J. gen. Virol. (1989), 70, 1505-1512.
Printed in Great Britain
1505
Key words: HIV-1/antigenic determinant/nonapeptides
Specificity and Function of the Individual Amino Acids of an Important
Determinant of Human Immunodeficiency Virus Type 1 that Induces
Neutralizing Activity
,By R O B H. M E L O E N , 1. R O B M. L I S K A M P 2 AND J A A P G O U D S M I T 3
1Central Veterinary Institute, P.O. Box 65, 8200 AB Lelystad, 2Department of Organic
Chemistry, Gorleaus Laboratory, P.O. Box 9502, 2300 RA Leiden and 3Human Retrovirus
Laboratory, Virology Department of the University of Amsterdam, Meibergdreef 15, 1105 AZ
Amsterdam, The Netherlands
(Accepted 20 March 1989)
SUMMARY
An important antigenic determinant of human immunodeficiency virus type 1 that
induces neutralizing activity in infected humans and chimpanzees was previously
mapped with nonapeptides between amino acids 307 and 320 on the external envelope
glycoprotein (gp120) of strain HTLV-IIIB (molecular clone BH 10) and amino acids 320
to 330 of strain HTLV-IIIRF. Using different sera we found different reactive
nonapeptides that overlapped and shared a tetrapeptide, G P G R . This tetrapeptide,
which is the same in HTLV-IIIB and HTLV-IIIRF, is flanked by amino acids that vary
between virus strains. Because G P G R is predicted to form a fl-turn and is flanked by
two cysteine residues that may form a disulphide bridge, a hairpin-like structure is
suggested for this part of gpl20. The tetrapeptide G P G R and the reactive peptides are
located on top of this structure, well exposed to antibodies. We determined the role of
the individual amino acids in antibody binding using three sets of peptide analogues
derived from three reactive nonapeptides (two of strain HTLV-IIIB which overlapped
and one of strain HTLV-IIIRF). Each set contained peptide analogues in which each
amino acid was replaced, one at a time, by all genetically encoded amino acids. At least
five consecutive amino acids in each nonapeptide were essential for antibody binding.
They include amino acids of G P G R and potentially provide the virus with ample
opportunity to escape immune surveillance.
INTRODUCTION
Human immunodeficiency virus type 1 (HIV-1) induces the acquired immunodeficiency
syndrome (AIDS). Soon after infection, when neutralizing antibodies appear, free virus antigens
disappear from the serum (Goudsmit et al., 1986). After an asymptomatic period of variable
duration, HIV-1 antigenaemia may reappear even in the presence of high neutralizing antibody
activity (Goudsmit et al., 1988d). A similar sequence of events occurred when sheep were
infected with visna virus (Narayan et al., 1981), a related virus of the lentivirus subfamily.
During a visna virus infection neutralizing antibodies were induced that have an increasing
neutralization range. Eventually, new pathogenic virus mutants occurred that evaded
neutralizing antibodies. Similar events occur during an HIV-1 infection. It is suggested that
HIV-1 might be able to 'outrun' the immune response by rapidly diverging to a population of
related but distinct virus mutants (Coffin, 1986).
As treatment and prevention of AIDS may depend on our understanding of the inability of
neutralizing antibodies to stop the reappearance of virus, we studied in detail an important
antigenic determinant of HIV-1, which induces neutralizing activity. This determinant resides
on the C-terminal portion of the external envelope glycoprotein (gpl20) (Putney et al., 1986;
Rusche et al., 1987, 1988; Goudsmit et al., 1988a, d). Using sera from HIV-l-infected humans
0000-8570© 1989 SGM
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R. H. M E L O E N , R. M. L I S K A M P AND J. G O U D S M I T
and chimpanzees we identified reactive nonapeptides between amino acids 307 and 320 of strain
HTLV-IIIB (molecular clone BH10) and between amino acids 320 and 330 of strain HTLVI I I R F with the PEPSCAN method (Geysen et aL, 1984, 1985). Polyclonal and monoclonal
antibodies raised against whole or parts of gp 120 or raised against gp 160 reacted also with these
nonapeptides. The polyclonal antibodies were raised in goats with purified gp120, with
baculovirus-expressed gp 160, with C-terminal gp 120 expressed in Escherichia coli and in rabbits
with C-terminal gp120 expressed in E. coil (Goudsmit et al., 1988a, d, e). The monoclonal
antibodies were raised with viral lysate (Bahraoui et al., 1988 ; Thomas et aL, 1988). The reactive
nonapeptides were located in an area that had been predicted to be antigenic, and named V3
(Modrow et aL, 1987). Although both strains share the tetrapeptide G P G R in this
immunodominant area, antibodies induced by strains of HIV-1 related to BH10 (HTLV-IIIB
and LAV-1) react with nonapeptides from the 307 to 320 amino acid region of BH10 but do not
cross-react with nonapeptides of the corresponding 320 to 330 region of HIV-1 strain HTLVI I I R F and vice versa (Goudsmit et aL, 1988a, b, c). Thus the strain specificities of the antibodies
induced by this part of gp 120 are defined by the amino acids that directly flank the tetrapeptide
GPGR. Antibodies raised against the nonapeptides from this area, or against larger peptides
that overlap this area, neutralize the virus and show cell fusion-inhibiting activities (Ho et aL,
1987; Goudsmit et aL, 1988d; Palker et aL, 1988).
As the tetrapeptide G P G R is predicted to form a fl-turn and because it is flanked by two
cysteine residues (amino acids 296 and 331 of BH 10 and amino acids 309 and 343 of RF) which
may form a disulphide bridge, we suggested a hairpin-like structure for this part of gp120;
G P G R is at the head of the loop and the arms are linked by a disulphide bridge. We studied the
role of the individual amino acids in antibody binding with three sets of peptide analogues
derived from three reactive nonapeptides (two of strain BH 10 that overlapped and one of strain
RF). Each set was made of peptide analogues in which each amino acid, one at a time, was
replaced by all other genetically encoded amino acids.
METHODS
Sera. Serum samples (Gajdusek et aL, 1985a, b; Goudsmit et aL, 1987) were taken from three chimpanzees
(A3D, A86B and A251) inoculated with HTLV-IIIB and from one chimpanzee (A22) inoculated with LAV-1.
Two chimpanzees (A3A and A243B) were inoculated with whole blood taken from chimpanzee A22. Chimpanzee
A304 was inoculated with whole blood taken from chimpanzee A243B. Chimpanzee A233 was inoculated with
HTLV-IIIRF and chimpanzee A3 was inoculated with brain tissue from an AIDS patient with progressive
encephalopathy. Sera from humans originated from homosexual males participating in a prospective study (De
Wolf et al., 1987).
H 1 V cell fusion inhibition assay. Sup-T1 cells (1.5 x 105 to 2 x l05 cells/well) were attached to 96-well microtitre
plates as described before (Goudsmit et al., 1988c). The medium was removed and B24 + cells (5 x 103 to 25 × 103
cells/well), washed twice with phosphate-buffered saline, were added to each well in 100 ~tl of Iscove's modified
medium plus 10% foetal bovine serum and threefold dilutions of heat-inactivated (for 30 min at 56 °C) human
serum. Reciprocal dilutions were 20, 60, 180 and 540. After incubation at 37 °C for 20 min the plates were
centrifuged at 1000 r.p.m, for 10 min at 37 °C. All experiments were done in duplicate and each plate included
immune and negative control sera. Syncytia were counted after 24 h of incubation at 37 °C. Ninety percent
reduction of syncytia formation at a certain serum dilution after 24 h was taken as the titre of the syncytium
formation-inhibiting antibody in the particular serum. A titre of 1:20 was considered significant.
P E P S C A N . Peptides were synthesized on solid supports and tested as described previously (Geysen et al., 1984,
1985). To prepare a set of replacement peptides (RNET) for R G P G R A F V T , I Q R G P G R A F or I T K G P G R V I
[these peptides start at positions 312 and 310 of gpl20 of strain HTLV-IIIB (molecular clone BH10) and at position
320 of gp120 of strain HTLV-IIIRF], each of the nine amino acids in the appropriate peptide was replaced, one at
a time, by the other 19 natural amino acids, while the rest of the sequence remained the same. An R N E T consisted
of 180 peptides, including nine copies of the parent sequence as controls. The scans were tested at a 100-fold
dilution; a peroxidase-labelled anti-antibody was used as the second antibody at a 1000-fold dilution.
RESULTS
The reactive nonapeptides that we studied in detail were located previously on gpl20 of
HIV-1 between the two cysteine residues at positions 296 and 331 of strain HTLV-IIIB and at
positions 309 and 343 of strain H T L V - I I I R F (Fig. 1 ; Goudsmit et al., 1988a), and were found
always to contain the amino acids of the tetrapeptide GPRP. In other words this tetrapeptide is
conserved between both strains.
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1507
(a)
I
G
296
-C-S
331
- S-
C-
HTLV-IIIB (BH10)
T
G
309
-C-S
343
- S-
C-
HTLV-IIIRF
Fig. l. Structure of the immunodominant area on gpl20 of HIV-1 strains HTLV-IIIB (to the left) and
HTLV-IIIRF (to the right). (a) Schematic illustration of the area under investigation in each HIV-1
strain. We assumed that the cysteine residues which flank this area form a disulphide bridge between
the arms of the hairpin-like structure. At the head of the haripin-like structure the locations are shown
of the nonapeptides of HTLV-IIIB maximally reactive with chimpanzee serum A3A (RIQRGPGRA)
and with human serum 172/12 (RGPGRAFVT) and of the nonapeptide of HTLV-IIIRF maximally
reactive with chimpanzee A233 serum (KGPGRVIYA). (b) Molecular models (without the amino acid
side chain) of the immunodominant area. The structure of HTLV-IIIB (amino acids 293 to 334) and
HTLV-IIIRF (amino acids 306 to 343) were generated with Macromodel (R. M. Liskamp, unpublished
results). The amino acids important for immunological recognition are displayed with van der Waals'
dot surface.
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R . H . MELOEN, R. M. LISKAMP AND J. GOUDSMIT
Table 1. Serological reactivities o f sera o f one human and several chimpanzees infected with
HIV-1
Serum
Human
172/12
Chimpanzee
A3
A304
A3A
A3D
A243B
A233
HIV-1
inoculum
Unknown
Brain tissue
LAV-1
LAV-1
HTLV-IIIB
LAV-1
HTLV-IIIRF
HTLV-IIIB
titre*
60
540
180
60
60
-
Maximally reactive nonapeptidet
HTLV-IIIB ( B H 1 0 )
HTLV-IIIRF
RGPGRAFVT
RGPGRAFVT
RIQRGPGRA
IRIQRGPGR
IQRGPGRAF
PGRAFVTIG
-
KGPGRVIYA
* Titre is expressed as reciprocal dilution at which 90~ of syncytia occur; - indicates titre <20.
t Sequence according to Myers et al. (1987); IRIQIGKPGR of HTLV-1IIB starts at position 307 of gp120 and
KGPGRVIYA starts at position 322 of gpl20 of HTLV-IIIRF.
Table 1 shows that the serum of a human, infected with an unknown strain of HIV-1 (172/12),
reacted with nonapeptides of strain H T L V - I I I B but not with those of strain H T L V - I I I R F . The
cell fusion inhibition titres underestimated the actual neutralizing activities, because the cell
fusion inhibition test, although directly related to neutralizing activity, is less sensitive than a
test measuring neutralizing activity. Thus values in the range of 60 must be regarded as
intermediate titres and those in the range of 540 as high titres (see Palker et al., 1988). Sera of
chimpanzees (A304, A3A and A243B) infected with strain LAV-1 or with strain H T L V - I I I B
(A3D) reacted with nonapeptides of strain H T L V - I I I B but not with those of strain HTLVI I I R F . The serum from a chimpanzee (A233) infected with the latter strain reacted only with the
nonapeptides of that strain and not with those of strain HTLV-IIIB. One serum sample, of a
chimpanzee (A3) infected with brain tissue of a human who died of AIDS, did not neutralize the
virus nor did it react with the nonapeptides of either strain.
We determined the role of the individual amino acids in antibody binding using three sets of
peptide analogues derived from three reactive nonapeptides (two of strain H T L V - I I I B that
overlapped and one of strain HTLV-IIIRF). Each set was made of peptide analogues in which
each amino acid, one at a time, was replaced by all other genetically encoded amino acids; a set
of replacement peptides is named R N E T . In the P E P S C A N high densities of peptides are used.
Antibody binding with high densities of epitopes is thought to be independent of affinity
(Nimmo et al., 1984; Griswold, 1987). This could lead to an overestimation of the low affinity
bindings in the P E P S C A N as compared with other (affinity-dependent) methods. Therefore the
absence of reactivities found with this method and with peptides substituted at certain positions
seems rather significant.
One R N E T was made for the sequence R G P G R A F V T which reacted maximally with human
serum 172/12. The peptides of this R N E T were tested with sera 172/12, A304 and A3 (Fig. 2).
The results obtained using serum 172/12 show that four out of nine amino acids cannot be
replaced; three irreplaceable amino acids, P G R , are from the conserved tetrapeptide G P G R ,
the fourth irreplaceable amino acid is F. A fifth amino acid, A, can be replaced by amino acids
T, S or N only. The results obtained using serum A304 show that six out of nine amino acids are
irreplaceable. A seventh amino acid, T, can be replaced by S only.
Another R N E T was made for the sequence I Q R G P G R A F which reacted maximally with
serum A243B. The peptides of this R N E T were tested with sera A3A, A 3 D and A243B (Fig. 2).
The results with serum A3A show that five consecutive amino acids are irreplaceable: Q, R, G,
P and G. The results with serum A 3 D are the same (not shown). The results with serum A243B
show that six amino acids are irreplaceable : I, the first R, G, P, the second R and F. A seventh
amino acid, the second G, can be replaced only by M.
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Neutralizing determinant of HIV-1
R
G
1509
P
G
R
A
F
V
T
R
G
P
G
R
A
F
V
T
1
Q
R
G
P
G
R
A
F
I
Q
R
G
P
G
R
A
F
lllllqEll
m 1411l,l,i..
ltl
,I...
I
T
K
G
P
G
R
V
I
Fig. 2. Replacement set patterns of a serum from a human (172/12) infected with an unknown strain of
HIV-1, sera of three chimpanzees (A304, A3A, A243B) infected with LAV-1 (closely related to HTLVIIIB) and a serum of a chimpanzee infected with HTLV-IIIRF. The binding activity of each serum with
each peptide is shown as a vertical line proportional to the absorbance in the ELISA. Each group of 20
lines corresponds to the complete replacement set for one of the amino acid positions in a peptide.
Within each group of 20 lines the left-hand line corresponds to the substitution of the original residue by
S, and the following lines to its substitution by residues GDNEQKRYHWFMLIVCPAT. This
grouping accounts for the size and polarity of the side chain and is derived from the circle arrangement
described by Doolittle (1985), which places amino acids with similar properties near one other. To
facilitate presentation we have 'cut' the circle between T and S; thus some amino acids with similar
properties (i.e. small side chains) are shown at the left- and right-hand sides. The sequence of the parent
peptide is indicated.
A33
The last R N E T was made for the sequence I T K G P G R V I of strain H T L V - I I I R F which
reacted maximally with serum A233. The results with the peptides of this R N E T show that of six
consecutive amino acids, five are irreplaceable: K, G, P, G and V. Amino acid R, located
between the second G and V, can be replaced only with K which has properties similar to those
of R.
DISCUSSION
In this paper we studied an antigenic determinant of HIV-1 which induces neutralizing
activity. Although we do not claim that it is the sole one (Ho et al., 1988), it appears to play a
major role in the induction of neutralizing activity after infection with HIV-1 or vaccination
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1510
R . H . MELOEN, R. M. LISKAMP AND ]. GOUDSMIT
with gpl20 or gpl60 (Putney et al., 1986; Rusche et al., 1987, 1988 ; Goudsmit et al., 1988a, d, e).
The determinant induces antibodies in most if not all sera of infected humans and chimpanzees
which neutralize the virus or cause cell fusion inhibition (Goudsmit et al., 1988 a). Peptides that
span this determinant completely inhibit the fusion of HIV-infected cells (Rusche et al., 1988)
and readily induce neutralizing activity when used to vaccinate animals (unpublished results)
whereas a nonapeptide spanning G P G R is able to adsorb a substantial portion of the
neutralizing activity from sera of infected chimpanzees as well as from human sera (J. Goudsmit
& J. McKeating, unpublished results).
The reactive nonapeptides that we studied in detail were located previously on gp120 of
HIV-1 between the two cysteine residues, at positions 296 and 331 of strain HTLV-IIIB
(molecular clone BH 10) and at positions 309 and 343 of strain H T L V - I I I R F (Fig. 1 ; Goudsmit et
al., 1988 a). The peptides form an antigenic determinant which induces neutralizing activity in
the sera of primates. The determinant is located in an area on gpl20 designated V3 by Modrow
et al. (1987). The determinant spans the tetrapeptide G P G R which is conserved between
European and American strains of HIV-1 and is predicted to be a fl-turn. The amino acids that
flank G P G R vary between strains of HIV-1. We assumed that the two cysteine residues located
approximately 10 amino acids to the left and to the right of the antigenic determinant are linked
by a disulphide bridge. Fig. 1 (b) shows molecular models based on this assumption and on the
assumption that G P G R forms a fl-turn (R. M. Liskamp, unpublished results). The models for
strains HTLV-IIIB and H T L V - I I I R F are very similar. They have a hairpin-like structure in
which the arms are linked with a disulphide bridge and the head is the antigenic determinant.
The results of the R N E T experiments (Fig. 2) show, in general, similar patterns irrespective
of the species, human or chimpanzee, or of the HIV-1 strain used. Five or more consecutive
amino acids appear essential for antibody binding; the sequence always includes amino acids of
the tetrapeptide G P G R , the predicted fl-turn. Geysen et al. (1988) made the assumption that if a
certain amino acid can be replaced by a limited number of other amino acids, it too is essential
for antibody binding. In the case of human serum 172/12, we found that A can be replaced in the
R G P G R A F V T sequence by three other amino acids, T, S or N. Therefore this amino acid is
regarded as essential for antibody binding. The R N E T results show minor differences between
the human and chimpanzee sera. This agrees with observations in other systems (Geysen et al.,
1986; R. H. Meloen, unpublished results) and may reflect species-dependent differences in the B
cell repertoire.
As the amino acids of the tetrapeptide G P G R are the same in strains HTLV-IIIB and HTLVI I I R F of HIV-1, and that the antibodies raised against either strain react with homologous but
not heterologous peptides, we suggest that the amino acids of G P G R do not interact directly
with the antibody but play an indirect, although essential, role in antibody binding perhaps by
maintaining the hairpin-like structure.
There are several reasons to argue that peptides reacting with antibodies may not properly
represent the native structures reacting with antibodies. First, in the PEPSCAN, amino acid
substitutions allowed at a given position may not be aUowed in the native structure because they
are lethal to the virus. This we cannot rule out and if it is the case, it emphasizes even more the
irreplaceability of the amino acids of this determinant. The second argument is that amino acid
substitutions not allowed in the PEPSCAN are allowed in the native structure. In general the
assumption is that free peptides have a fixed structure (PEPSCAN peptides sitting at the end of
long polyacrylic strands surrounded by water), Available data suggest that this assumption may
be wrong because small free peptides do not usually have fixed or strongly favoured
conformations and are expected to adapt to the more structured antibody binding site (Getzoff
et al., 1987). To what extent the substitutions reported here will be matched by substitutions of
virus variants that escape the action of neutralizing antibodies remains to be seen. However,
limited data obtained with escape variants of another virus (foot-and-mouth disease virus) and
replacement studies did not show any incongruities (unpublished results). Thus we feel
confident that the strict structural requirements of the determinant suggested by the R N E T data
are real.
RNA viruses mutate easily and because a single amino acid change is sufficient to mask the
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Neutralizing determinant of HIV-1
1511
determinant, the presence in the determinant of five or more amino acids which are essential for
antibody binding gives the virus ample opportunity to escape immunosurveillance.
Strain specificity of the neutralizing activity in serum of HIV-1-infected individuals broadens
when the infection progresses (Goudsmit et al., 1988c) and is a general phenomenon for
lentiviruses (Kono et al., 1973 ; Henson & McGuire, 1974; Ishii & Ishitani, 1975; Narayan et al.,
1981). It has been suggested that during infection with a lentivirus mutants arise that escape the
action of the existing antibodies. The new virus mutants may induce additional antibodies and
thus broaden the strain specificity of the antisera. However antibody activity against the HIV-1
determinant, described in this report, remains strain-specific as infection progresses (J.
Goudsmit, C. Thiriart & R. H. Meloen, unpublished observation); the broadening of the
neutralizing activity is not paralleled by more extensive recognition of peptides in the V3 area.
This indicates that the broadened neutralizing activity is not induced by peptides in the V3 area.
We speculate that the determinant in the V3 area is important for protection and that the
immune system produces antibodies that recognize only the determinant on the virus that
initiated the infection. When subsequent virus mutants arise the immune system is unable to
produce changed antibodies that protect against the changed determinant. This could explain
why AIDS occurs in the presence of neutralizing activity.
We would like to thank Drs J. van Bekkum and W. M. M. Schaaper for critical reading of the manuscript. Drs
F. de Wolf, J. Lange, D. M. Asher, D. C. Gajdusek and C. J. Gibbs are thanked for the supply of serum and
clinical information. We thank W. C. Puyk and D. K u p e r u s for their excellent technical assistance.
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(Received 18 July 1988)
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