Download Assembly of conformation-dependent neutralizing domains on

Journal of General Virology (1992), 73, 2913-2921. Printed in Great Britain
2913
Assembly of conformation-dependent neutralizing domains on
glycoprotein B of human cytomegalovirus
Ishtiaq Qadri, David Navarro,t Pedro Paz and Lenore Pereira*
Division o f Oral Biology, School of Dentistry, University o f California San Francisco, San Francisco,
California 94143-0512, U.S.A.
We analysed the antigenic properties of human
cytomegalovirus (CMV) glycoprotein B (gB) by constructing a set of deletion derivatives lacking different
portions of the carboxy terminus and reacting them
with a panel of monoclonal antibodies with neutralizing activity. We found that two novel antigenic
domains that bind neutralizing antibodies were
assembled on truncated forms ofgB, one in the aminoterminal half and one that spans the midregion of the
molecule. Assembly of the conformation-dependent
epitopes occurred independently of residues in the
carboxy-terminal half of the molecule and did not
depend on proteolytic cleavage of the molecule between
amino acids 460 and 461. Ten antibodies recognized a
derivative with 447 amino-terminal residues; their
failure to recognize a derivative 411 residues long
suggested that the amino acids required for assembly of
these epitopes either were incorrectly folded, or had
been totally or partially deleted in this derivative.
Epitopes for three antibodies with complementindependent neutralizing activity were assembled when
amino acids from the midregion of gB between residues
447 and 476 were present. Two other antigenic
domains were formed by the addition of residues 476 to
618 and 619 to 645 from the carboxy-terminal half of
gB. Our results underscore the importance of conformation in the antigenic structure and functional
properties of both the amino- and carboxy-terminal
portions of gB.
Introduction
inducing neutralizing antibodies in infected patients and
in animals immunized with the glycoprotein (Britt et al.,
1988; Gonczol et al., 1986; Rasmussen et al., 1985).
CMV gB is made as a single polypeptide chain of
approximately 120K which undergoes glycosylation and
cleavage between residues 460 and 461 during transport
through the exocytic pathway (Pereira et aL, 1984;
Radsak et al., 1990; Rasmussen et al., 1988, Spaete et al.,
1988). Cleavage of gB occurs in the Golgi compartment,
and the specificity of the calcium-dependent protease for
the target site is dependent on basic amino acids at or
near this site (Spaete et al., 1990). The gB heterodimer is
composed of the amino- and carboxy-terminal fragments, which remain disulphide-bonded (Britt, 1984)
and are anchored in the membranes of infected cells by
the hydrophobic transmembrane sequence (Spaete et al.,
1988). The amino-terminal portion, having 15 N-glycosylation sites, is heavily modified and is approximately
110K; the carboxy-terminal portion, having five glycosylation sites, is approximately 58.5K (Meyer et al., 1990;
Pereira et al., 1984; Rasmussen et al., 1988). Nucleotide
sequence analyses ofgB genes from CMV strains AD169
and Towne show sequence variation at the extreme
amino terminus between residues 28 and 67 (Cranage et
Human cytomegalovirus (CMV) is a pathogen that
causes significant morbidity and mortality in immunocompromised patients, including organ transplant recipients (Meyers et al., 1986; Singh et al., 1988) and
congenitally infected newborns (Pass et al., 1980; Stagno
et al., 1983). Among those at risk from life-threatening
CMV pneumonia and CMV chorioretinitis are patients
with AIDS (Drew, 1988; Jacobson & MiUs, 1988;
Pepose, 1989). CMV infections elicit neutralizing antibodies that react with the viral glycoproteins in infected
cells, particularly with a major virion envelope glycoprotein (Britt & Vugler, 1990; Pereira et al., 1982a, 1983,
1992; Rasmussen et al., 1991) that has been designated
glycoprotein B (gB) on the basis of sequence similarity
with herpes simplex virus 1 (HSV-1) glycoprotein B
(Chee et al., 1990; Cranage et al., 1986; Pellett et al.,
1985).
CMV gB is a major component of the virion envelope
(Farrar & Greenaway, 1986). It is highly immunogenic,
t Present address: Universityof Valencia, Facultyof Medicine,
Valencia, Spain.
0001-1092 © 1992SGM
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I. Qadri and others
al., 1986; Spaete et al., 1988). A n a l y s i s of several clinical
strains also showed sequence v a r i a b i l i t y in other regions
of the molecule, i n c l u d i n g residues 440 to 460 (Chou,
1992; C h o u & D e n n i s o n , 1991).
O u r studies o n C M V gB have focused o n m a p p i n g its
a n t i g e n i c a n d f u n c t i o n a l regions using a p a n e l of
m o n o c l o n a l a n t i b o d i e s (MAbs). A n a l y s i s of the epitopes
in the extreme a m i n o t e r m i n u s of gB revealed a region of
c o n t i n u o u s a m i n o acids t h a t b i n d s several antibodies,
i n c l u d i n g one with c o m p l e m e n t - d e p e n d e n t n e u t r a l i z i n g
activity (Basgoz et al., 1992; M e y e r et al., 1992; P e r e i r a et
al., 1991). I n the p r e s e n t study, we m a d e a set of deletion
constructs in the C M V (AD169) gB gene e n c o d i n g
derivatives that lacked c a r b o x y - t e r m i n a l sequences of
various lengths. Epitopes assembled o n these derivatives
were identified by t r a n s i e n t l y expressing the products i n
COS-1 cells a n d detecting t h e m by i m m u n o f l u o r e s c e n c e ,
a n d in some instances by i m m u n o p r e c i p i t a t i o n , using
n e u t r a l i z i n g a n t i b o d i e s to gB. W e f o u n d four conform a t i o n - d e p e n d e n t regions o n gB that b i n d n e u t r a l i z i n g
a n t i b o d i e s a n d identified the a m i n o acids required for
assembly of d i s c o n t i n u o u s epitopes in these regions. O u r
studies show that the a m i n o - t e r m i n a l half of gB b e t w e e n
residues 411 a n d 447 is required to assemble a n a n t i g e n i c
region recognized by a n t i b o d i e s with c o m p l e m e n t i n d e p e n d e n t a n d - d e p e n d e n t n e u t r a l i z i n g activity.
A s s e m b l y of the epitopes in this region is i n d e p e n d e n t of
the c a r b o x y - t e r m i n a l half of the molecule. W e also
identified the a m i n o acids r e q u i r e d to assemble epitopes
i n the m i d r e g i o n of gB that s p a n the proteolytic cleavage
site. Lastly, we m o r e precisely defined the a m i n o acids
required to assemble epitopes i n the c a r b o x y - t e r m i n a l
half of gB that had b e e n identified in a n earlier study
(Banks et al., 1989).
Methods
Construction of deletion derivatives in CMV (AD169) gB. A panel of
deletion derivatives of CMV (AD169) gB was constructed. BamHI
fragment T containing the gB gene ofCMV (AD169) was excised from
cosmid 7091 (Fleckenstein et al., 1982) and cloned into the BamHI site
of pUC18. The EagI fragment (3125 bp) encoding gB was cloned into
the SmaI site of the eukaryotic expression vector pMT2 (Wong et al.,
1985) and designated pMTgB. A derivative, pMT3, was constructed by
inserting a synthetic linker, SpeI, with stop codons in all three frames at
the blunt-ended EcoRI site. Subfragments expressing gB of various
lengths were then cloned into this plasmid at the appropriate restriction
sites. The deletion constructs were obtained by using convenient
restriction endonuclease sites within the gB gene. Plasmids p258, p411,
p447 and p476 were obtained by excising the BgllI and blunt-ended
BssHII, NdeI, ClaI and ApaLI fragments, respectively, from pMTgB
and then cloning them into the BgllI and Sinai restriction sites of
pMT3. Plasmids p618, p645 and p687 were obtained by excising the
BglII-BgllI, SalI-SalI and EcoRI-EcoRI fragments, respectively,
from pMTgB and cloning them into the compatible sites of pMT3. To
construct plasmid p760, a 907 bp EcoRI subfragment of pMTgB was
clv
NH21
1
1
1
1
100
200
300
1
1
1
258
1 ~ I
400
500
I
I[]
600 700
I
ICO,HCMV (ADI69) gB
800
906
.............
CMV gB-(1-258)
411- . . . . . . . . . .
.........
CMV gB-(1-411)
CMV gB-(1-447)
447
1
1
i
1
1
"
476. . . . . . . .
618 . . . . . .
645- . . . . .
687-76ff - - .
.
.
.
CMV gB-(1-476)
CMV gB-(1-618)
CMV gB-(1-645)
CMV gB-(1-687)
CMV gB-(l-760)
Fig. 1. Deletion constructs ofCMV (ADI69) gB. Designations for the
products of the deletion constructs of the gB gene are shown on the
right. The dashed region represents the deleted portion of the
glycoprotein. The number of amino acids contained in the mutated
forms ofgB is shown at the junction between the solid and dashed lines.
The cleavage site in the intact gB molecule is indicated by an arrow
(clv) (Spaete et al., 1988). The box indicates the transmembrane region.
first cloned into the EcoRI site of pUC18 and a SpeI linker containing
stop codons in all three frames was added to the Bali site of the gB
sequence. This subfragment, which contained a stop codon at residue
760, was cloned into the EcoRI site of the EagI fragment in the correct
orientation.
Cells and medium. COS-1 cells were obtained from the American
Type Culture Collection and were grown in Dulbecco's modified
Eagle's minimum essential medium, supplemented with 10~ foetal
bovine serum.
MAbs. The properties of the panel of MAbs to CMV (AD169) gB
produced in this laboratory and used in this study have been reported
(Banks et al., 1989; Pereira et al., 1982b, 1984). The mechanism of
neutralization by these antibodies will be described elsewhere (D.
Navarro et at., unpublished data). Cells were fixed in acetone and
stained with MAbs after 48 h as previously described (Pereira et al.,
1989). The pool of MAbs used for immunofluorescence reactions
consisted of CH45-1, CH86-3, CH408-1 and CH386-3 which recognize
continuous epitopes mapping in domain DClv (Table 2).
DNA transfections. Plasmid DNAs (10 to 20 ktg/106 cells) were
precipitated on COS-I cells using calcium phosphate. Cells were
exposed to DNA precipitates in medium containing chloroquine
(100 ~tM)for 5 h, then rinsed and fresh medium was added.
Radiolabelling, immunoprecipitation and PAGE. Plasmid DNAs were
precipitated on COS-1 ceils as described above and were labelled with
[35S]methionine(5 ~tCi/ml;DuPont New England Nuclear) from 36 to
48 h post-transfection. The cells and culture medium were extracted
with 1~ sodium deoxycholate, 1~ Nonidet P-40 and 0.1 ~ SDS. The
extracts were reacted with the antibodies and the precipitates formed
were eluted in SDS, boiled and electrophoresed in a denaturing
polyacrylamide gel (9~ polyacrylamide cross-linked with diallyltartardiamide).
Results
Transient expression o f deletion constructs o f C M V g B
T h e structure of the deletion derivatives in the gB gene of
C M V (AD169) is s h o w n in Fig. 1. D e r i v a t i v e s that
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2915
Antigenic domains assembled on C M V gB
i
i
i
i
i
i
200K-
iii iii
ii,i ¸
¸....
..... ~' : 5 . ~
Fig. 2. Immunofluorescence reactions of a pool of MAbs to CMV gB
with products of deletion constructs o f the CMV gB gene. Cells were
fixed in acetone at 48 h post-transfection and stained with a pool of
MAbs to continuousepitopesmapping in domain DClv (Table 2). (a)
Wild-type CMV gB, (b) gB-(1-258),(c) gB-(1-411),(d) gB-(1-447),(e)
gB-(1-645), (f) gB-(1-760).
spanned the amino-terminal half of gB expressed the
residues from positions I to 258, 411 and 447. The longer
derivatives expressed the residues from positions 1 to
476, 618, 645, 687 and 760 in the carboxy-terminal half
beyond the cleavage site between amino acids 460 and
461. In the first series of experiments, the derivatives of
gB were expressed by transfecting COS-1 cells with
plasmid DNAs containing the deletion constructs.
Expression of the truncated glycoproteins was first
monitored by immunofluorescence with a pool of MAbs
to continuous epitopes mapping at the extreme amino
terminus within residues 28 to 67 (Basgoz et al., 1992;
Pereira et al., 1991). Each construct expressed a protein
reactive with the antibody pool at levels approximately
equivalent to that detected in cells transfected with the
intact gB gene (Fig. 2). In general, the staining pattern
exhibited by these mutated proteins did not differ
significantly from the pattern observed in cells expressing wild-type gB. The exception was gB-(1-411) (Fig. 2c),
which showed a reticular distribution when compared
with gB and the other derivatives.
To determine whether the actual sizes of the proteins
agreed with estimates of their size based on the number
: :
Fig. 3. Immunoprecipitates of selected truncated forms of CMV gB
transiently expressed in COS-1 cells. Aliquots of radiolabelled cell
extracts were reacted with the pool of MAbs to continuous epitopes
mapping in domain DClv (Table 2). Lane M, Mr markers, gB bands
contained in the precipitates are indicated by dots.
of residues contained in the derivatives, immunoprecipitation reactions were performed with the pool of
antibodies to the extreme amino terminus of gB.
Transfected COS-1 cells were radiolabelled and precipitated as described in Methods. Fig. 3 compares the
electrophoretic mobilities of the mutated forms with that
of intact gB. Analysis of the precipitates formed from the
cell extracts showed that the truncated products migrated more slowly than the predicted size and appeared
to be modified by the addition of sugars. The shift in
mobility for each product depended on the number of
N-glycosylation sites retained after truncation of the
molecule, based on the predicted Asn-X-Ser/Thr consensus sequences in the AD169 gB gene (Cranage et al.,
1986). The apparent Mr of the derivatives was as follows:
gB-(1-258), 46K; gB-(1-411), 72K; gB-(1-447), 82K; gB(1-476), 91K; gB-(1-618), 101K; gB-(1-645), 106K; gB(1-687), 110K; gB-(1-760), 116K. Analysis of precipitates
formed after the reaction of antibodies with the culture
medium indicated that the truncated forms of gB were
poorly released from cells (data not shown).
We had noted previously that trace amounts of cleaved
gB were generated in transient expression of the CMV
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2916
L Qadri and others
Table 1. Grouping of neutralizing MAbs to CMV (AD169) gB by immunofluorescence and
immunoprecipitation reactions with truncated derivatives
Reaction with truncated derivatives
Antigenic
region
DClv
D1
D2a
D2b
D3
MAb
CH408-1
CH177-3,CH253-1, CH358-5,
CH382-2, CH388-4, CH395-1,
CH87-1t, CH92-1t,
CH105-7~, CH112-1~
CH244-4,CH130-9, CH143-13
CH424-1,CH432-1, CH434-1,
CH436-1, CH442-1, CH446-2
CH51-4, CHl14-5, CH409-2
258*
411
447
476
618
645
687
760
+
--
+
-
+
+
+
+
+
+
+
+
+
+
+
+
-
-
-
+
-
+
+
+
+
+
+
+
+
+
+
+
.
.
.
.
.
* Numbers indicate the length (no. of amino acids) of CMV (AD169) amino-terminal derivatives.
t Immunofluorescencereactions were weak but reproducible with CMV gB-(1-447).
strain Towne gB gene (Banks et al., 1989). In the present
study, the carboxy-terminal cleavage fragments expected
to be generated from intact gB were again barely
detectable. Cleavage of the longer derivatives gB(1-645), gB-(1-687) and gB-(1-760) was not detected even
though the size of these fragments would have been
within the separation limits of this gel and others of
higher acrylamide concentration. Insofar as intact gB
and the longer derivatives are not cleaved in COS-1 cells
(Fig. 3), it appears that this cell type does not contain
significant amounts of the calcium-dependent protease
found in C H O cells stably expressing a truncated form of
the gB gene (Spaete et al., 1990). We have reported that
the panel of MAbs to CMV gB immunoprecipitates both
the intact form of gB and the faster migrating cleavage
products (Pereira & Hoffman, 1986; Pereira et al., 1984);
thus cleavage does not affect the conformation of these
epitopes. In order to confirm that uncleaved gB
molecules do not differ significantly in conformation
from cleaved forms, a C H O cell line expressing the
mutant CMV gB pXgB24clv4, which contains three
altered residues near the cleavage site (Spaete et al.,
1990), was reacted with a subset of MAbs to domains D1,
D2a, D2b and D3 (Table 2) spanning the length of the
molecule (D. Spaete, unpublished observations). It was
found that the antibodies recognized the uncleaved
derivative ofgB, indicating that lack of cleavage does not
affect these epitopes.
MAbs recognize epitopes assembled by truncated
derivatives of gB
In the next series of experiments, we located the amino
acids required to assemble the conformation-dependent
neutralizing epitopes on gB by reacting each truncated
glycoprotein in immunofluorescence tests with the
individual MAbs. Results of these experiments (Table 1)
are summarized as follows. (i) The shortest glycoproteins, gB-(1-258) and gB-(1-411), did not assemble any of
the discontinuous epitopes detected by the antibody
panel. However, gB-(1-258) contained the continuous
neutralizing epitope recognized by antibody CH408-1,
which had been shown previously to map in domain
DC 1v in the extreme amino terminus (Basgoz et al., 1992;
Meyer et al., 1992; Pereira et al., 1991). (ii) In contrast to
the results obtained with gB-(1-411), analysis of the next
longer form gB-(1-447), comprising almost the entire
amino-terminal half of gB, showed that 10 conformationdependent epitopes were present. Of these epitopes, five
(CH177-3, CH253-1, CH358-5, CH382-2 and CH388-4)
had complement-independent neutralizing activity and
five had complement-dependent activity (Table 2) (D.
Navarro et al., unpublished data). Four antibodies
(CH87-1, CH92-1, CH105-7 and CHII2-1) reacted
weakly but reproducibly with gB-(1-447), indicating that
their epitopes, grouped here in domain D1, may be only
partially assembled on this form of the molecule. The
neutralizing domain assembled when amino acids 411 to
447 are present, designated domain D1, is the first
discontinuous antigenic region to be identified in the
amino-terminal half of CMV gB. (iii) Three additional
discontinuous epitopes, recognized by neutralizing antibodies CH244-4, CH130-9 and CH143-13, were assembled on gB-(1-476). These epitopes were grouped into
domain D2a and are assembled when residues 447 to 476
are present. (iv) Six more discontinuous epitopes,
recognized by antibodies CH424-1, CH432-1, CH434-1,
CH436-1, CH442-1 and CH446-2, were expressed on gB(1-618). These epitopes were grouped into domain D2b,
which is assembled by the addition of residues 476 to 618
in the carboxy-terminal half of gB. (v) gB-(1-645)
contained epitopes for three neutralizing antibodies,
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Antigenic domains assembled on C M V g B
2917
Table 2. Grouping o f 36 epitopes into antigenic domains on C M V (AD169) gB
Antigenic domain/
epitope location
MAb
Neutralizing
activity*
DCI~, amino terminus: 28 to 67
N-1
CH408-1
N-2
CH45-1
N-3
CH86-3
N-4
CH386-3
N-5
CH396-3
N-6
CH404-4
N-7
CH412-2
DI, midregion: 411 to 447
M-1
CH177-3
M-2
CH253-1
M-3
CH358-5
M-4
CH382-2
M-5
CH388-4
M-6
CH395-I
M-7
CH87-1
M-8
CH92-1
M-9
CHI05-7
M-10
CHl12-1
D2a, midregion: 447 to 476
M-11
CH244-4
M-12
CH130-9
M-13
CH143-13
D2b, carboxy-terminal half: 476 to 618
C-1
CH424-1
C-2
CH432-1
C-3
CH434-1
C-4
CH442-1
C-5
CH446-2
C-6
CH436-1
D3, carboxy-terminal half: 618 to 645
C-7
CH51-4
C-8
CH114-5
C-9
CH409-2
DC2, intracellular: 716 to 906
I-1
CH158-2
I-2
CH216-2
I-3
CH380-4
1-4
CH381-1
1-5
CH385-3
1-6
CH402-5
1-7
CH410-3
DC3, intraceUular: 833 to 898
I-8
CH405-1
1-9
CH421-5
1-10
CH28-2
-
Epitopes expressed on gB deletion derivatives,
fusion proteins and synthetic peptides
Present on AD169 gB-(I-258) and on fl-galactosidase
fusion protein, amino acids 27 to 84t
Assembled on gB-(1-447) but not gB-(1-411)
+
+
+
+
+
Assembled on gB-(1-476) but not gB-(1-447)
+
+
+
+
+
+
+
-
Assembled on gB-(1-618) but not gB-(1-476)
(antigenic region identified by others:~)
Assembled on gB-(1-645) but not gB-(l-618)
(antigenic region identified by otbers:~)
Present on gB-(716-906)t but not gB-(1-832)
Present on gB-(716-906)t but not gB-(1-832)
Peptide (833 to 852)
Peptide (833 to 852)
Peptide (878 to 898)
Complement-independent (D. Navarro et al., unpublished data).
t (Basgoz et al., 1992; Meyer et al., 1992; Pereira et al., 1991).
:~(Kniess et al., 1991; Liu et al., 1991; Utz et al., 1989; Spaete et al., 1988).
*
CH51-4, C H l 1 4 - 5 a n d CH409-2, s h o w n i n a n earlier
study to be a s s e m b l e d w h e n residues 619 to 680 are
p r e s e n t (Banks et al., 1989). T h i s result i n d i c a t e d t h a t the
residues required for assembly of these d i s c o n t i n u o u s
epitopes are located b e t w e e n a m i n o acids 619 a n d 645 in
the carboxy t e r m i n u s , in a d o m a i n w h i c h we have
d e s i g n a t e d D3. (vi) gB-(1-687) a n d gB-(1-760) expressed
all of the d i s c o n t i n u o u s epitopes c o n t a i n e d i n gB-(1-645),
b u t failed to express the c o n t i n u o u s epitopes i n d o m a i n s
D C 2 a n d D C 3 , w h i c h h a d b e e n previously m a p p e d i n the
intracellular carboxy t e r m i n u s (Basgoz et al., 1992;
Pereira et al., 1991).
Discontinuous epitopes assembled by residues in the
amino-terminal half and midregion o f g B
Some of the epitopes f o u n d i n the p r e s e n t study to
assemble i n d o m a i n s D1 a n d D 2 were reported to require
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I. Qadri and others
CMV gB-(1-447)
7
CMV gB-(1-618)
CMV gB-(1-411)
on
t-~
: i¸¸ '
eq
"7,
i¸¸~:~: :iJ
97K-
~,~,
,
............
69K--
46K--
Fig. 4. Immunoprecipitates of truncated CMV (AD169) gB-(l-411)
and gB-(1-447) reacted with individual MAbs to gB. COS-1 cells were
prepared as described in Methods. The pool contained MAbs to
continuous epitopes. Lane M, Mr markers, gB bands contained in the
precipitates are indicated by arrows.
residues closer to the carboxy terminus of gB for their
formation in an earlier study in which we used two
deletion constructs of CMV strain Towne gB expressing
618 and 680 amino acids (Banks et al., 1989). Immunofluorescence analysis of the shorter AD 169 gB constructs
now indicated that these epitopes are formed by
assembling residues closer to the amino terminus of the
molecule. To confirm the location of these epitopes in the
amino-terminal half of gB, we performed two immunoprecipitation experiments. In the first experiment, we
reacted selected antibodies from domain D 1 by immunoprecipitation with the short derivatives gB-(1-411) and
gB-(1-447) (Fig. 4). Antibody CH408-1, recognizing the
amino-terminal domain DClv, and antibody CH442-1,
recognizing domain D2b between residues 476 and 618,
served as positive and negative control antibodies,
respectively. We found that antibodies CH177-3 and
CH382-2, which had and had not reacted with the 618residue Towne construct respectively, precipitated
gB-(1-447) but failed to recognize the shorter mutant
gB-(1-41t), confirming the immunofluorescence experiments showing that these epitopes map in the aminoterminal half of gB. As expected, antibody CH408-1
recognized both of the AD169 gB derivatives and
antibody CH442-1 failed to react with either.
In the second experiment, several of the antibodies
Fig. 5. Immunoprecipitates of truncated CMV (AD169) gB-(1-618)
reacted with selected MAbs to gB. COS-I cells were prepared as
described in Methods. The pool contained MAbs to continuous
epitopes. Lane M, Mr markers, gB bands contained in the precipitates
are indicated by an arrowhead.
that had failed to react by immunofluorescence with the
618-residue Towne gB construct (Banks et al., 1989), but
did react by immunofluorescence with the AD169
construct of the same length (Table 1), were tested in
immunoprecipitation reactions with this AD169 construct. Of these antibodies, CH382-2, CH395-1,
CH105-7 and CH112-1 recognized antigenic region D1,
CH244-4 and CH130-9 recognized region D2a, and
CH434-1, CH436-1 and CH446-2 recognized region
D2b. We also included three antibodies (CH409-2,
CH51-4 and CHl14-5) that were non-reactive by
immunofluorescence with both the Towne and AD169
constructs, and one antibody (CH412-2) to domain DC l v
as a positive control (Pereira et al., 1991). The results
obtained by immunoprecipitation (Fig. 5) agreed with
the results obtained by immunofluorescence with three
exceptions. Antibodies CH105-7 and CHll2-1, which
were positive by immunofluorescence, failed to precipitate the derivative. In this case, it is likely that the
epitopes present in the native form were lost from the
detergent-solubilized derivative. In contrast, CH244-4,
which was negative in immunofluorescence tests, reacted with the mutant construct by immunoprecipiration, suggesting that this epitope was either not
properly assembled or not accessible to the antibody on
the folded form of the derivative. It is possible that
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A n t i g e n i c d o m a i n s a s s e m b l e d on C M V g B
N
1
NH21
M
100
200
300
400
D1, ~)2 and D2b ~J
Major functionalregion: ~
promotesvlnon entry, I
cell-to-censpread of
I
infectionand fusion
J
Neutralizingdomain
Var ab e ep topes
C
500
II
[
I
[
[
lmmunodominant
neutralizingdomain
I
700
600
~
800
906
CO2H
DC2 DC3
~Continuous epitopes
I
Neutralizingdomain
Diseontinousepitopes
Fig. 6. Topographic map of the antigenic and functional domains
mapped on CMV (AD169) gB. Antigenic domains are shown as
ellipses. Domains DCIv, DC2 and DC3 containing continuous
epitopes were mapped previously(Basgozet al., 1992; Pereira et aL,
1991). Boxesshowour designationsfor the antigenic regions of gB and
list the functional properties that have been identified for certain
neutralizing domains (D. Navarro et al., unpublished data). Four
antigenic regionsof the gB moleculeare indicated: N (aminoterminus;
positions 28 to 67), M (midregion; 411 to 476), C (carboxyterminus;
476 to 645) and I (intracellular; 719 to 906). The cleavage site is
indicated by an arrow (clv) (Spaete et al., 1988). The shaded box
designates the hydrophobic transmembrane domain (Cranage et aL,
1986). Immunodominant neutralizing domain D2b and neutralizing
domain D3 contain antigenic sites mapped by other laboratories
(Kniess et al., 1991; Liu et al., 1991 ; Spaete et aL, 1988;U tz et al., 1989).
different results obtained with the Towne construct in
the previous study (Banks et al., 1989) were due to small
differences in the amino acid sequence between the
strains; however, this seems unlikely since the antibodies
that failed to react with the Towne derivative nevertheless recognized epitopes shared by the intact gB of both
strains AD169 and Towne. The most likely explanation
is that the differences in reactivities with the Towne
construct may have resulted from a small spontaneous inframe internal deletion generated during the construction of this mutant gB. Particularly relevant to the
folding of the truncated forms of gB are four cysteine
residues mapping between amino acids 411 and 618. A
mutation that altered a cysteine residue might have led to
mispairing of the cysteines, causing formation of
mismatched disulphide bonds, partial malfolding of the
molecule and loss of selected epitopes. The detailed
analysis of the set of eight deletion derivatives in the
AD169 gB gene that we have now completed enables us
to locate correctly the amino acids required to assemble
the conformation-dependent epitopes in the various
antigenic regions of the molecule.
Discussion
In this study, we generated from the CMV gB gene a set
of deletion constructs that lacked different lengths of the
sequences specifying the carboxy terminus, in order to
2919
locate residues that confer reactivity with our panel of
MAbs. Table 2 summarizes our findings to date
regarding the antigenic regions of CMV (AD169) gB,
which have been analysed by identifying amino acids
required to assemble the epitopes recognized by neutralizing antibodies. The locations of these regions are
summarized in a schematic diagram (Fig. 6). We have
divided the gB molecule into four antigenic regions: the
amino (N) terminus residues from 28 to 67, the
midregion (M) residues from 411 to 476, the carboxy (C)
terminus residues from 476 to 645, and the intracellular
(I) residues from 716 to 906. Epitopes of antibodies
recognizing each antigenic region have been grouped in
Table 2 by their location in the gB molecule, e.g. N-I,
M-l, etc.
We have identified three regions of continuous
epitopes: DClv, a neutralizing domain in the amino
terminus region which contains strain-specific epitopes,
and DC2 and DC3, located in the intracellular region of
the molecule (Basgoz et al., 1992; Pereira et al., 1991).
Our work and that of others (Meyer et al., 1990) has
shown that nucleotide sequence differences in the
extreme amino terminus of gB specified by strains
AD169 and Towne (Spaete et al., 1988) affect their
antigenic properties. Both conserved and variable
antigenic sites are contained in the region encompassing domain DClv (Meyer et al., 1992). Other investigators have identified continuous neutralizing epitopes
mapping between residues 589 and 645 (Kniess et al.,
1991) and between 608 and 625 (Utz et al., 1989). Discontinuous neutralizing epitopes are assembled in four
domains, D1, D2a and D2b, and D3. Domain D1, one of
two novel conformation-dependent domains identified
in this study, is assembled in part by the folding of
residues 411 to 447 in the amino-terminal half of gB.
Domain D2a, the second novel domain, is assembled in
the midregion by residues 447 to 476, spanning the
cleavage site. Domain D2b, also in the midregion, is
assembled by amino acids 476 to 618, and domain D3 by
amino acids 619 to 645 in the carboxy-terminal half of
the molecule. The antigenic region designated AD-1 by
others (Kniess et al., 1991), which contains a continuous
epitope mapping between residues 589 and 645, partially
overlaps domains D2 and D3. In locating the recognition
sites of a large number of neutralizing MAbs to the
region of gB between residues 476 and 645, our studies
and those of others suggest that this region is immunodominant (Banks et al., 1989; Kniess et al., 1991 ; Liu et
al., 1991; Spaete et al., 1988; Utz et al., 1989).
Since most of the neutralizing epitopes in our panel of
antibodies are dependent on the conformation of gB, the
formation of the antibody recognition sites may require
the juxtaposition not only of discontinuous amino acids
composing those sites but also of other discontinuous
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2920
I. Qadri and others
residues in the amino-terminal half of gB that promote
the proper folding of the antigenic domains identified in
the present study. We are in the process of constructing
mutant gB molecules with small deletions spanning the
antigenic domains of the molecule to distinguish more
precisely the amino acids that affect the epitope folding
from those which are assembled into the antibody
recognition site (I. Qadri & L. Pereira, unpublished
data). Preliminary analysis of the antigenic properties of
these mutated forms showed that deletion of residues 170
to 411 and of residues 411 to 447 precludes assembly of
domain D1 epitopes. This finding shows that amino
acids 170 to 411 also play a role in assembling these
epitopes. In addition, domain D3 was perturbed by
deleting residues 411 to 447, which supports our finding
that folding of the carboxy-terminal half of gB depends
on certain residues in the amino-terminal half of the
molecule. Epitopes in domain D3 are lost when residues
548 to 618 are deleted, suggesting that adjacent residues
participate in the antigenic structure of this region. No
antigenic changes were induced by deleting residues 645
to 702, suggesting that this region is not directly involved
in assembling epitopes in the extracellular region of gB,
as indicated in the present study. We recently carried out
a detailed analysis of the neutralizing activities of
complement-independent antibodies to CMV gB, which
revealed that they prevent virion entry by blocking
fusion of the viral envelope with the cell membrane, the
spread of infection from cell to cell, and the formation of
syncytia by infected glioblastoma cells (D. Navarro et al.,
unpublished data). The detailed mutational analysis of
CMV gB now underway will enable us to formulate a
model for the folding of the molecule that is consistent
with its antigenic structure and its function in virion
infectivity and the cell-to-cell transmission of infection.
We thank Richard Spaete for analysing the CHO cell line expressing
the CMV gB mutant pXgB24clv4 with the panel of MAbs and Scott
Frank for assisting in the analysis of neutralizing epitopes. These
studies were supported by Public Health Service grants A123592 and
AI30873 from the National Institute of Allergy and Infectious Diseases
and by grant HL33713 from the National Heart and Lung Institute to
Stanford University. D.N. was supported by a fellowship from the
Spanish Ministry of Education and Science.
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