1. health and fitness
2. disease
3. aids and hiv

JOURNAL OF VIROLOGY, May 1991, p. 2393-2401
Vol. 65, No. 5
0022-538X/91/052393-09$02.00/0
Copyright © 1991, American Society for Microbiology
Characterization and Sequence Analyses of Antibody-Selected
Antigenic Variants of Herpes Simplex Virus Show a
Conformationally Complex Epitope on Glycoprotein H
U. A. GOMPELS,t* A. L. CARSS, C. SAXBY, D. C. HANCOCK,: A. FORRESTER, AND A. C. MINSON
Division of Virology, Department o Pathology, Cambridge University, Cambridge CB2 IQP, United Kingdom
Received 2 November 1990/Accepted 30 January 1991
penetrate cells (8), while a temperature-sensitive gH mutant,
tsQ26, at the nonpermissive temperature produces extracellular virus which lacks gH and is rendered noninfectious
(17). gB has been extensively studied by use of temperaturesensitive mutants, antibody-resistant mutants, and insertion
or deletion mutants (8, 9, 16, 25, 26, 31, 45, 48). Results
indicate a role in virus penetration involving fusion (8, 9),
although possible interactions with other glycoproteins during the adsorption process have been suggested (32, 45). In
contrast, less is known concerning the function of gH as this
glycoprotein has been defined only relatively recently (7, 22,
42). Furthermore, unlike gB or gD, few gH-specific monoclonal antibodies (MAbs) have been generated for the study
of gH function (20, 22). However, the pronounced biological
properties of gH-specific antibodies on virus replication in
vitro suggest that gH is at least as important as gB in the
process of virus entry. Like some antibodies specific for the
nonconserved HSV type 1 (HSV-1) gD, the MAbs to gH
strongly neutralize virus infectivity in the absence of complement and can inhibit cell fusion by syncytial virus strains
Herpes simplex virus (HSV) encodes at least seven virion
glycoproteins, gB, gC, gD, gE, gG, gH, and gI, which are
located on both the virion and the infected cell surface where
they can act to mediate infectivity (1, 3, 22, 29, 35, 47, 53).
Glycoproteins homologous to HSV gB and gH have been
identified in every herpesvirus examined including other
human herpesviruses representing the a, 1B, and y subgroups-varicella-zoster virus, human cytomegalovirus
(HCMV), and Epstein-Barr virus, respectively (13-15, 20,
36). Both gB and gH appear to be essential for virus
replication; only the nonconserved gD has similar properties
(34). A gB-null virus has been shown to bind to but not
* Corresponding author.
t Present address: Department of Medicine, Addenbrooke's Hospital, Level 5, Cambridge University, Hills Road, Cambridge CB2
2QQ, United Kingdom.
t Present address: Biochemistry of the Cell Nucleus Laboratory,
Imperial Cancer Research Fund Laboratories, Dominion House,
Bartholomew Close, London EClA 7BE, United Kingdom.
2393
Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest
Thirteen antigenic variants of herpes simplex virus which were resistant to neutralization by monoclonal
antibody 52S or LP11 were isolated and characterized. The antibodies in the absence of complement potently
neutralize infectivity of wild-type virus as well as inhibit the transfer of virus from infected to uninfected cells
("plaque inhibition") and decrease virus-induced cell fusion by syncytial strains. The first variant isolated
arose in vivo. Of 66 type 1 isolates analyzed from typing studies of 100 clinical isolates, one was identified as
resistant to neutralization by LP11 antibody. The glycoprotein H (gH) sequence was derived and compared
with those of wild-type and syncytial laboratory strains SC 16, strain 17, and HFEM. The sequences were highly
conserved in contrast to the diversity observed between gH sequences from herpesviruses of different
subgroups. Only four coding changes were present in any of the comparisons, and only one unique coding
change was observed between the laboratory strains and the clinical isolate (Asp-168 to Gly). These sequences
were compared with those of antigenic variants selected by antibody in tissue culture. Twelve variants were
independently selected with antibody LP11 or 52S from parent strain SC16 or HFEM. For each variant, the
gH nucleotide sequence was derived and a point mutation was identified giving rise to a single amino acid
substitution. The LP11-resistant viruses encoded gH sequences with amino acid substitutions at sites
distributed over one-half of the gH external domain, Glu-86, Asp-168, or Arg-329, while the 52S-resistant
mutant viruses had substitutions at adjacent positions Ser-536 and Ala-537. One LP11 mutant virus had a point
mutation in the gH gene that was identical to that of the clinical isolate, giving rise to a substitution of Asp-168
with Gly. Both LP11 and 52S appeared to recognize distinct gH epitopes as mutant virus resistant to
neutralization and immunoprecipitation with LP11 remained sensitive to 52S and the converse was shown for
the 52S-resistant mutant virus. This is consistent with previous studies which showed that while the 52S epitope
could be formed in the absence of other virus products, virus gene expression was required for stable
presentation of the LP11 epitope, and for transport of gH to the cell surface (Gompels and Minson, J. Virol.
63:4744-4755, 1989). All mutant viruses produced numbers of infectious particles that were similar to those
produced by the wild-type virus, with the exception of one variant which produced lower yields. The
antibody-resistant phenotype of the LP11 mutant viruses could be partially complemented by growth or
titration on wild-type gH-producing cell lines, but the mutants differed in decreased resistance to plaque
inhibition or neutralization of infectivity. Taken together, these results show that gH has a conformationally
complex epitope which is part of a domain which has an essential function in virus infectivity and spread. This
epitope is not stable when gH is expressed in the absence of virus infection, and this epitope may be
differentially presented at the infected cell surface or on the viral envelope.
2394
GOMPELS ET AL.
MATERIALS AND METHODS
Antibodies. Anti-HSV-1 gH MAbs 52S (49) and LP11 (7)
used as hybridoma supernatants or ascites fluid as
described previously (22, 23). Hybridoma supernatants were
concentrated 15 times by using a Centriprep 30 concentrator
(Amicon, Danvers, Mass.) and supplemented with 0.02%
azide.
Viruses and cells. BHK, Vero, and HEp-2 cells were
grown in Glasgow modified Eagle medium supplemented
with 10% tryptose phosphate broth and 10% newborn calf
serum. Cell lines F6 and B1.3 are Vero cells which have
stably integrated multiple copies of the HSV-1 gH coding
region under the control of the early gD promoter. The gH
gene is expressed only after superinfection with virus, and
levels of gH produced exceed that achieved during highmultiplicity infections analyzed 16 to 20 h postinfection. The
cell lines were derived by cotransfection of the gH gene with
the neomycin resistance gene followed by selection with the
drug G418 (Geneticin; GIBCO) and will be described further
elsewhere. The virus strains used were HSV-1 HFEM (56),
SC16 (27), and 103/65 (6). Virus stocks were prepared by
infections at a multiplicity between 0.01 and 0.1 PFU per cell
of HEp-2 cells, and titers were determined by assay on BHK
and Vero cells. HSV-1 103/65 is a clinical isolate which is
resistant to the effects of MAb LP11 (6).
Virus neutralizations. Dilutions of virus in a 100-,u volume
were mixed with 5 pul of antibody and incubated at room
temperature for 1 h before infection. Virus was then adsorbed to monolayers of cells for 1 h at 37°C and then
washed twice with medium and overlaid with fresh medium
with or without antibody diluted 1:200 and incubated for 2
were
days at 37°C.
Selection of antibody-resistant mutant virus. Antibodyresistant mutant viruses VAI, VAII, NillA, and NillB were
selected independently by prior incubation of virus with
antibody followed by infection of HEp-2 cells. A total of 106
PFU of HSV-1 strain HFEM were incubated with antibody
LP11 as ascites diluted 1:20 for 1 h at room temperature and
then mixed with 1 ml of medium and allowed to adsorb to
monolayers of 5 x 106 HEp-2 cells for 1 h at 37°C. The
inoculum was removed, and the cells were washed twice
with 5 ml of medium and then overlaid with 5 ml of fresh
medium and incubated at 37°C for 2 days. The virus was
harvested and titers were determined in the presence or
absence of antibody. This procedure was repeated until the
virus population gave a similar titer in the presence or
absence of antibody (three to four rounds of selection) and
then was repeated twice more. Antibody-treated virus was
cloned twice by limiting dilution.
Antibody-resistant mutant viruses AD1, NiPIIIA, NiPIIIB, LP11A, and LP11B were independently selected as
above except that antibody LP11 diluted 1:200 was also
included in the medium overlay after virus neutralization and
adsorption. Mutants LP11A and LP11B were derived from
strain SC16. The virus populations thus obtained were
completely resistant to infectivity neutralization by antibody
after one round of selection. The procedure was repeated
before virus was cloned twice as described above.
Similarly, antibody-resistant mutant viruses 52SR, 5RA,
and SRB were independently selected and cloned in the
presence of 52S antibody before and during infection. Mutants 52SR and SRB were selected from strain HFEM, and
mutant 5RA was selected from SC16. For each selection, 106
PFU of virus were incubated with an equal volume of 52S
hybridoma supernatant. Antibody was included in the medium at a 1:20 dilution.
Two additional VAI mutant viruses were selected from
HFEM and SC16 parent strains by cotransfection of plasmid
DNA containing the VAI mutant gH gene together with
infected cell DNA of wild-type HFEM or SC16, using the
calcium coprecipitation method in BES buffer (25 mM BES,
140 mM NaCl, 0.75 mM Na2HPO4) as described previously
(23). After virus was harvested, antibody-resistant virus was
selected and cloned as detailed above.
Immunoprecipitations. HEp-2 cells were infected with
each virus at a multiplicity of infection (MOI) of 5 PFU per
cell. After 4 h, cells were labeled with [35S]methionine (800
Ci/mmol; Amersham) at 25 ,uCi/ml in methionine-free medium containing 1% fetal calf serum by incubation for a
further 12 h at 37°C. Cells were lysed in immunoprecipitation
buffer and incubated with antibody and then protein A-Sepharose, and immunoprecipitation products were separated on
a 10% polyacrylamide gel with sodium dodecyl sulphate
(SDS). The gel was then dried down and exposed to film as
described previously (23).
Plasmid clones. Infected cell DNA was extracted from
proteinase K-SDS lysates with phenol and chloroform as
described previously (22). The Bgl m fragment from each
infected cell DNA was purified and ligated with BglIIdigested and phosphatased plasmid vector PKC7 (22). Preparations of plasmid DNA were made by the alkaline-SDS
method (5).
Derivation of nucleotide sequence and analyses. The nucleotide sequence of the gH gene for each virus was determined
by the Sanger dideoxy-chain termination method from sequences cloned into bacteriophage M13mpl8 (38) by using
methods detailed by Bankier and Barrell (2) and 14 synthetic
17-mer primers from the HFEM BglII m sequence (22)
spanning the gH sequence at the following positions from the
BglII recognition sequence: 1435, 1626, 1728, 1904, 2044,
2240, 2435, 2647, 2841, 2978, 3228, 3456, 3676, and 3837. Nru
Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest
(7, 22, 40, 42, 43). Efficient neutralization of infectivity of
HCMV or Epstein-Barr virus has also been achieved with
antibodies specific for the respective gH homologs (reviewed
in reference 20). One of these MAbs specific for EpsteinBarr virus gH appears to prevent fusion rather than adsorption (39), and a similar result has been recorded for interactions of HSV antibodies with HSV gH (19). Unlike most
other neutralizing antibodies, MAbs to HSV-1 gH also
inhibit the transfer of virus from infected to uninfected cells
during plaque formation by both syncytial and nonsyncytial
strains. It has been proposed that the interaction of antibody
with gH expressed at the cell surface prevents its function in
mediating cell-to-cell spread through formation of intercellular bridges.
Studies of gH in mammalian cell expression systems have
shown that gH requires interactions with other virus products for cell surface localization and formation of an antigenic structure important for its function in mediating infectivity (23). To identify protein domains involved in functions
of gH, we are studying interactions of HSV-1 mutants with
the type 1-specific MAbs 52S and LP11, which neutralize
both infectivity and cell-to-cell spread. MAb 52S recognizes
gH expressed in the absence of other virus proteins, whereas
MAb LP11 only recognizes gH in the presence of other virus
proteins (23). In the present study, 13 antibody-resistant
mutants independently selected in the presence of either
antibody were characterized. We show that LP11 recognizes
a conformationally complex epitope in gH which spans
one-half of the external domain.
J. VIROL.
VOL. 65, 1991
ANTIBODY-RESISTANT MUTANTS OF HSV-1 gH
CD
In
,O
%a
%
40
u,
_ V
v.
.
I
I
I
I
HFEM
CT
C
GA
SC16
f c
f
I
4.n
C
.n
'0
N
in
m0
_
.
(,.-
0Wp
in
r~-_
-0'~
a
r-
a
2395
4 O)
I
I
I
v i rus
17
i c
103/65
C
R R
T
I
G
C
iT
G
C
I
Q G
C
I
Gi
C
G C
G
CC
Q
T
T
T
T R
C
C G
R
R
P
RT
D
R
A
T
SR
6
U
T
FIG. 1. Comparisons of the gH nucleotide sequence from different HSV strains. Nucleotide sequence was derived for the gH gene from
each strain. Comparisons were made to the strain HFEM nucleotide sequence and encoded amino acid sequence. Coding differences are
underlined. Nucleotide and amino acid sequences are numbered from the initiating codon. Strain 17 and HFEM sequences are from references
37 and 22, respectively. A, Alanine; V, valine; P, proline; T, threonine; D, aspartic acid; G, glycine.
subfragments of 2.3 and 2.0 kb were purified from plasmid
DNA and ligated with SmaI-digested and phosphatased
bacteriophage M13mpl8 DNA. The sequence was then
determined by using universal and synthetic primers with
35S-dATP (Amersham) as a radioactive label, and the products were fractionated by Tris-borate-EDTA buffer gradient
electrophoresis (4). The reaction products obtained with
each individual chain-terminating dideoxynucleotide were
electrophoresed in parallel to allow rapid and accurate
identification of any nucleotide substitution.
RESULTS
Strain variation of HSV-1 gH sequences. The gH gene is a
member of the subset of conserved herpesvirus genes. As
determined by comparisons of encoded amino acid sequences, it is apparent that the homologous gH sequences
from different herpesvirus subgroups (a, or -y) form one of
the most divergent of these related protein families (15, 20,
21). To further examine this divergence and as a starting
point to evaluate functional domains and the relevance of
mutations which arise during selection with neutralizing
antibody, we investigated the variation in gH sequence
between strains of HSV-1. The gH gene coding sequence
was derived for two recent clinical isolates, SC16 (27) and
103/65 (6), and compared with the sequence previously
described for passaged laboratory strains HFEM (22, 56) and
17 (37) of syncytial and nonsyncytial plaque phenotypes,
respectively. Strain SC16 was chosen for comparison since it
is a virulent strain used to study pathogenicity in a mouse
model (27). Strain 103/65 was selected from 100 clinical
isolates which were serotyped by using MAbs to HSV-1 or
HSV-2. Of 66 type 1 isolates, one was resistant to neutralization with the HSV-1 virus gH-specific MAb LP11. Examination of the nucleotide sequence of the gH genes of these
viruses showed that, in contrast to the divergence seen
between herpesviruses of different subgroups, the gH seI,
quence was highly conserved between strains of HSV-1. For
each comparison with the HFEM gH sequence, only 10
nucleotide differences were found, of which only 4 were
coding changes (Fig. 1). The coding changes were clustered
at the amino-terminal half of the external domain, a region
not conserved between homologous gH sequences from
herpesviruses of different subgroups (20). In comparisons
between the LP11 antibody-resistant clinical isolate 103/65
and strain HFEM, only one of the four coding changes in the
gH sequence was not present in strain 17. This change,
adenine 503 to guanine, results in the substitution of glycine
for aspartate 168. This was a potential site which conferred
resistance to the virus-neutralizing effects of antibody LP11.
Anti-gH antibody-resistant mutant virus. To further identify amino acids which may interact with neutralizing antibody and to determine sites on the protein molecule which
function in mediating infectivity, we selected antibodyresistant mutant viruses in tissue culture for subsequent
sequence analysis of their gH genes. Twelve independently
selected antibody-resistant mutant viruses were isolated by
treatment with antibody followed by limiting dilution cloning
from infected tissue culture cells. Antibody-resistant viruses
were selected in three ways. In the first method, antibodies
LP11 and 52S were used only to neutralize initial infectivity,
and the infected monolayer was then washed to dilute
remaining antibody. In the second method, the ability of
these antibodies to prevent infected cell-to-cell spread, (i.e.,
"plaque inhibition" [7, 22]) was used as an additional
selection pressure by including antibody in the medium
overlay of the infected cells after infection with antibodytreated virus. A third method was used to create two
additional mutants by marker transfer. Cells were cotransfected with a mixture of cloned mutant gH DNA and DNA
from cells infected with wild-type virus. Antibody-resistant
mutants were selected from the resulting progeny and were
characterized to confirm that antibody resistance was conferred by the mutation in the gH coding sequence. Each
Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest
amino acid changes
2396
J. VIROL.
GOMPELS ET AL.
244
LP1 1 Resistant mutants
aa
86
1
Ila
168
ntd (2514)
(838)
E-[K
D-G
256
G-A
503 A--G
9
VAl
tsQ26
(HFEM)
. NiP3B (HFEM)
1- I
.103/65 clinical isolate
lib
D-N
502
G-A
. LP11A (SC16)
E-K
LP 1 1 B (SC 16)
86
Ilia
IIIb
329
R-W
R- Q
985
986
Ni2B
(HFEM)
(HFEM)
(HFEM)
. Ni2A
(HFEM)
C-T
* AD1
G-A
VA2
*
T
III
D-G
D*N
168
R-'W
LP 1 1 R
R-sQ
329
T
800
IV
S-L
A-V
536,537
52SR
FIG. 3. Summary of sites of amino acid substitutions found on
the gH sequence of HSV gH mutant viruses. LP11R and 52R are
LP11 resistant and 52S resistant, respectively. The single amino acid
code is used as follows: A, alanine; C, cysteine; D, aspartic acid; E,
glutamic acid; G, glycine; K, lysine; L, leucine; N, asparagine; Q,
glutamine; V, valine; W, tryptophan.
52S Resistant mutants
IVa
IVb
536 S-L
537 A-V
1607 C -T
161 0 C-'T
* 52SR
(HFEM)
* 5RA
(SC 16)
* 5RB
(HFEM)
Temperature-sensitive mutant
244 W-C
731 G-T
ts 026 (KOS)
FIG. 2. HSV gH mutants. Coding changes in antibody-selected
antigenic variants and the temperature-sensitive mutant tsQ26 are
shown. Sites are designated I through V. The names of the mutant
viruses are indicated at the right. Viruses marked with a black dot
were used as representatives for each group in all subsequent
V
assays.
mutant virus isolated by one of these methods was completely resistant to neutralization by the MAb LP11 or 52S
used for the selection. Antibody-resistant mutant virus,
selected by using both antibody-mediated neutralization of
infectivity and plaque inhibition, arose at a frequency of
10-6. Progeny virus obtained from cotransfection of plasmid
DNA containing the mutant gH gene with wild type-infected
cell DNA gave rise to resistant virus at a frequency of 10'3.
Coding sequence of gH gene from antibody-resistant mutant
viruses. The nucleotide sequence was derived for each
mutant gH gene and compared with the gH sequence of the
wild-type strains SC16 and HFEM. For each virus, there
was a single point mutation in the gH coding sequence which
gave rise to an amino acid substitution (Fig. 2 and 3).
Mutants resistant to MAb LP11 had amino acid substitutions
in their gH sequence at one of three sites, 86, 168, and 329,
designated sites I, II, and III, respectively. Mutant NiP3B
selected from wild-type strain HFEM had a point mutation
in the gH gene that was identical to the nonconservative
coding change found in the LP11-resistant clinical isolate
103/65 (Fig. 2). Mutant virus selected in the presence of 52S
antibody had single point mutations which gave rise to amino
acid substitutions at one of two adjacent amino acids at 536
and 537 (Fig. 2), designated sites IVa and IVb, respectively.
Multiple independently derived mutants defined sites II, III,
and IV. Mutants at sites II and III additionally have either of
two different amino acid substitutions, designated a or b. A
total of eight different gH mutant viruses from strains HFEM
and SC16 were isolated and renamed mutant viruses I, Ila,
Ilb, Illa, ITIb, IVa, and IVb depending on the amino acid
substitution or antibody used (Fig. 2). Only one antibodyresistant virus was isolated with a point mutation at site I,
but the resistance conferred by this encoded amino acid
substitution was confirmed by marker transfer. Plasmid
DNA containing the mutant VAI gH gene (site I) was
cotransfected with infected cell DNA of wild-type strain
SC16 or HFEM. Virus was selected with antibody LP11 and
cloned by limiting dilution. Both these recombinant site I
niutant viruses contained the identical site I mutation as
determined by nucleotide sequence analysis.
Properties of gH antibody-resistant mutant virus. The properties of the gH mutant viruses were examined. Because the
mutants resistant to MAb 52S or LP11 had amino acid
substitutions at distinct sites, the interactions of the mutant
viruses with these antibodies were studied by virus neutralization and immunoprecipitation. Mutant virus resistant to
LP11 could be neutralized by 52S but not by LP11, and
conversely 52S-resistant virus could be neutralized by LP11
but not by 52S (Table 1). The ability of mutation at each site
to confer resistance to neutralization appears to be the
consequence of reduced affinity of the selecting antibody for
the mutant gH molecule. Thus, Fig. 4 shows that each
mutant gH could not be immunoprecipitated by the selecting
antibody but remained precipitable by the nonselecting
antibody. The result obtained with the HFEM IVa mutant is
ambiguous because in this experiment, immunoprecipitation
by the nonselecting antibody was weak. However, the
equivalent mutant in strain SC16 (final two lanes in Fig. 4)
gave an unambiguous result.
To find whether mutations to antibody resistance resulted
in growth impairment, HEp-2 cells were infected with parental or mutant viruses at high (MOI = 10) or low (MOI =
0.1) multiplicity and incubated at various temperatures
(340C, 370C, 400C). Cultures were harvested after 16 h (high
MOI) or 48 h (low MOI), and the virus yields were assayed.
In some instances, intracellular and extracellular yields were
assayed separately. Table 2 gives the results of a high MOI
at 370C. None of the mutants with the exception of mutant I
exhibited noticeable impairment of growth by comparison
with the parental virus. Mutant I gave the lowest intracellu-
Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest
NiP3A (HFEM)
I
11
400
2397
ANTIBODY-RESISTANT MUTANTS OF HSV-1 gH
VOL. 65, 1991
TABLE 2. Yield of wild-type and gH antibody-resistant
mutant virus
TABLE 1. Neutralization of antibody-resistant mutant virusa
PFU after antibody treatment
Virus
Total PFU'
52S
LP11
Strain
10
5
8
5
11
130
123
1
90
85
90
93
1
0
HFEM
I
Ila
Illa
IlIb
IVa
IVb
Wild type
2.4
4.1
8.6
5.9
6.3
8.2
1.3
SC16
Ilb
IVa
Wild type
1.3 x 109
9.3 x 108
1.7 x 109
2.0 x 107
2.5 x 107
8.8 x 106
65 (Ila)
4.4 x 108
3.6 x 107
Intracellular
HFEM (syn)
Wild type
I
IIa
Illa
IlIb
IVa
IVb
SC16
Wild type
lIb
IVa
0
70
0
1
0
97
WT
mab
11
52
I
11
Ila
52
11
52
lila
11
52
x
x
x
x
x
x
x
105
106
107
107
106
107
107
to account for the altered growth phenotype of mutant I, and
this is under further investigation.
Properties of gH antibody-resistant mutant virus grown on
gH-producing ceH lines. The phenotype of gH antibodyresistant mutant virus was further examined in the presence
of wild-type gH. These rescue experiments tested whether
the mutant gH could be complemented by wild-type gH in
either the role of mediating infectivity or that of cell-to-cell
spread. The available evidence suggests that gH functions in
the fusion of the virion envelope with the plasma membrane
during virus penetration and is also involved in the fusion of
cellular plasma membranes in syncytium formation and
cell-to-cell spread of virus. Antibodies to gH, in particular
antibody LP11, inhibit both processes, but we do not know
whether the mechanism of inhibition by LP11 is the same for
both processes, nor indeed whether gH behaves similarly at
the virion surface and the cell surface. We therefore decided
SC 6
VW
lVa lVb WT
Illb
H FEM
vi rus
109
3.2
4.0
3.4
1.0
3.0
1.7
3.8
a HEp-2 cells (2 x 106) were infected at an MOI of 5, and virus was
harvested 16 h postinfection and titrated.
lar yield and gave a dramatically reduced yield of extracellular virus (underlined in Table 2). Experiments at other
temperatures and at low multiplicity confirmed this result.
Mutant I always gave the poorest yield (5- to 20-fold lower
than wild type) and greatly reduced yields of extracellular
virus (100-fold decrease). To determine whether the impaired growth of mutant I could be compensated by supplying wild-type gH, a high MOI experiment was repeated with
two cell lines, Vero-F6 and Vero-B1.3, each of which
provides wild-type gH in trans following infection (see next
section). Table 3 shows that the growth of mutant I is
impaired by comparison with wild-type virus regardless of
the cell type used for infection or for assay of the progeny.
Marker transfer of the site I mutation into the strain SC16
background yielded a virus which was resistant to neutralization with antibody LP11 but which exhibited no reproducible growth impairment. Thus, second-site mutations seem
st rain
108
108
108
108
108
108
Illb
1 1
52
_W41
a.
11
52
11
52
11
5;2
11
52
1\Va
11
52
_
FIG. 4. Immunoprecipitation of 35S-labeled gH from mutant and wild-type virus-infected cells. Cells were infected at an MOI of 5, and
lysates were prepared 16 h after infection. The lysates were treated with antibody LP11 or 52S, antibody-antigen complexes were purified with
protein A-Sepharose, and the products were separated by electrophoresis on a 10% polyacrylamide gel with SDS. Mutant virus are designated
by site of coding change. The gH from LP11-resistant mutant virus-infected cells is immunoprecipitated with 52S antibody, and the gH from
the 52S-resistant mutant virus was immunoprecipitated with LP11 antibody.
Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest
a Vero cells were infected with 250 PFU of virus treated with or without
antibody (1:20 dilution of ascites fluid) for 1 h at room temperature. The
virus-antibody mixture was removed, cells were washed twice with 5 ml of
medium, and the overlay was replaced with 5 ml of fresh medium. The number
of plaques are normalized from 100% for mock neutralizations. Assays were
done in triplicate.
x
x
x
x
x
x
x
Extracellular
2398
GOMPELS ET AL.
J. VIROL.
TABLE 3. Growth and titration of antibody-resistant mutant
virus I on gH-producing cell lines F6 and B1.3
Virus-infected
cells
HFEM in:
Vero
PFU as titrated in cell
Vero
Vero-F6
TABLE 4. Neutralization and plaque inhibition of antibodyresistant mutant virus grown on wild-type gH-producing cell lines
% PFU after antibody treatment"
line':
Vero-B1.3
Vero-F6
Vero-B1.3
3.2 x 108
3.3 x 107
2.2 x 107
3.3 x 108
3.6 x 107
2.4 x 107
2.6 x 108
3.8 x 107
3.8 x 107
Mutant I in:
Vero
Vero-F6
Vero-B1.3
3.8 x 107
2.4 x 106
4.7 x 106
3.5 x 107
5.5 x 106
3.7 x 106
4.5 x 107
4.3 x 106
3.5 x 106
to ask whether the antibody-resistant phenotype of gH
mutants was dominant or recessive to the wild-type phenotype and whether the same relationship would pertain at the
virion surface and the cell surface. The experiments required
virions and infected cells that contained mixtures of mutant
and wild-type gH molecules, and this was achieved using a
cell line, Vero-F6, that supplies wild-type gH in trans. The
derivation and characterization of this cell line will be
described elsewhere. Briefly, Vero-F6 cells contain multiple
copies of the gH gene under the control of an HSV-1
promoter, and when these cells are infected with HSV, gH is
synthesized from these resident genes. The following evidence shows that functional gH is produced by Vero-F6
cells: (i) a temperature-sensitive mutant (tsQ26 [17]) and a
null mutant in gH both grow to normal yields on these cells,
and (ii) HSV-2 grown on Vero-F6 cells is neutralized (>50%)
by LP11, a type 1 gH-specific antibody. The rationale of our
experiments is as follows. Mutant virus prepared on Vero-F6
cells and titrated on Vero cells can be used to measure
wild-type gH-induced sensitivity to antibody-mediated neutralization because wild-type gH will be incorporated into
mutant virions. On the other hand, virus prepared on Vero
cells and titrated on Vero-F6 cells in the presence of antibody can be used to measure wild-type gH-induced sensitivity to antibody-mediated plaque inhibition because wild-type
gH will be present on the cell surface. Vero or Vero-F6 cells
were therefore infected with mutant or wild-type virus at an
MOI of 1, and after 16 h, the virus was titrated on Vero or
Vero-F6 cells with or without antibody treatment. Mutant
IVa or IVb virus prepared on Vero or Vero gH cells showed
no change in resistance to antibody 52S, although virus
titrated on Vero gH cells showed reduced plaque size in the
presence of 52S antibody. However, mutant I, IIa, Ilb, IIIa,
and Illb viruses (the LP11-resistant mutant viruses) showed
differing sensitivities to antibody-mediated virus neutralization or plaque inhibition in the presence of wild-type gH.
Table 4 shows that these mutants yielded different results in
these experiments and, in particular, that the same phenotype did not predominate in both assays. Thus, to take the
extreme cases, for mutant IlIb the wild-type (sensitive)
phenotype predominates in neutralization, whereas the mutant (resistant) phenotype predominates in plaque inhibition.
By contrast, the results for mutant I (on a syncytial background) suggest that the mutant phenotype predominates in
neutralization, while there is a strong influence of the wildtype phenotype in plaque inhibition. For mutant IIb, the
wild-type (sensitive) phenotype is dominant in both assays.
Vero/
Vero/
Vero-gH
Vero-gH/
Vero
Vero-gH/
Vero
HFEM (syn)
Wild type
I
Ila
lIla
IlIb
0
101
93
104
121
0
55+
56+
93+
106+
0
69
58
19
15
0
29+
37+
29+
28+
SC16
Wild type
Ilb
0
115
0
22+
0
8
0
3+
Vero-gH
a Vero or Vero-gH (Vero-F6 or Vero-B1.3) cells were infected with 250
PFU of virus treated with or without antibody LP11 (1:20 ascites fluid) for 1
h at room temperature. Infected cells were overlaid with medium containing
antibody (1:200). The % PFU are normalized from 100% without antibody
treatment. Plaque inhibition is indicated as + when plaque size was greatly
reduced. Vero/Vero, cells for growth/cells for titration.
These results imply that the effect of antibody LP11 at the
virion surface and the cell surface is not the same and
suggest that the function or presentation of gH at the cell and
virion surface is different.
DISCUSSION
The results presented here show that gH has a conformationally complex epitope which is part of a domain important
for the function of gH in mediating infectivity and spread
from infected to uninfected cells. The gH-specific MAbs
LP11 and 52S, in the absence of complement, neutralize
infectivity, cell-to-cell spread, and virus-induced cell fusion
(7, 22, 49). The antibodies are specific for conformationdependent epitopes and do not recognize denatured gH.
Multiple independent isolates were selected in the presence
of antibody 52S or LP11 from syncytial and nonsyncytial
wild-type strains HFEM and SC16, respectively, and were
characterized. These antibodies appeared to recognize distinct epitopes, as variants were resistant to neutralization
only by the selecting antibody and the mutant gH could not
be immunoprecipitated by the selecting antibody but remained sensitive to the other antibody. The genes for the
MAb-selected antigenic variants were sequenced. Each mutant virus selected with MAb LP11 had a gH gene with a
point mutation encoding a single amino acid substitution at
one of three sites (residues 86, 168, and 329, designated sites
I, II, and III) which span the N-terminal half of the external
domain of the gH molecule (Fig. 2 and 3). Site II and III
mutant viruses were confirmed by multiple independent
isolates, and site II mutants were selected from strains
HFEM and SC16. The validity of the site I mutant virus was
confirmed by recombination of the mutant gH gene from
HFEM mutant I with a different parent strain, SC16. Mutant
virus selected with MAb 52S from both strains HFEM and
SC16 encoded gH sequences with single amino acid substitutions at adjacent residues (536 and 537), site IV. It is
implicit in our interpretation of the data that those mutations
that confer resistance to LP11 identify sequences that contribute to the LP11 epitope. This view is supported by the
results of immunoprecipitation experiments which show that
mutations at any one of these noncontiguous sites (site I, II,
or III) greatly reduce LP11 binding. Of course, it is conceiv-
Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest
a Cell lines Vero-F6 and Vero-B1.3 are inducible for gH expression. Cells (2
x 106) were infected at an MOI of 5, and virus was harvested 16 h
postinfection and titrated on monolayers of the indicated cell lines.
Virus
VOL. 65, 1991
ANTIBODY-RESISTANT MUTANTS OF HSV-1 gH
able that a mutation distant from an epitope will result in a
conformational change that acts at long distance to modify
the epitope, but there is no precedent for this scenario from
studies of other viral transmembrane glycoproteins and it
seems likely that such long-range effects would modify
glycoprotein function. None of the observed mutations
significantly modified the growth properties of the virus, and
we consider that the simple interpretation of the data is that
sites I, II, and III all contribute to the LP11 epitope.
Conformationally complex sites, in which different parts of a
polypeptide chain contribute to an antigenic site, have been
identified in other glycoproteins, notably in the hemagglutination molecule of influenza virus (reviewed in reference
51). Nevertheless, the LP11 epitope of HSV gH seems
unusually complex in that a single antibody can select
mutations in three distant regions of the polypeptide over a
span of 243 amino acid residues. In contrast, the 52S
epitope, which is also conformation dependent, appears
more simple in that resistant mutants contained mutations at
a single site. It may be that examination of a larger number
of mutants would reveal additional, distant, sites. However,
we have previously shown that whereas the 52S epitope can
form when gH is expressed in the absence of other HSV-1
proteins, the epitope recognized by LP11 is formed only
when other HSV-1 proteins are expressed (23). It is possible
that interaction of gH with a second viral protein is required
to stabilize the complex epitope seen by LP11, but direct
evidence for such an interaction has yet to be demonstrated.
We also know that expression of other viral products is
required for transport of gH to the cell surface; thus,
interactions that are necessary for gH conformation may be
related to cell surface transport (23). For pseudorabies virus
gB and CMV gB, oligomerization is a requisite for export
from the endoplasmic reticulum to the Golgi apparatus,
where subsequent modifications are followed by transport to
the cell surface (52, 55). Some antigenic sites are dependent
on this oligomerization (10). For HSV-1 gH, it follows that
interactions with another virus product or virus-induced
cellular product may allow export to the Golgi apparatus and
cell surface with concomitant conformational change leading
to formation of the LP11 epitope. Although sequences which
confer retention in the endoplasmic reticulum have been
described (44, 54), none of these are applicable to HSV-1
gH. Furthermore, although gH appears to be distributed
throughout the cytoplasm as expressed in the temperaturesensitive COS cell and vaccinia virus systems (18a, 23), the
prominent localization around the nuclear membrane is
visually more akin to the nuclear membrane lamin proteins
than to the resident endoplasmic reticulum protein markers,
and this may imply a subcompartmentalization of the endoplasmic reticulum (28).
gH, like other HSV glycoproteins, is acquired by the
virion envelope and by the infected cell plasma membrane
by different routes. HSV capsids appear to be enveloped at
the inner nuclear membrane, while viral glycoproteins are
expressed at the cell surface by the more familiar pathway
from the endoplasmic reticulum via the Golgi apparatus. It is
therefore conceivable that the presentation of gH at the
virion membrane and the plasma membrane is different.
Some support for this view comes from studies of the gH
mutants for growth, neutralization, and titration on cell lines
producing wild-type gH. LP11-resistant mutant viruses prepared on gH cell lines and titrated on normal Vero cells
varied in resistance to antibody neutralization. HFEM site I
and II mutant viruses remained more resistant to neutralization of infectivity than site III mutant viruses. In contrast,
LP11-resistant mutant virus prepared on Vero cells and
titrated on gH cell lines varied in resistance to antibodymediated plaque inhibition. In this system, wild-type gH is
provided in the cell postinfection and thus effects on plaque
inhibition can be separated from those on infectivity as
described above. Here, site III mutant viruses were more
resistant to plaque inhibition in the presence of wild-type gH
than site I or II mutant viruses (Table 4). Thus, antigenic
variants may differ depending on where the selecting antibody interacts with gH, either on the cell surface or on the
viral envelope. Changes at any of the three sites cause
changes in the LP11 epitope giving rise to resistance to
neutralization, but in the presence of wild-type gH these
effects may vary. Further studies of mutant gH in expression
systems will be required to investigate the mechanism giving
rise to these different phenotypes.
The envelope glycoproteins of many viruses are targets for
antibody-mediated neutralization, and in some cases these
proteins can vary to evade the immune response. This has
been shown for influenza viruses (51) and is strongly suggested for gpl20 of human immunodeficiency virus (11, 12,
18, 33, 41, 46). Neutralizing antibodies to HSV protect
animals from experimental infection and may contribute to
the control of recurrences (e.g., see reference 50), but there
is little evidence for antigenic variation of HSV glycoproteins in response to immune selection. Antibodies to gH
have been shown to be strongly protective in experimental
infection (50), but gH appears to be a poor immunogen (at
least in mice), and we know nothing about the response to
gH in natural infection of humans. Our data suggest that gH
exhibits little variation since only four conservative changes
were identified in comparisons between four isolates, two of
which have a long passage history. This is in contrast to the
extreme variation in gH sequences from herpesviruses of
different subgroups (20), in which gH is one of the most
divergent of subgroup-common proteins and similarities are
found only around cysteine residues in the C-terminal half of
the external domain. Nevertheless, it is interesting that 1 of
66 clinical isolates contained a mutation identical to that
obtained by in vitro selection with antibody LP11, and it
would be worth screening a large number of isolates with this
antibody. As the gH homologs of varicella-zoster virus,
HCMV, and Epstein-Barr virus are also targets for strong
neutralization of infectivity by respective gH-specific antibodies, a common function in virus entry is implicated;
however, gH in these systems may interact with different
cellular or viral proteins to function in mediating virus
infectivity and spread. We showed here that conformational
epitopes in the nonconserved N-terminal half of the external
domain of HSV-1 gH are recognized by neutralizing antibodies and previously showed a requirement for interactions
with virus or virus-induced products to form one of these
sites and to allow transport of gH to the cell surface (23). In
this regard, it is of interest that some evidence for interactions with cellular or viral proteins with CMV gH (gp86K)
have been presented (24, 30). Whether this is related to
observations for HSV gH or any other herpesvirus gH
remains to be determined.
2399
United Kingdom.
Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest
ACKNOWLEDGMENTS
We are grateful to Christine Lelliott for expert cell culture
assistance, Susanne Bell for purifying and testing oligonucleotide
primers, and both Anita Hancock and Mary Wright for professional
typing of the manuscript.
This work was supported by the Medical Research Council of the
2400
GOMPELS ET AL.
19. Fuller, A. O., R. E. Santos, and P. G. Spear. 1989. Neutralizing
antibodies specific for glycoprotein H or herpes simplex virus
permit viral attachment to cells but prevent penetration. J.
Virol. 63:3435-3443.
20. Gompels, U. A., M. A. Craxton, and R. W. Honess. 1988.
Conservation of glycoprotein H (gH) in herpesviruses: nucleotide sequence of the gH gene from herpesvirus saimiri. J. Gen.
Virol. 69:2819-2829.
21. Gompels, U. A., M. A. Craxton, and R. W. Honess. 1988.
Conservation of gene organization in the lymphotropic herpesviruses herpesvirus saimiri and Epstein-Barr virus. J. Virol.
62:757-767.
22. Gompels, U., and A. Minson. 1986. The properties and sequence
of glycoprotein H of herpes simplex virus type 1. Virology
153:230-247.
23. Gompels, U. A., and A. C. Minson. 1989. Antigenic properties
and cellular localization of herpes simplex virus glycoprotein H
synthesized in a mammalian cell expression system. J. Virol.
63:4744-4755.
24. Gretch, D. R., B. Kari, L. Rasmussen, R. Gehrz, and M. F.
Stinski. 1988. Identification and characterization of three distinct families of glycoprotein complexes in the envelope of
human cytomegalovirus. J. Virol. 62:875-881.
25. Highlander, S. L., W. Cai, S. Person, M. Levine, and J. C.
Glorioso. 1988. Monoclonal antibodies define a domain as herpes simplex virus glycoprotein B involved in virus penetration.
J. Virol. 62:1881-1888.
26. Highlander, S. L., D. J. Dorney, P. J. Gage, T. C. Holland, W.
Cai, S. Person, M. Levine, and J. C. Glorioso. 1989. Identification of mar mutations in herpes simplex virus type 1 glycoprotein B which alter antigenic structure and function in virus
penetration. J. Virol. 63:730-738.
27. Hill, T. J., H. J. Field, and W. A. Blyth. 1975. Acute and
recurrent infection with herpes simplex virus in the mouse: a
model for studying latency and recurrent disease. J. Gen. Virol.
28:341-353.
28. Holtz, D., R. A. Tanaka, J. Hartwig, and F. McKeon. 1989. The
CaaX motif of lamina A functions in conjunction with the
nuclear localisation signal to target assembly to the nuclear
envelope. Cell 59:969-977.
29. Johnson, D. C., M. C. Frame, M. W. Ligas, A. M. Cross, and N.
Stow. 1988. Herpes simplex virus immunoglobulin G Fc receptor activity depends on a complex of two viral glycoproteins, gE
and gI. J. Virol. 62:1347-1354.
30. Keay, S., T. C. Merigan, and L. Rasmussen. 1989. Identification
of cell surface receptors for the 86-kilodalton glycoprotein of
human cytomegalovirus. Proc. Natl. Acad. Sci. USA 86:1010010103.
31. Kousoulas, K. G., H. Bin, and L. Pereira. 1988. Antibody
resistant mutations in cross-reactive and type-specific epitopes
of herpes simplex virus type 1 glycoprotein B map in separate
domains. Virology 166:423-431.
32. Kuhn, J. E., M. D. Kramer, W. Willenbeacher, H. Wieland,
E. U. Lorentzen, and R. W. Braun. 1990. Identification of herpes
simplex virus type 1 glycoproteins interacting with the cell
surface. J. Virol. 64:2491-2497.
33. Lasky, L., G. Nakamura, D. Smith, C. Fennie, C. Shimasaki, E.
Patzer, P. Berman, T. Gregory, and D. Capon. 1987. Neutralisation of the AIDS retrovirus by antibodies to a recombinant
envelope glycoprotein. Cell 50:975-985.
34. Ligas, M. W., and D. C. Johnson. 1988. A herpes simplex virus
mutant in which glycoprotein D sequences are replaced by
,-galactosidase sequences binds to but is unable to penetrate
into cells. J. Virol. 62:1486-1494.
35. Longnecker, R., S. Chatterjee, R. J. Whitley, and B. Roizman.
1987. Identification of a herpes simplex virus 1 glycoprotein
gene within a gene cluster dispensable for growth in cell culture.
Proc. Natl. Acad. Sci. USA 84:4303-4307.
36. McGeoch, D. J., M. A. Dalrymple, A. J. Davison, A. Dolan,
M. C. Frame, D. M. McNab, L. Perry, J. E. Scott, and P. Taylor.
1988. The complete DNA sequence of the long unique region in
the genome of herpes simplex virus type 1. J. Gen. Virol.
69:1531-1574.
Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest
REFERENCES
1. Ackerman, M., R. Longnecker, B. Roizman, and L. Pereira.
1986. Identification, properties and gene location of a novel
glycoprotein specified by herpes simplex virus 1. Virology
150:207-220.
2. Bankier, A. T., and B. G. Barrell. 1983. Shotgun DNA sequencing, p. 1-33. In R. A. Flavell (ed.), Techniques in the life
sciences, vol. B5. Elsevier/North-Holland Publishing Co., Amsterdam.
3. Bell, S., M. Cranage, L. Borysiewicz, and A. Minson. 1990.
Induction of immunoglobulin G Fc receptors by recombinant
vaccinia virus expressing glycoproteins E and I of herpes
simplex virus type 1. J. Virol. 64:2181-2186.
4. Biggin, M. D., T. J. Gibson, and C. F. Hong. 1983. Buffer
gradient gels and 35S-label as an aid to rapid DNA sequence
determination. Proc. Natl. Acad. Sci. USA 80:3963-3965.
5. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction
procedure for screening recombinant plasmid DNA. Nucleic
Acids Res. 7:1513-1523.
6. Buckmaster, E. A., M. P. Cranage, C. S. McLean, R. R. A.
Coombs, and A. C. Minson. 1984. The use of monoclonal
antibodies to differentiate isolates of herpes simplex types 1 and
2 by neutralisation and reverse passive haemagglutination tests.
J. Med. Virol. 13:193-202.
7. Buckmaster, E. A., U. Gompels, and A. C. Minson. 1984.
Characterisation and physical mapping of an HSV-1 glycoprotein of approximately 115 x 103 molecular weight. Virology
139:408-413.
8. Cai, W., G. Baohua, and S. Person. 1988. Role of glycoprotein B
of herpes simplex virus type 1 in viral entry and cell fusion. J.
Virol. 62:2596-2604.
9. Cai, W. Z., S. Person, C. Debroy, and B. H. Gu. 1988.
Functional regions and structural features of the gB glycoprotein of herpes simplex virus type 1. An analysis of linker
insertion mutants. J. Mol. Biol. 201:575-588.
10. Chapsal, J. M., and L. Pereira. 1988. Characterisation of
epitopes on native and denatured forms of herpes simplex virus
glycoprotein B. Virology 164:427-434.
11. Cheng-Mayer, C., M. Quiroga, J. W. Tung, D. Dina, and J. A.
Levy. 1990. Viral determinants of human immunodeficiency
virus type 1 T-cell or macrophage tropism, cytopathogenicity,
and CD4 antigen modulation. J. Virol. 64:4390-4398.
12. Cordonnier, A., L. Montagnier, and M. Emerman. 1989. Single
amino acid changes in HIV envelope affect viral tropisms and
receptor binding. Nature (London) 340:571-574.
13. Cranage, M. P., T. Kouzarides, A. T. Bankier, S. Satchwell, K.
Weston, P. Tomlinson, B. Barrell, H. Hart, S. E. Bell, A. C.
Minson, and G. L. Smith. 1986. Identification of the human
cytomegalovirus glycoprotein B gene and induction of neutralising antibodies via its expression in recombinant vaccinia virus.
EMBO J. 5:3057-3063.
14. Cranage, M. P., G. L. Smith, S. E. Bell, H. Hart, C. Brown,
A. T. Bankier, T. Tomlinson, B. G. Barrell, and A. C. Minson.
1988. Identification and expression of a human cytomegalovirus
glycoprotein with homology to the Epstein-Barr virus BXLF2
product, varicella-zoster virus gpIII, and herpes simplex virus
type 1 glycoprotein H. J. Virol. 62:1416-1422.
15. Davison, A. J., and P. Taylor. 1987. Genetic relations between
varicella-zoster virus and Epstein-Barr virus. J. Gen. Virol.
68:1067-1079.
16. DeLuca, N., S. Person, D. J. Bzik, and W. Snipes. 1984. Genome
locations of temperature-sensitive mutants in glycoprotein gB of
herpes simplex virus type 1. Virology 137:382-389.
17. Desai, P. J., P. A. Schaffer, and A. C. Minson. 1988. Excretion
of non-infectious virus particles lacking glycoprotein H by a
temperature sensitive mutant of herpes simplex virus type 1:
evidence that gH is essential for virion infectivity. J. Gen. Virol.
69:1147-1156.
18. Dowbenko, D. G., G. Nakamura, C. Fennie, C. Shimasaki, L.
Riddle, R. Harris, T. Gregory, and L. Lasky. 1988. Epitope
mapping of the human immunodeficiency virus type 1 gpl20
with monoclonal antibodies. J. Virol. 62:4703-4711.
18a.Forrester, A., and A. C. Minson. J. Gen. Virol., in press.
J. VIROL.
VOL. 65, 1991
ANTIBODY-RESISTANT MUTANTS OF HSV-1 gH
37. McGeoch, D. J., and A. J. Davison. 1986. DNA sequence of the
herpes simplex virus type 1 gene encoding glycoprotein H and
identification of homologues in the genomes of varicella-zoster
virus and Epstein-Barr virus. Nucleic Acids Res. 14:4281-4292.
38. Messing, J. 1983. New M13 vectors for cloning. Methods
Enzymol. 10:20-78.
39. Miller, N., and L. M. Hutt-Fletcher. 1988. A monoclonal antibody to glycoprotein gp85 inhibits fusion but not attachment of
Epstein-Barr virus. J. Virol. 62:2366-2372.
40. Minson, A. C., T. C. Hodgman, P. Digard, D. C. Hancock, S. E.
Bell, and E. A. Buckmaster. 1986. An analysis of the biological
properties of monoclonal antibodies against glycoprotein D of
herpes simplex virus and identification of amino acid substitutions that confer resistance to neutralisation. J. Gen. Virol.
67:1001-1013.
41. Myers, G., S. F. Josephs, J. A. Berzofsky, A. B. Rabson, T. F.
Smith, and F. Wong-Staal (ed.). 1989. Human retroviruses and
AIDS 1989-compilation and analysis of nucleic acid and amino
acid sequences. Los Alamos National Laboratory, Los Alamos,
N.M.
42. Noble, A. G., G. T.-Y. Lee, R. Sprague, M. L. Parish, and P. G.
Spear. 1983. Anti-gD monoclonal antibodies inhibit cell fusion
induced by herpes simplex virus type 1. Virology 129:218-244.
43. Para, M. F., M. L. Parish, A. G. Noble, and P. G. Spear. 1985.
Potent neutralizing activity associated with anti-glycoprotein D
specificity among monoclonal antibodies selected for binding to
herpes simplex virions. J. Virol. 55:483-488.
44. Pelham, H. R. B. 1989. Control of protein exit from the
endoplasmic reticulum. Annu. Rev. Cell Biol. 5:1-25.
45. Pereira, L., M. Ali, K. Kousoulas, B. Huo, and T. Banks. 1989.
Domain structure of herpes simplex virus 1 glycoprotein B:
neutralising epitopes map in regions of continuous and discontinuous residues. Virology 172:11-24.
46. Reitz, M. S., Jr., C. Wilson, C. Naugle, R. C. Gallo, and M.
Robert-Guroff. 1988. Generation of a neutralisation-resistant
variant of HIV-1 is due to selection for a point mutation in the
envelope gene. Cell 54:57-63.
47. Richman, D. D., A. Buckmaster, S. Bell, C. Hodgeman, and
A. C. Minson. 1986. Identification of a new glycoprotein of
herpes simplex virus type 1 and genetic mapping of the gene that
codes for it. J. Virol. 57:647-655.
48. Sarmiento, M., and P. G. Spear. 1979. Membrane proteins
specified by herpes simplex viruses. IV. Conformation of the
virion glycoprotein designated VP7 (B2). J. Virol. 34:1159-1167.
49. Showalter, S. D., M. Zweig, and B. Hampar. 1981. Monoclonal
antibodies to herpes simplex virus type 1 proteins, including the
immediate-early protein ICP4. Infect. Immun. 34:684-692.
50. Simmons, A., and A. A. Nash. 1985. Role of antibody in primary
and recurrent herpes simplex virus infection. J. Virol. 53:944948.
51. Skehel, J. J. 1986. Antigenic variation in Hong Kong influenza
virus haemagglutinins, p. 19-24. In T. H. Birkbeck and C. W.
Penn (ed.), Antigenic variation in infectious diseases. IRL
Press, Oxford.
52. Spaete, R. R., A. Saxena, P. I. Scott, G. I. Song, W. S. Probert,
W. J. Britt, W. Gibson, L. Rasmussen, and C. Pachl. 1990.
Sequence requirement for proteolytic processing of glycoprotein B of human cytomegalovirus strain Towne. J. Virol. 64:
2922-2931.
53. Spear, P. G. 1984. Glycoproteins specified by herpes simplex
virus, p. 315-356. In B. Roizman (ed.), The herpesviruses, vol.
3. Plenum Publishing Corp., New York.
54. Stirzaker, S. C., and G. W. Both. 1989. The signal peptide of the
rotavirus glycoprotein VP7 is essential for its retention in the
ER as an integral membrane protein. Cell 56:741-747.
55. Whealey, M. E., A. K. Robbins, and L. W. Enquist. 1990. The
export pathway of the pseudorabies virus gB homolog glI
involves oligomer formation in the endoplasmic reticulum and
protease processing in the Golgi apparatus. J. Virol. 64:19461955.
56. Wildy, P., W. C. Russell, and R. W. Horne. 1960. The morphology of herpesviruses. Virology 12:204-222.
2401
Downloaded from http://jvi.asm.org/ on January 21, 2015 by guest