidence suggests that gB is recruited to the complex
first, followed by gH-gL (32, 62). The gB protein functions as
a trimer and appears to undergo a conformational change
during entry but lacks features characteristic of a number of
viral fusion proteins (35). The gH protein contains se-
quences similar to known fusion proteins, including a pep-
tide fusion loop and two heptad repeats, suggesting that gH
may be the actual fusion protein (31). HSV-1 gB reportedly
binds to cell surface receptors, but the identity of these
receptors is unknown, and their significance for fusion and
entry is not clear (10). HSV-1 gH has also been reported to
bind to a cell surface receptor, ?v?3integrin, but the signif-
icance of this binding is unknown (59, 67). It is clear that
more needs to be learned about the function of gD, gB, and
gH in fusion and entry.
Viral envelope glycoproteins are synthesized and processed
through the cellular exocytic pathway and modified through
glycosylation by host cell enzymes. These modifications include
the addition of sialic acid residues in the trans-Golgi compart-
ment. The predominant terminal carbohydrate on glycans in
mammalian cells are ?2,3- or ?2,6-linked sialic acids. Sialic
acids have a stabilizing effect on glycoproteins and enzymatic
desialylation often results in significant changes in the struc-
ture and function of these proteins (13, 27, 40, 46, 47, 58, 82).
Previous studies have shown that gC, gD, and gH are sialylated
but the specific linkages and possible functions of sialic acids
on these proteins have not been determined (11, 22, 25, 51, 61,
68). The goal of the present study was to determine whether
sialylation of HSV-1 envelope proteins is important for infec-
tivity. The data suggest that ?2,6-linked sialic acids on one or
more HSV entry proteins are required for viral entry into cells.
* Corresponding author. Mailing address: Department of Ophthal-
mology and Visual Sciences, University of Wisconsin School of Med-
icine and Public Health, 6630 Medical Sciences Center, 1300 Univer-
sity Avenue, Madison, WI 53706. Phone: (608) 262-8054. Fax: (608)
262-0479. E-mail: email@example.com.
?Published ahead of print on 17 January 2007.
MATERIALS AND METHODS
Cell culture and viruses. All studies were carried out in Vero cells (ATCC
CLL-81) or Hep-2 cells (ATCC CCL-23) cultured in Dulbecco modified Eagle
medium supplemented with 5% calf serum and 5% fetal bovine serum (34).
Experiments requiring prolonged incubations at 4°C were performed with cells
on poly-L-lysine (P4707; Sigma-Aldrich, Inc., St. Louis, MO)-coated plates. Mi-
croscopic examination was used to confirm the presence of stable cell layers
throughout each experiment. For some experiments the growth medium was
buffered with 25 mM HEPES (pH 7.3) in place of carbonate.
HSV-1 KOS and a ?-galactosidase-expressing variant, hrR3, were used for the
majority of the studies (33, 34). For studies involving the role of gC, ?gC2-3, a
mutant virus expressing ?-galactosidase in place of gC, and ?gC2-3rev, a rescued
virus, were used (38, 87, 88). High-titer viral stocks were produced in Vero or
Hep-2 cells as described previously (34). Purification of virions was carried out
with sucrose gradients as we described previously (87) with minor modifications
(50). The titers of viral stocks were determined by plaque assay on Vero cells.
Enzymatic digestion of virions and cells. The carbohydrate-digesting enzymes
used in these studies and their linkage specificities are shown in Table 1. The
?2,3 specific neuraminidase (NEB 2-3) has a 260-fold preference for ?2,3 com-
pared to ?2,6 linkages (42). The Vibrio cholerae and Arthrobacter ureafaciens
enzymes digest both linkages with perhaps a slight preference for ?2,6 linkages
(1, 85, 86). For digestion, high-titer viral stocks were diluted into identical final
volumes in 1? enzyme buffer as specified by the manufacturer. The amount of
neuraminidase used was 0.01 to 0.04 U for V. cholerae, 0.1 to 0.4 U for A.
ureafaciens, and 500 to 2,000 U for NEB 2-3 as determined by the suppliers. Each
digestion contained 2 ? 106to 2 ? 108PFU in 200 to 600 ?l of buffer. The virions
were then incubated at 37°C for either 1 h or 4 h as noted in the text and then
diluted 1,000-fold prior to determining the titers on Vero or Hep-2 cells. Con-
trols included mock-treated virus or enzymes added just prior to the assay to
minimize digestion. For cell treatments, confluent Vero cell monolayers in six-
well plates were exposed to 0.2 U of A. ureafaciens, 0.02 U of V. cholerae, or 1,000
U of NEB 2-3 for 1 h at 37°C in Dulbecco modified Eagle medium with 2%
serum. The cells were rinsed once with medium and then infected with virus.
Viral attachment. Viral attachment to cells was measured by using a cell-based
enzyme-linked immunosorbent assay (CELISA). Vero cells were plated in poly-
L-lysine-coated 96-well culture plates at a density of 104cells per well. Three days
later, the cells were incubated with control virions, digested virions, or virions
incubated with either Maackia amurensis lectin (MAL1) or Elderberry bark
lectin (ELD) (Table 1) at 4°C for 1 h. The MAL1 lectin has a 40-fold preference
for ?2,3-linked sialic acid over ?2,6 linkages (89). The ELD lectin has a 50- to
125-fold preference for ?2,6 over ?2,3 linkages (69). The cells were then rinsed
with ice-cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde
in PBS for 10 min at room temperature, and rinsed three times with PBS. The
wells were blocked with 1% bovine serum albumin (catalog no. 160069; ICN
Biochemicals, Cleveland, OH) in PBS and incubated with rabbit HSV-1 specific
polyclonal antiserum (B 0114; Dakocytomation, Glostrup, Denmark) for 1 h at
22°C, followed by a rinse with PBS. The cells were incubated with an alkaline
phosphatase-conjugated goat anti-rabbit immunoglobulin G (A3687; Sigma-Al-
drich, St. Louis, MO) for 1 h and rinsed three times with PBS. The alkaline
phosphatase substrate para-nitrophenyl phosphate (H1007; Sigma-Aldrich) was
then added, and the absorbance at 405 nm in the linear range of the assay was
determined by using an ELX-800 plate reader (BioTek Instruments, Winoski,
VT). To control for the possibility that the desialylation of virions altered anti-
body binding in the CELISA, equal amounts of mock- or neuraminidase-di-
gested virions were serially diluted and adsorbed to 96-well plates, and the
amount of antibody binding was determined as described above. To control for
the possibility of altered binding of virions to the plates, mock- and neuramini-
dase-digested virions were adsorbed to plates and then subjected to BCA protein
determination (Pierce, Rockford, IL). The amount of binding in the CELISA
was corrected for the differences. The polyclonal rabbit anti-HSV antiserum had
a 10% greater binding preference to virions digested with the NEB 2-3 enzyme
and was 40% less effective in binding to V. cholerae- or A. ureafaciens-digested
samples (data not shown). All CELISA values reported were corrected for the
difference in binding of the antibody to mock- and enzyme-treated virions.
VP16 translocation to the nucleus. Vero cells were exposed to mock- and
enzyme-treated virions at a multiplicity of infection of 2. The cells were incu-
bated for 3 h at 37°C, harvested by centrifugation, and resuspended in Laemmli
buffer. Nuclear fractions were isolated by using the NucBuster extraction kit
(71183-3; Novagen, Inc., San Diego, CA). Whole-cell and nuclear fractions were
then sonicated with 10 pulses at a 30% duty cycle using a Branson cell disruptor
200 (Branson Ultrasonics, Danbury, CT), and the amount of protein was deter-
mined by using the BCA assay (Pierce). Nuclear fractions and whole-cell samples
were normalized for protein content and then electrophoresed in 10% denatur-
ing polyacrylamide gels and transferred to nitrocellulose. Immunoblotting was
carried out as previously described (75, 87). The blots were probed with primary
mouse monoclonal anti-VP16 antibody (V4388; Sigma-Aldrich) and developed
by using goat alkaline phosphatase-conjugated anti-mouse immunoglobulin
G (A3562; Sigma-Aldrich) and alkaline phosphatase substrate (B5655; Sigma-
Neuraminidase digestion reduces infectivity of HSV-1. To
test the hypothesis that sialylation of viral envelope glycopro-
teins was important for maintaining infectivity, gradient-puri-
fied virions were digested with V. cholerae, A. ureafaciens, or
NEB 2-3 neuraminidases at 37°C for either 1 h or 4 h, and the
titer of infectious virus was determined by plaque assay. Con-
trols included virions incubated in buffer only, addition of
enzyme just before the titer was determined, and virions di-
gested with Endo H (endo-?-N-acetylglucosaminidase H),
which cleaves high mannose side chains that are not sialylated.
Digestion with NEB 2-3 for 1 h (Fig. 1A) or 4 h (data not
shown) had no effect on viral infectivity. This was not due to a
lack of these linkages on the HSV envelope proteins (Fig. 2) or
a failure of the enzyme to digest the samples (Fig. 3 and 6). In
contrast, digestion of virions with V. cholerae or A. ureafaciens
neuraminidases for 1 h reduced infectivity 10-fold, and diges-
tion for 4 h reduced infectivity by 1,000-fold (Fig. 1A). Immu-
noblotting for gC, gB, gD, and gH indicated that contamina-
tion of the neuraminidases with protease did not explain the
loss in infectivity (Fig. 3 and 6). There was no reduction in titer
for either mock-treated virions or samples when enzyme was
added just prior to the titer being determined. Incubation of
cells for 1 h with a concentration of neuraminidases 1,000-fold
higher than what they were exposed to in the titer reduction
assays did not alter infectivity with virions (data not shown).
These results indicate that removal of ?2,3-linked sialic acid on
virions has no effect on infectivity, implying that ?2,6-linked
sialic acids on viral envelope proteins are critical for efficient
infection by HSV-1.
Reduced infectivity is not specific for Vero cells. To ascer-
tain whether the requirement for sialic acid was specific for
TABLE 1. Neuraminidases and lectins used in this study
Enzyme or lectinDescriptionSpecificityManufacturerCatalog no.
V. cholerae neuraminidase
A. ureafaciens neuraminidase
New England Biolabs
11 080 725 001
10 269 611 001
P0728LS. enterica serovar Typhimurium
Sialic acid binding lectin
Sialic acid binding lectin
3732TEUTON AND BRANDT J. VIROL.
Vero cells, we measured the infectivity of desialylated virions
in Hep-2 cells. We also compared viral stocks prepared in
either Vero or Hep-2 cells to determine whether the cell line
used for preparation of viral stocks was important. As shown in
Fig. 1B, virus grown in Hep-2 cells showed the same loss of
infectivity when digested with V. cholerae neuraminidase seen
with virus prepared in Vero cells. Identical results were ob-
tained with A. ureafaciens neuraminidase (data not shown).
Digestion with NEB 2-3 neuraminidase had no effect. Virions
prepared in either Vero or Hep-2 cells and whose titers were
determined on the other cell type after digestion with V. chol-
erae or A. ureafaciens also showed the same pattern of infec-
FIG. 1. Digestion of HSV-1 with neuraminidases reduces infec-
tious titer. (A) HSV-1 KOS produced in Vero cells was mock digested
(?) or digested with the indicated neuraminidases (■) for either 1 or
4 h, and the remaining infectious virus was determined by plaque assay
in Vero cells. Mock-digested virus was also mixed with the indicated
enzyme immediately prior to the plaque assay to minimize the time for
digestion (o). (B) HSV-1 KOS produced in Hep-2 cells was mock
digested or digested with NEB 2-3 neuraminidase or V. cholerae neur-
aminidase for 4 h before the titers were determined in Hep-2 cells.
Identical results were obtained with A. ureafaciens neuraminidase
(data not shown). The data presented represent the means and stan-
dard deviations of three independent assays. All values are reported as
percentages with the “mock” value defined as 100%. NEB 2-3, Salmo-
nella enterica serovar Typhimurium LT2 neuraminidase; VC, V. chol-
FIG. 2. Exposure of HSV-1 to sialic acid-binding lectins reduces
viral attachment. HSV-1 hrR3 grown in Vero cells was exposed to
MAL1 or ELD for 45 min prior to exposure to cells. Attachment was
then measured by using a CELISA, with results given as the percent-
age of the signal intensity with the untreated samples defined as 100%
(s). The open bars represent background signal with lectins only and
no virus. The solid bars indicate binding of virions in the presence of
the lectins. The data represent the means and standard deviations of
three independent assays.
FIG. 3. Neuraminidase digestion of HSV-1 virions alters the mo-
bility of HSV-1 glycoproteins B, H, and D in sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (PAGE). HSV-1 stocks were di-
gested for 4 h at 37°C with the indicated enzymes. The digested virions
were mixed with Laemmli buffer, boiled for 5 min, and subjected to
PAGE. After transfer to nitrocellulose, the blots were developed with
antibodies specific for gB (A), gH (B), and gD (C) (Virusys, Sykesville,
MD; Advanced Biotechnologies, Columbia, MD). The blots shown are
representative examples of multiple independent assays. NEB 2-3, S.
enterica serovar Typhimurium LT2 neuraminidase; VC, V. cholerae
neuraminidase; AU, A. ureafaciens neuraminidase. The positions of
the molecular weight markers are denoted on the left.
VOL. 81, 2007VIRION SIALIC ACID AND HSV INFECTION3733
tivity loss (data not shown). These results indicate that the loss
of infectivity after desialylation is not specific for Vero cells
and that potential cell-specific differences in sialylation pat-
terns between Vero and Hep-2 cells are inconsequential.
HSV-1 virions contain both ?2,6- and ?2,3-linked sialic
acids. One possible explanation for the observation that diges-
tion with NEB 2-3 neuraminidase did not reduce infectivity is
that HSV-1 envelope proteins lack ?2,3-linked sialic acid. To
determine whether ?2,3- and ?2,6-linked sialic acids were
present on virions, a CELISA measuring virion attachment to
cells was carried out in the presence or absence of MAL1 or
ELB lectins that are specific for ?2,3- and ?2,6-linked sialic
acids, respectively (Fig. 2). Incubation of virions with either
lectin reduced viral attachment to background levels. Incuba-
tion of virions with either lectin also reduced the infection of
cells by 100- to 1,000-fold (data not shown). These results
suggest that HSV-1 virions contain both ?2,3- and ?2,6-linked
sialic acids on envelope glycoproteins.
?2,6- and ?2,3-linked sialic acids are present on gB, gD, and
gH. Having shown that both types of linkages were present on
virions, we sought to determine whether individual glycopro-
teins involved in entry contained both types of linkages. Viri-
ons were enzymatically digested for 4 h with NEB 2-3, V.
cholerae, or A. ureafaciens neuraminidase, electrophoresed,
and immunoblotted with antiserum specific for gB, gH, or gD
(Fig. 3). Mock-digested virions were included as controls. Di-
gestion with any of the enzymes shifted the mobility of gB and
gH to the same extent (Fig. 3A and B). Digestion with any of
the enzymes also altered the mobility of gD (Fig. 3C), but the
shift was greater for the V. cholerae and A. ureafaciens enzymes
compared to NEB 2-3-digested gD. These results confirm the
lectin-binding results and show that gB, gD, and gH contain
both ?2,6- and ?2,3-linked sialic acids.
Neuraminidase digestion does not reduce viral attachment.
To determine whether the loss of infectivity was due to re-
duced attachment of virions to cells, the binding of mock-
treated and desialylated virions to cells was measured by using
the CELISA-based attachment assay. As shown in Fig. 4, the
amount of virus attached to cells was not significantly different
between the controls and the digested virions, suggesting that
viral attachment was not affected by desialylation.
Glycoprotein C is not involved in the loss of infectivity. The
loss of infectivity after V. cholerae or A. ureafaciens digestion
was not due to a defect in attachment, which is mediated
primarily by gC. To rule out a role for gC in the loss of
infectivity after neuraminidase digestion, we repeated these
studies with ?gC2-3, a gC-null virus, and ?gC2-3rev, in which
the gC gene was reinserted. Digestion with NEB 2-3 neuramin-
FIG. 4. Attachment of HSV-1 hrR3 is not affected by digestion
with neuraminidase or Endo H. HSV-1 hrR3 grown in Vero cells was
digested with NEB 2-3, V. cholerae neuraminidase, or Endo H (■) for
4 h at 37°C. For each enzyme condition the virus was mock digested in
the same buffer (?). After digestion, attachment was measured by
using a CELISA, with the results given as percentages with mock-
digested controls defined as 100% for each sample pair. Digestion with
A. ureafaciens neuraminidase gave the same results as for V. cholerae
neuraminidase (data not shown). The data represent the means and
standard deviations of triplicate independent assays. NEB 2-3, S. en-
terica serovar Typhimurium LT2 neuraminidase; VC, V. cholerae neur-
FIG. 5. The absence of gC does not affect the titer reduction seen
after neuraminidase digestion. HSV-1 ?gC2-3 (A) or ?gC2-3rev
(B) was mock digested (?) or digested with the indicated neuramini-
dase (■) for 4 h before the titer was determined by plaque assay.
Values are reported as percentages, with “mock” defined as 100%. The
data represent the means and standard deviations of triplicate inde-
pendent assays. Digestion with A. ureafaciens neuraminidase gave re-
sults identical to those for the V. cholerae enzyme (data not shown).
NEB 2-3, S. enterica serovar Typhimurium LT2 neuraminidase; VC, V.
3734TEUTON AND BRANDT J. VIROL.
idase did not reduce the infectivity of either the gC-null virus
(Fig. 5A) or the revertant virus (Fig. 5B). In contrast, digestion
with V. cholerae or A. ureafaciens enzymes reduced the infec-
tivity of both ?gC2-3 and ?gC2-3rev to a level similar to that
seen with the wild-type virus, indicating that gC is not involved
in the reduction of infectivity (Fig. 5A and B). As shown in Fig.
6, digestion of gC with each of the neuraminidases resulted in
a slight decrease in mobility. The decreased mobility of neur-
aminidase-digested gC has been seen previously (G. Cohen,
unpublished data), but the reason for the apparent increase in
molecular weight is not clear. These results suggest that gC
contains sialic acids with both linkages and confirms reports
that gC is sialylated, although the types of linkages present had
not been determined previously (51, 55).
Sialic acid is required for efficient viral entry. We next
sought to determine whether the removal of sialic acids af-
fected viral entry into cells by quantifying the amount of VP16
translocated to the nucleus. VP16 is a tegument protein that
traffics to the nucleus shortly after entry and is an accepted
marker for viral entry (16). Vero cell monolayers were cooled
to 4°C and then exposed to either mock- or V. cholerae neur-
aminidase-digested virions for 2 h at 4°C. The cells were ex-
posed to increasing concentrations of virus ranging from 5 ?
105to 1 ? 108PFU per well increasing in half-log steps. After
attachment, the cultures were shifted to 37°C for 3 h. The cells
were then harvested, and the amount of VP16 was measured
by immunoblotting. For one set of samples, nuclear fractions
were isolated and analyzed. A duplicate set of samples was
lysed and electrophoresed without fractionation to measure
total VP16. As shown in Fig. 7, in the whole-cell lysates VP16
was first detected when 5 ? 106virions were loaded for either
mock- or V. cholerae-digested samples (left panels). In the
example shown, there appeared to be slightly more VP16 sig-
nal in the V. cholerae-digested whole-cell samples (twofold).
These results confirm that attachment of the virus to cells was
not significantly altered by V. cholerae digestion. When the
amount of VP16 in the nuclear fractions was compared, we
found that VP16 was first detected in the lane loaded with 5 ?
107virions. There was a faint VP16 signal in the nuclear frac-
tions from cells exposed to V. cholerae-treated neuraminidase
in the lane loaded with 108virions, suggesting a 5- to 10-fold
decrease in entry compared to the mock-digested samples
The HSV-1 glycoproteins involved in entry are posttransla-
tionally modified in the exocytic pathway by glycosylation, with
the terminal step being the addition of sialic acid in the trans-
Golgi compartment. For most mammalian cell types, sialic
acids can be added either in ?2,3 or ?2,6 linkages. It is com-
mon to find both types of linkages on an individual glycopro-
tein. Although the HSV-1 proteins involved in attachment and
entry have been extensively characterized, the significance of
FIG. 6. Neuraminidase digestion of HSV-1 virions decreases the
mobility of gC in sodium dodecyl sulfate-PAGE. HSV-1 stocks were
digested as indicated with the indicated enzymes for 4 h at 37°C. The
digested virions were mixed with Laemmli buffer, boiled for 5 min, and
subjected to PAGE. After transfer to nitrocellulose, the blot was de-
veloped with polyclonal anti-gC antibody. The blot shown is a repre-
sentative example of multiple independent blots. NEB 2-3, S. enterica
serovar Typhimurium LT2 neuraminidase; VC, V. cholerae neuramin-
idase; AU, A. ureafaciens neuraminidase.
FIG. 7. Digestion of HSV-1 with V. cholerae neuraminidase reduces nuclear localization of VP-16 without reducing viral attachment. In
replicate experiments HSV-1 hrR3 was either mock digested (top panels) or digested with V. cholerae neuraminidase (bottom panels). After the
treatment, Vero cells in confluent six-well plates were infected in increasing in half-log steps with 5 ? 105to 1 ? 108PFU/well. The amount of
virions added was based on the starting titer before digestion. After 3 h at 37°C, the cells were processed in two ways. To measure the total VP16,
whole-cell samples were centrifuged, mixed with Laemmli buffer, subjected to BCA protein determination to ensure equal loading, and subjected
to PAGE (left panels). To determine the amount of nuclear VP16, the cells were fractionated, and the nuclear fractions were subjected to BCA
protein concentration to ensure equal loading and subjected to PAGE (right panels). All blots were developed with an antibody specific for VP16.
VC, V. cholerae neuraminidase.
VOL. 81, 2007 VIRION SIALIC ACID AND HSV INFECTION3735
sialylation has not been studied. Our data provide the first
evidence that sialic acids on one or more viral glycoproteins
are critical for maintaining the infectivity of the virions and
that gB, gD, and gH contain both ?2,3 and ?2,6 linkages. In
addition, we have shown that the maintenance of infectivity
appears to be specific for ?2,6-linked sialic acids. The data also
show that the reduction in infectivity was not due to reduced
attachment or to an effect on gC but instead was due to inef-
ficient entry of virions into cells.
Digestion of virions with NEB 2-3, which has a 260-fold
preference for ?2,3-linked sialic acid (41), had no effect on
infectivity, whereas digestion with V. cholerae or A. ureafaciens
neuraminidases reduced infectivity. These results suggest that
?2,6-linked sialic acids are critical for virion infectivity, but this
conclusion is tentative because neuraminidases with the re-
quired degree of specificity for ?2,6 linkages are not currently
available. The apparent requirement for ?2,6 linkages is not
due to selective ?2,6 sialylation of gB, gD, or gH since diges-
tion with each of the neuraminidases resulted in a mobility
shift. These observations are consistent with previous studies
showing that gD and gH are sialylated (61, 68). One potential
explanation for the specificity is that ?2,6-linked sialic acids are
located at critical positions in gB, gD, or gH. This implies that
domain-specific sialylation may be occurring and will require
further studies to determine the location of ?2,3- and ?2,6-
linked sialic acids on the viral glycoproteins.
The electrophoretic mobility of gB and gH after neuramin-
idase digestion was similar whether V. cholerae, A. ureafaciens,
or NEB 2-3 neuraminidase was used. In contrast, digestion of
gD with NEB 2-3 resulted in only a slight shift compared to gD
from virions digested with V. cholerae or A. ureafaciens neur-
aminidases. It is possible that some of the ?2,3-linked sialic
acids on gD may not be accessible for digestion. Alternatively,
gD may contain a higher ratio of ?2,6- to ?2,3-linked sialic
acid. If gD were to contain more ?2,6-linked sialic acid, re-
moval could significantly affect gD-mediated entry functions.
HSV-1 gD contains three N-linked and two O-linked glyco-
sylation sites that are modified (19, 20, 44, 71). Mutation of the
three N-linked sites in gD results in a conformationally altered
but functional protein, suggesting that glycan side chains play
a role in maintaining gD structure (72). The removal of sialic
acids from the O-linked side chains could alter the structure
and therefore the function of gD. Confirmation that desialyla-
tion of gD is involved in the reduced infectivity and whether
desialylation alters the interaction of gD with cellular receptors
or affects assembly of the fusion complex will require further
The HSV-1 gC is heavily glycosylated with at least eight N
linkages and numerous O linkages, and up to 80% of these side
chains are sialylated (11, 22, 44, 50, 56, 57, 66), so it was
surprising that the reduction in infectivity was not due to re-
duced attachment. Digestion of gC with each of the enzymes
resulted in a similar increase in the apparent molecular weight
for reasons that are not clear. It is possible that gC is resistant
to enzymatic desialylation, which would explain the lack of
effect on attachment. However, the migration of gC did shift
after neuraminidase digestion, suggesting that the protein was
altered by exposure to the enzymes. Further studies on the
effect of desialylation on gC structure and function will be
needed to explain the apparent increase in molecular weight.
Sialic acids on cells are known to serve as receptors for a
number of viruses, including influenza virus; respiratory syncy-
tial virus; adeno-associated virus types 1, 4, 5, and 6; adenovi-
rus type 37; polyomaviruses; minute virus of mice; feline cali-
civirus; and avian infectious bronchitis virus (2, 3, 8, 15, 24, 26,
29, 45, 76, 77, 93, 95). For influenza virus, polyomaviruses, and
adenovirus type 37, the crystal structures of the sialic acid
binding sites have been determined, and the structural features
important for the stereoselectivity of binding to specific sialic
acids are known (14, 15, 26, 77, 78). These differences in
receptor specificity play an important role in pathogenesis for
several viruses (55, 76). Notably, for influenza virus, acquisition
of the ability to bind ?2,6-linked sialic acid is required for
infection of humans with avian strains (29, 79). When we in-
cubated cells for 1 h with 1,000 times the amount of neuramin-
idase they would have seen in the titer assays, we saw no
reduction in infection (data not shown), suggesting that sialic
acids on cells do not play a role in infection. This is consistent
with the fact that sialic acid has not been reported to function
as a receptor for HSV-1.
The role of sialic acids on virions is less well understood, but
two general effects have been reported. Neuraminidase diges-
tion of some viruses, including lentiviruses, vesicular stomatitis
virus, respiratory syncytial virus, and influenza virus, results in
enhanced infectivity (8, 42, 53, 63, 80). For HIV-1, sialic acids
may sterically hinder attachment and entry of the virus (43,
81). In contrast, neuraminidase digestion of porcine reproduc-
tive and respiratory syndrome virus inhibits infection of por-
cine alveolar macrophages by reducing attachment to cells
(23). Based on our studies, HSV-1 can be added to the list of
viruses that require sialylation of envelope proteins for efficient
infection and is the first example of sialic acids on virion gly-
coproteins specifically affecting entry into cells.
Our observation that sialic acid is required for efficient entry
of HSV-1 into cells raises the possibility that sialic acid-binding
agents could be effective antivirals; thus, we have identified a
new target for the development of drugs to prevent HSV-1
infection. Recently, we described a novel peptide, TAT-C, that
blocks HSV-1 entry (13a), and preliminary studies suggest that
TAT-C binds to sialic acid on virions (unpublished data). Thus,
TAT-C may be inhibiting entry by interfering with sialic acid-
mediated entry functions. Other carbohydrate-binding agents
have been shown to have antiviral activity. A modified theta
defensin, RC-2, blocks the attachment and entry of HSV-1 (96)
and HIV-1 (91) and has been shown to act as a minilectin (90).
Lactoferrin, which blocks HSV-1 attachment, binds to cellular
glycosaminoglycans (52). Cyanovirin-N binds to carbohydrates
and inhibits the infection of several viruses, including HIV-1
and hepatitis C virus and is currently in clinical trials as a
microbicide to block sexually transmitted viral infection (9, 12,
36). Mannose-binding proteins from several plants inhibit
HIV-1 infection and can select for HIV-1 with mutations in
glycosylation sites within gp120 (4–7). These results clearly
indicate that carbohydrates, and sialic acid in particular for
HSV-1, are valid targets for antiviral drug development.
In summary, we report for the first time that sialic acids on
one or more HSV-1 envelope proteins are required for effi-
cient infection of cells. The observation that infectivity was
reduced by digestion with V. cholerae or A. ureafaciens neur-
aminidase, but not NEB 2-3 neuraminidase, suggests that ?2,6-
3736TEUTON AND BRANDTJ. VIROL.
linked sialic acids are involved. The effect of neuraminidase
digestion is not specific for Vero cells, nor did it depend on the
cell line used for viral propagation. We have also shown that
enzymatic desialylation does not affect attachment to cells and
that gC is not involved in the reduced infectivity. The reduced
infectivity of neuraminidase-digested virions is due to ineffi-
cient entry of the virus into cells, suggesting that the fusion
proteins, gB, gD, or gH are involved. Our results also suggest
that sialic acids on HSV-1 envelope proteins may be valid
targets for antiviral drug development. Further studies on the
role of sialic acid in HSV-1 entry will likely provide novel
insights into the function of gB, gD, and gH in entry.
We thank Sharon Altmann, Aaron Kolb, Gilbert Jose, Radeekorn
Akkarawongsa, Hermann Bultmann, Stacey Schulz-Cherry, and
Donna Peters for helpful comments on these studies and the manu-
script. We also thank Elizabeth Froelich for administrative assistance.
This study was supported in part by NIH grants PO1-AI52089,
RO1-EY07336, and P30 EY016665 to C.R.B.; the Consortium for
Functional Glycomics (GM-62116); and by an unrestricted grant from
Research to Prevent Blindness to the Department of Ophthalmology
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