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J Virol, July 1998, p. 6218-6222, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Polymorphisms of the Hepatitis A Virus Cellular
Receptor 1 in African Green Monkey Kidney Cells Result in Antigenic
Variants That Do Not React with Protective Monoclonal
Antibody 190/4
Dino
Feigelstock,
Peter
Thompson,
Pravina
Mattoo, and
Gerardo G.
Kaplan*
Laboratory of Hepatitis Viruses, Division of
Viral Products, Center for Biologics Evaluation and Research, Food
and Drug Administration, Bethesda, Maryland 20892
Received 20 January 1998/Accepted 30 March 1998
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ABSTRACT |
Monoclonal antibody (MAb) 190/4 blocks binding of hepatitis A virus
(HAV) to the HAV cellular receptor 1 (havcr-1) and protects African
green monkey kidney (AGMK) clone GL37 cells (GL37 cells) against HAV
infection. BS-C-1 and CV-1 cells, two widely used AGMK cell lines, did
not react with MAb 190/4 but expressed havcr-1, as judged by Western
blot analysis. The cDNA coding for havcr-1 was amplified from BS-C-1
and CV-1 total cellular RNA by reverse transcription-PCR. Alignment of
the amino acid sequences inferred from the cDNA nucleotide sequences
showed that BS-C-1 and CV-1 havcr-1 differed from GL37 havcr-1 by
having two substitutions in the Cys-rich region, N48H and K108Q, and 10 to 11 additional substitutions plus the insertion of 18 to 22 amino
acids in the mucin-like region. Studies with chimeras of GL37 havcr-1
and BS-C-1 havcr-1 showed that the K108Q substitution was responsible
for the lack of reaction of MAb 190/4 with BS-C-1 and CV-1 cells. Binding studies indicated that HAV bound to dog cell transfectants expressing the BS-C-1 havcr-1 as well as the GL37/BS-C-1 havcr-1 chimeras. These results indicate that antigenic variants of havcr-1 are
expressed in AGMK cells and that binding of HAV to these havcr-1 variants tolerates changes in protective epitope 190/4.
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TEXT |
Hepatitis A virus (HAV), the
causative agent of acute hepatitis in humans, is the only member of the
hepatovirus genus of the Picornaviridae, a family of small,
nonenveloped, positive-strand RNA viruses that include human pathogens
such as poliovirus and rhinovirus (for a review, see reference
2). We recently identified the glycoprotein encoded
by the HAV cellular receptor 1 gene (HAVcr-1) as an African green
monkey kidney (AGMK) cellular receptor for HAV (3).
Nucleotide sequence analysis revealed that the HAVcr-1 cDNA codes
for a novel mucin-like class I integral-membrane glycoprotein, which was termed havcr-1, whose extracellular domain contains four
putative N-glycosylation sites and two distinctive regions: an
N-terminal, Cys-rich region that displays homology to members of the
immunoglobulin superfamily and a mucin-like, C-terminal region
containing 27 repeats of the consensus sequence PTTTTL. Our knowledge
about the interaction of HAV with havcr-1 is limited; however, Thompson
et al. (10) have recently shown that the havcr-1 Cys-rich
region and its first N-glycosylation site are required for HAV receptor
function. Monoclonal antibody (MAb) 190/4, which was raised
against the cell surfaces of AGMK clone GL37 cells (GL37 cells),
protected these cells against HAV infection and was used as a probe to
molecularly clone the HAVcr-1 cDNA. The lack of cross-reaction of MAb
190/4 with human HeLa cells (3) was puzzling; however, more
intriguing was our preliminary result suggesting that this MAb did not
react with the cell surfaces of other AGMK cell lines.
Protective epitope 190/4 is not conserved among AGMK cell
lines.
To confirm that the protective epitope 190/4 was not
conserved among AGMK cell lines, we performed a highly sensitive cell surface radioimmunoassay (Fig. 1) of
BS-C-1 and CV-1 cells obtained from the American Type Culture
Collection (ATCC). Briefly, duplicate wells of unfixed cells grown in
96-well plates were treated with twofold dilutions of ascites fluid of
MAb 190/4 or control anti-human fibronectin MAb FN-15 (Sigma Chemical
Co.) for 1 h at room temperature, washed extensively, and treated
with 0.1 µCi of 125I-labeled sheep anti-mouse antibody
(Ab) (Amersham, Inc.) per well in 100 µl of phosphate-buffered
saline-1% bovine serum albumin for 1 h at room temperature.
After being washed extensively, the plates were
autoradiographed at 
70°C for 48 h with an intensifying screen. The control MAb FN-15 reacted with GL37, BS-C-1, and CV-1 cells, whereas MAb 190/4 reacted only with GL37 cells, which indicated that the 190/4 epitope was not present in the other two cell lines.
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FIG. 1.
Cell surface radioimmunoassay of the expression of
epitope 190/4 in AGMK cell lines. Duplicate wells of GL37, BS-C-1, and
CV-1 cells grown in 96-well plates were treated with dilutions of MAb
190/4 or anti-human fibronectin MAb FN-15 ascites fluid, washed
extensively, and treated with 125I-labeled sheep anti-mouse
Ab. After being washed extensively, the plates were autoradiographed at
70°C for 48 h with an intensifying screen.
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The lack of reaction with MAb 190/4 raised the possibility that the
HAVcr-1 gene was not present in BS-C-1 and CV-1 cells.
Southern blot
analysis of genomic DNA probed with
32P-labeled,
full-length HAVcr-1 cDNA showed similar patterns of
HAVcr-1-specific
bands in BS-C-1, CV-1, and GL37 cells (data not
shown). Northern blot
analysis of total cellular RNA probed with
32P-labeled,
full-length HAVcr-1 cDNA (
3) showed that GL37, BS-C-1,
and
CV-1 cells expressed a 2.1-kb HAVcr-1-specific message (data
not
shown). To further confirm that havcr-1 was expressed in the
AGMK cell
lines, a Western blot analysis (
10) of cell extracts
from
BS-C-1, CV-1, and GL37 cells was done with the anti-GST2
Ab, which was
raised against the mucin-like region of havcr-1
expressed in
Escherichia coli (Fig.
2). Dog
cells transfected
with the GL37 HAV cr-1 cDNA, which were termed cr5
cells, or vector
pDR2 (
7,
9), which were termed DR2 cells,
were included
as controls (
10). BS-C-1 and CV-1 cells
expressed prominent
68-kDa havcr- 1-specific bands (lanes 1 and 2),
whereas GL37 cells
expressed a smaller major havcr-1 band with a
molecular mass of
65 kDa (lane 3). The cr5 cells (lane 4) expressed a
prominent
65-kDa band that comigrated with the major band expressed in
GL37
cells. The DR2 cells (Fig.
2, lane 5) did not react with the
anti-GST2
Ab, which indicated that the bands observed in the blot were
havcr-1
specific. The remaining smaller and less conspicuous bands
observed
in the blot are probably different glycosylation forms or
degradation
products of havcr-1.
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FIG. 2.
Western blot analysis of cytoplasmic extracts of AGMK
cell lines. Cytoplasmic extracts of AGMK CV-1 (lane 1), BS-C-1 (lane
2), and GL37 (lane 3) cells and control dog cells transfected with GL37
HAVcr-1 cDNA (cr5 cells [lane 4]) and vector alone (DR2 cells [lane
5]) were prepared in RSB-1% Nonidet P-40. Cytoplasmic extracts were
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(10% gel), transferred to nylon membranes, and probed with rabbit
anti-GST2 Ab. The positions and sizes of prestained molecular mass
markers are shown on the right.
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Molecular cloning of HAVcr-1 from BS-C-1 and CV-1 cells.
To
further analyze the molecular basis for the lack of reaction of MAb
190/4 with BS-C-1 and CV-1 cells, we amplified the HAVcr-1 cDNAs
from these two cell lines by reverse transcription (RT)-PCR.
To do so, total RNA was extracted from mouse Ltk
cells
(ATCC) and from GL37, BS-C-1, and CV-1 cells by using the RNASTAT-60
kit as suggested by the manufacturer (Tel-Test "B", Inc.).
First-strand cDNA was synthesized from 10 µg of total RNA with
oligo(dT) and avian myeloblastosis virus reverse transcriptase as
suggested by the manufacturer (Promega Corp.). The HAV cr-1 cDNAs were
amplified by PCR with 10% of the RT reaction and a mixture of
Taq and Pwo DNA polymerases in 30 cycles as
recommended by the manufacturer (Expand High Fidelity PCR System;
Boehringer Mannheim). Synthetic oligonucleotides (1 µg)
HAVcr-15'end
(5'-CGGATACGCGGATCCGCGCGTAGGTTTAGTTTTTGAAGTTCTTCTGTG-3'), which is positive sense and codes for a BamHI site
adjacent to nucleotides (nt) 1 to 36 of the HAV cr-1 cDNA, and
HAVcr-13'end (5'-AGAGCCTAGTCTAGA
TTTTTAGGGTGAATTAAACTCACTTTATTTCCCCAT-3'), which is negative sense
and codes for an XbaI site followed by five T residues
complementary to the poly(A) tract and the complement of nt 2071 to
2035 of the HAVcr-1 cDNA, were used as PCR primers. The PCR was
initiated by a hot start technique in a 50-µl reaction mixture
without MgCl2 but containing wax beads which, upon melting, provided a final concentration of 1.5 mM MgCl2 (HotWax
Mg+ beads; Invitrogen). HAVcr-1 cDNA PCR fragments of
approximately 2.1 kb were amplified from BS-C-1, CV-1, and GL37 cells
but not from Ltk cells. The nucleotide sequences of the
PCR fragments were determined as described previously (10)
with positive- and negative-sense synthetic oligonucleotides spaced 300 to 400 bases apart, which revealed that BS-C-1 and CV-1 cells coded for
HAVcr-1 cDNA variants of 2,127 and 2,139 bp, respectively, that shared
approximately 95% identity with the 2,076-bp GL37 HAVcr-1 cDNA.
Alignment of the nucleotide sequences of the AGMK HAVcr-1 cDNAs showed
that the difference in the lengths of the cDNAs were mainly due to nucleotide insertions in the repeat area of the mucin-like region (data
not shown).
Due to ambiguities in the 5' end sequences, we amplified the 5' ends of
the AGMK HAVcr-1 cDNAs by RT-PCR by using the conditions
mentioned
above and PCR primers cr63-83
+
(5'-GGTGGGAGACAGAGGAAACA-3'), a positive-sense
synthetic oligonucleotide
coding for nt 63 to 83 of the
HAVcr-1 cDNA, and cr403-425
(5'-TAGCGTGTCTCCTTCCGATAGG-3'), a negative-sense synthetic
oligonucleotide
coding for the complement of nt 425 to 403 of the
HAVcr-1 cDNA.
This RT-PCR analysis resulted in the amplification of a
single
band of 352 nt in GL37 cells and two bands of 352 and 404 nt in
BS-C-1 and CV-1 cells. Nucleotide sequence analysis revealed that
the 404-nt band contained an insertion of 52 nt at position
182
of the 5'-UTR of the HAVcr-1 cDNA. These data indicated that BS-C-1
and CV-1 cells coded for two HAVcr-1 cDNA forms: a short HAVcr-1
cDNA
form containing a 5'-UTR similar to that of the GL37 HAVcr-1
cDNA and a
long HAVcr-1 cDNA form containing an insertion of 52
nt in the 5'-UTR.
Alignment of the inferred amino acid sequences from the cDNA sequences
(Fig.
3A) revealed that the short forms
of the BS-C-1
HAVcr-1 and CV-1 HAVcr-1 cDNAs coded for receptors of 469 and
473 amino acids, respectively, which shared 99% identity. The
BS-C-1 havcr-1 had 92% identity with the GL37 havcr-1 and contained
2 substitutions in the Cys-rich region (N48H and K108Q) and the
insertion
of 18 amino acids corresponding to three extra repeats
plus 11 additional substitutions, i.e., T165M, M169T, M203T, R211M,
T215M,
L219I, M223T, M227T, M262T, N296D, and P307A, in the TSP-rich
region.
The CV-1 havcr-1 was similar to the BS-C-1 havcr-1; however,
it
contained a four-amino-acid insertion corresponding to an additional
repeat and did not have the L219I substitution in the mucin-like
region. No differences were found in the havcr-1 transmembrane
and
cytoplasmic domains of the three AGMK cell lines. The insertion
of 52 nt in the long forms of the BS-C-1 HAVcr-1 and CV-1 HAVcr-1
cDNAs
resulted in the putative insertion of five extra amino acids,
MADPI, at
the N terminus of the havcr-1, which increased the size
of the putative
signal sequence from 17 to 22 residues (Fig.
3B).
Since the signal
sequences are cleaved intracellularly, we expect
that the mature
havcr-1 encoded by the long and short forms of
the HAVcr-1 mRNA are
identical.
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FIG. 3.
Alignment of havcr-1 from different AGMK cell lines. (A)
Alignment of amino acid sequences predicted from the HAVcr-1 cDNAs of
BS-C-1, CV-1, and GL37 cells was done with the Clustal W program. Gaps
introduced in the sequences for the alignment are indicated by dashes;
numbers of residues starting with the respective initiating methionine
codons are indicated. Shading, identical amino acids; white letters,
the six Cys residues of the Cys-rich region; asterisk, the end of the
signal sequence; arrowhead, the beginning of the mucin-like region;
underlining, the transmembrane region. (B) Alignment of the putative
signal sequences predicted from the short and long forms of the BS-C-1
and CV-1 HAVcr-1 cDNAs compared to the GL37 havcr-1 signal sequence.
The dashes indicate untranslated sequences. The amino acid sequences
were inferred from the HAVcr-1 cDNA nucleotide sequences deposited in
GenBank (see text).
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The 2.1-kb HAVcr-1 cDNA PCR fragment amplified from BS-C-1 cells was
gel purified, cut with
XbaI, and cloned into pDR2 cut
with
BamHI, filled in with DNA polymerase I Klenow fragment
(Pharmacia
Biotech), and cut with
XbaI. The nucleotide
sequences of three
clones were obtained; one clone, whose sequence was
identical
to that of the PCR fragments and contained a 52-nt insertion
at
the 5'-UTR, was termed pDR2BS-C-1 and was used as the source of
BS-C-1 HAVcr-1 cDNA for further constructions.
The K108Q substitution in havcr-1 is responsible for the lack of
reaction with MAb 190/4.
To determine which changes in the BS-C-1
havcr-1 and the CV-1 havcr-1 were responsible for the lack of reaction
with protective MAb 190/4, we constructed chimeras between the GL37
havcr-1 and the BS-C-1 havcr-1 (Fig.
4a). Since the
anti-GST2 Ab did not react with havcr-1 expressed at the cell
surfaces of the AGMK cells and dog cell transfectants
(10), we used a FLAG-tagged GL37 havcr- 1
(10), termed GL37 flag, to construct some of the chimeras and monitor their expression at the cell surface. The unique
PvuII and BstBI cleavage sites at nt 482 and 607 of the GL37 HAVcr-1 cDNA were conserved in the BS-C-1 HAVcr-1 cDNA;
therefore, we used them to swap cDNA fragments between the two cDNAs
using standard methods as described previously (10). These
cDNA constructs were cloned into the pDR2 vector, verified by automatic
nucleotide sequence analysis, and transfected into dog cells.
Hygromycin-resistant dog cell transfectants were selected as described
previously (10). Western blot analysis with the anti-GST2 Ab
showed that all chimeras were expressed in the dog cell transfectants
and migrated with the expected molecular weights (data not shown). A
cell surface enzyme-linked immunosorbent assay (ELISA) (10)
of the dog cell transfectants with MAb 190/4 or anti-FLAG MAb M2 (Kodak
Co.) (Fig. 4b) showed that cr5 cells reacted only with MAb 190/4, that
cells expressing the GL37 flag construct reacted with MAb 190/4 and anti-FLAG MAb M2, and that DR2 cells did not react with either MAb.
These data indicated that both MAbs reacted specifically against their
corresponding epitopes at the cell surfaces of the dog cells. As
expected, dog cells expressing the BS-C-1 havcr-1 did not react with
either MAb. Dog cells expressing the q1 chimera, which differed from
the BS-C-1 havcr-1 in only a H48N substitution, did not react with
either MAb, indicating that this change did not result in the
expression of epitope 190/4. Moreover, dog cells expressing the
reciprocal construct, chimera q6, which differed from the GL37 havcr-1
in an N48H substitution, reacted with MAb 190/4, suggesting that amino
acid 48 of the Cys-rich region probably does not form part of the 190/4
epitope. However, dog cells expressing chimera q7, which contained the
whole Cys-rich region of the GL37 havcr-1 in the BS-C-1 havcr-1
background, resulted in expression of the 190/4 epitope. Dog cells
expressing the reciprocal construct, chimera q8, in the background of
the FLAG-tagged GL37 havcr-1 reacted with MAb M2, which indicated that
this chimera was expressed at the cell surfaces of the dog cell
transfectants. Because dog cells expressing the q8 chimera did not
react with MAb 190/4, we concluded that this epitope was located in the
Cys-rich region of the GL37 havcr-1. Since the N48H substitution failed
to destroy the 190/4 epitope, the only other change present in the
Cys-rich region which could account for the lack of reaction of MAb
190/4 with BS-C-1 havcr-1 was the K108Q substitution. To verify this, we constructed chimera q9 (containing a K108Q substitution in the
FLAG-tagged GL37 havcr-1 background) and chimera q10 (containing a
Q108K substitution in the BS-C-1 havcr-1 background). The cell surface ELISA showed that dog cells expressing
chimera q9 contained the M2 but not the 190/4 epitope, which indicated
that K108Q substitution was responsible for the lack of reaction with
MAb 190/4. Dog cells expressing chimera q10 reacted with MAb 190/4,
which clearly indicated that a Q108K substitution was sufficient to
induce expression of the 190/4 epitope in BS-C-1 havcr-1.
Considered together, these data showed that the K108Q
substitution in BS-C-1 and CV-1 havcr-1 was responsible for the lack of
reaction with MAb 190/4 and, therefore, suggested that amino acid
residue 108 of the Cys-rich region of havcr-1 probably forms part of
the 190/4 epitope.
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FIG. 4.
Chimeras between BS-C-1 havcr-1 and GL37 havcr-1. (a)
Schematic drawing of BS-C-1/GL37 havcr-1 chimeras. Chimeras between the
BS-C-1 havcr-1 (black box with hatching) and the GL37 havcr-1 (grey
box) containing an inserted FLAG peptide in the mucin-like region (GL37
flag) were constructed. The signal sequence and transmembrane domain
are indicated by solid black boxes. The havcr-1 amino acid residues
N48, K108, N296, and P307 of GL37 flag are in grey letters, and H48,
Q108, D296, and A307 of BS-C-1 havcr-1 are in black. Additional
substitutions and insertions in the repeat area of the mucin-like
region were not marked (see text). Chimeras q1, q6, q7, q8, q9, and q10
contain different arrangements of the residues 48, 108, 296, and 307 of
havcr-1. Chimera q6 was constructed without a FLAG tag. Restriction
sites for PvuII and BstBI endonucleases used in
the construction of the chimeras are indicated in boldface. (b)
Expression of protective epitope 190/4 at the cell surfaces of dog
cells transfectants. Expression of the 190/4 and M2 epitopes at the
surfaces of dog cells expressing GL37 havcr-1 (cr5 cells), FLAG-tagged
GL37 havcr-1 (flag cells), BS-C-1 havcr-1, and chimeras q1, q6, q7, q8,
q9, and q10 was determined by ELISA with twofold dilutions of MAb 190/4
(circles) or anti-FLAG MAb M2 (triangles). Dog cells transfected with
vector pDR2 alone (DR2 cells) were used as a negative control for the
ELISA. Absorbance at 450 nm was plotted versus the MAb dilution
starting at 0.4 µg/ml. Plotted values are means of triplicate
wells ± standard errors of the means. The results correspond to
one experiment which was repeated at least two times, with
approximately 5 to 10% experimental error. O.D., optical density.
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Binding of HAV is not disrupted by the K108Q substitution in BS-C-1
havcr-1.
To determine whether the K108Q substitution that
destroyed the 190/4 epitope also affected binding of HAV to BS-C-1
havcr-1, we performed a binding assay using the dog cell transfectants (10). To do so, duplicate wells of cells grown in 96-well
plates were treated with different dilutions of tissue culture-adapted HAV strain HM175 for 1 h at 35°C and washed extensively, and
bound HAV was detected by using 125I-labeled human anti-HAV
Ab and autoradiography (Fig. 5). The control cr5 cells bound HAV in a concentration-dependent manner, whereas cells expressing GL37 havcr-1 containing a deletion of the
Cys-rich region (d1-cells) (10) and DR2 cells did not bind virus. Dog cell transfectants expressing the BS-C-1 havcr-1, chimera q8, and chimera q9 also bound HAV in a concentration-dependent manner,
which indicated that although the K108Q substitution destroyed the
190/4 epitope, it did not abrogate binding of HAV to BS-C-1 havcr-1.
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FIG. 5.
Binding of HAV to dog cell transfectants. Dog cells
transfected with vector pDR2 (DR2 cells) and dog cell transfectants
expressing GL37 havcr-1 (cr5 cells), GL37 havcr-1 containing a deletion
of the Cys-rich region (d1 cells), BS-C-1 havcr-1,
chimera q8, and chimera q9 were grown in 96-well plates and infected
with 1:10, 1:20, and 1:40 dilutions of purified HAV HM175 for 1 h
at 35°C or mock infected (). After extensive washing, monolayers
were fixed and HAV bound to the cells was detected with
125I-labeled human anti-HAV Ab. Autoradiography of the
96-well plate showing duplicate wells for each treatment is
presented.
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Concluding remarks.
Our results showed that the BS-C-1 havcr-1
and CV-1 havcr-1 contained a K108Q substitution in the Cys-rich region
that was responsible for the lack of reaction with MAb 190/4. Ashida
and Hamada (1) have recently identified an havcr-1 variant
in S.la/Ve-1 cells, a hybrid between marmoset liver and Vero cells, as
an HAV receptor using the independently derived protective MAb 2H4,
which suggested that havcr-1 is indeed a generic receptor for HAV in monkey cell lines. This S.la/Ve-1 havcr-1 has 95.7% identity with GL37
havcr-1, has some of the characteristic features of the BS-C-1/CV-1 havcr-1, and contains the K108Q substitution; therefore, it will probably not react with MAb 190/4. Unfortunately, our anti-GST2 Ab did
not react with havcr-1 expressed at the cell surfaces of AGMK cells
(10); therefore, it cannot be used to protect BS-C-1 and
CV-1 cells against HAV infection and further prove that havcr-1 is a
general HAV cellular receptor in AGMK and other primate cells. Since
African green monkeys (AGM) (Cercopithecus aethiops) are a
diverse group of animals that were classified into four geographically distinctive subspecies (5), it is possible that the
above-mentioned havcr-1 variants correspond to different alleles of
HAVcr-1 from one or more subspecies of AGM. This receptor variability
in AGM does not seem to be unique to havcr-1, since a high degree of polymorphism has also been reported for CCR5, the major coreceptor for
macrophage-tropic isolates of human immunodeficiency virus type 1 (4).
Anti-poliovirus receptor protective MAbs react with primate cells of
different origins, such as HeLa, BS-C-1, and CV-1 cells
(
6,
8), which indicates that the protective epitope is conserved
among primates. Our data indicated that the GL37 havcr-1 protective
epitope 190/4 is conserved neither in human cells (
3) nor in
different AGMK cell lines. The natural antigenic variability of
havcr-1
in AGM allowed us to map the 190/4 epitope to amino acid
108 of the
Cys-rich region of havcr-1. The lack of reaction of
MAb 190/4 with
havcr-1 in Western blots suggested the possibility
that the 190/4
epitope is discontinuous and that other residues
located far from amino
acid 108 also form part of this epitope.
Further mutagenesis will be
required to precisely map the 190/4
epitope and to determine which
residues of havcr-1 are crucial
for the HAV-havcr-1 interaction. Since
MAb 190/4 blocks binding
of HAV to havcr-1, it is possible that HAV
interacts with the
residues that form the 190/4 epitope. If this is the
case, the
K108Q change did not affect binding of HAV to havcr-1, which
suggested
that the HAV-havcr-1 interaction can withstand some degree
of
variability at this position. However, we cannot rule out the
possibility that binding of MAb 190/4 to havcr-1 esterically blocks
binding of HAV to a site different from epitope 190/4 or induces
conformational changes in havcr-1 that inhibit binding of HAV.
Nucleotide sequence accession numbers.
The sequences obtained
in this study have been assigned GenBank accession no. AF043446,
AF043447, AF043448, and AF043449.
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ACKNOWLEDGMENTS |
We thank Stephen Feinstone for encouragement and helpful advice and
Sara Gagneten, Barry Falgout, and Hira Nakhasi for comments on the
manuscript. We also thank Michael Klutch for automatic sequencing.
This research was supported in part by the appointment of D.F. to the
Postgraduate Research Participation Program at the Center for Biologics
Evaluation and Research administered by the Oak Ridge Institute for
Science and Education through an interagency agreement between the U.S.
Department of Energy and the U.S. Food and Drug Administration.
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FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Viral Products, CBER-FDA, 8800 Rockville Pike, Bldg. 29A-NIH, Room
1D10, HFM-448, Bethesda, MD 20892. Phone: (301) 827-1870. Fax: (301) 480-5326. E-mail: gk{at}helix.nih.gov.
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J Virol, July 1998, p. 6218-6222, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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