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Journal of Virology, May 1999, p. 4475-4480, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Monoclonal Antibodies against the Minimal
DNA-Binding Domain in the Carboxyl-Terminal Region of Human
Immunodeficiency Virus Type 1 Integrase
Tetsuya
Ishikawa,1,2
Nobuo
Okui,1,3
Noriko
Kobayashi,1
Ryuta
Sakuma,1
Tadaichi
Kitamura,3 and
Yoshihiro
Kitamura1,*
Division of Molecular Genetics, National
Institute of Infectious Diseases,
Musashimurayama,1 and Department of
Urology, Faculty of Medicine, University of Tokyo,
Bunkyo-ku,3 Tokyo, and Institute of
Biomedical Science, Terumo Corporation R. & D. Center,
Ashigarakami-gun, Kanagawa,2 Japan
Received 21 October 1998/Accepted 13 February 1999
 |
ABSTRACT |
Integrase of human immunodeficiency virus type 1 (HIVIN) consists
of 288 amino acids, and its minimum DNA-binding domain (MDBD) (amino
acids [aa] 220 to 270) is required for the integration reaction. We
produced and characterized four murine monoclonal antibodies (MAbs) to
the MDBD of HIVIN (strain LAI). Immunoblot and enzyme-linked
immunosorbent assays with truncated HIVINs showed that those MAbs
recognized sequential epitopes within the MDBD (aa 228 to 236, 237 to
252, 253 to 261, and 262 to 270). Their binding to HIVIN inhibited
terminal cleavage and strand transfer activities but not disintegration
activity in vitro. This collection of MAbs is useful for studying the
structure and function of the MDBD by complementing mutational analyses
and other biochemical studies.
| |
TEXT |
Integration of a DNA copy of the
viral RNA genome into a chromosome of the host cell is an essential
step in the retroviral life cycle (4, 21, 41). The viral
enzyme integrase (IN) catalyzes the process in three steps (5,
19). First, two nucleotides are removed from the 3' ends of the
viral DNA (in a process known as terminal cleavage [TC]). Second, the
recessed 3' ends of the viral DNA are then joined to 5' staggered sites in the target DNA in a concerted cleavage and ligation reaction (in a
process known as strand transfer [ST]). Finally, integration is
completed by repair of the short gaps flanking the viral DNA intermediate. The TC and ST reactions can be reproduced in vitro with
purified IN and double-stranded oligonucleotide substrates that mimic
the ends of viral DNA (6, 8, 23, 38). Furthermore, IN
catalyzes a reversal of the ST reaction in vitro (disintegration) with
a branched-DNA substrate (Y-mer) that mimics the product of the ST
reaction (11).
Biochemical analysis of IN from human immunodeficiency virus type 1 (HIV-1) has revealed that the C-terminal region (amino acids [aa] 160 to 288) contains nonspecific DNA-binding activity (18, 31, 40,
42), which is mapped to aa 220 to 270 (the minimum DNA-binding
domain [MDBD]) (30, 31). Analyses by nuclear magnetic
resonance also revealed that the MDBD consists of a five-stranded 
-barrel similar to that of Src homology region 3 domains forming a
homodimer (14, 29). Mutational analysis showed that the MDBD
is essential for TC and ST activities of HIV-1 IN (HIVIN) (7, 13,
15, 16, 26), whereas it is dispensable for disintegration
activity (13, 30, 37, 39, 40). Mutations in this region
abolish viral DNA synthesis (reverse transcription), implying that
HIVIN interacts with reverse transcriptase (RT) (17, 27,
32). Moreover, substitution of the W235 residue within the MDBD
does not affect in vitro TC and ST activities, whereas the virus
mutants carrying those substitution mutations cannot replicate (9,
10, 27, 28). But the function of the MDBD is not well
characterized by monoclonal antibodies (MAbs), partly because few MAbs
to the MDBD have been cloned (3, 33). This study presents a
collection of MAbs reactive against the MDBD and demonstrates the
effects of MAb binding on various in vitro activities, such as TC and
ST activities, and on the capability of HIV-1 to interact with RT.
Production of MAbs against IN.
Female BALB/c mice were
immunized primarily with HIVIN fused to Escherichia coli
maltose-binding protein (MBP) and thereafter with HIVIN, with the
N-terminal 20 aa residues containing a hexahistidine tag. MBP-HIVIN and
hexahistidine-HIVIN were expressed and purified as described in
references 7, 34, and 36, with
equipment from New England Biolabs, (NEB), Beverly, Mass., and Novagen, Madison, Wis. Spleen cells of the mice were fused with P3-X63-Ag8.653 mouse myeloma cells (22, 24). Screening culture supernatants by enzyme-linked immunosorbent assay (ELISA) with hexahistidine-HIVIN identified about 50 hybridomas. Twelve hybridomas were successfully subcloned by limiting dilution and then injected into the peritoneal cavity of pristane-primed female BALB/c mice to obtain ascites fluid
containing MAbs. Nine hybridomas produced enough ascites fluid for
further analyses. Each MAb was purified with a HiTrap protein A column
(Amersham Pharmacia Biotech Ltd., Little Chalfont, United Kingdom)
followed by subsequent dialysis against 20 mM HEPES-Na (pH 7.5).
Immunoblot analysis with MBP-HIVIN and six-His-tagged HIVIN (data
not shown) showed that six clones (7-19, 8-6, 2-19, 8-22, 4-20, and
6-19) were specific to HIVIN (Fig. 1),
whereas the others were specific to the hexahistidine tag.
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FIG. 1.
Schematic representation of epitopes recognized by the
MAbs. The upper part of the figure shows a linear map of HIVIN. The N
terminus (aa 1 to 49) with the HHCC motif, the central catalytic core
(aa 50 to 159) with the DD(35)E motif, and the C terminus (aa 160 to
288) with DNA-binding activity domains are shown. A hatched box shows
the MDBD (aa 220 to 270). The epitope regions recognized by the
individual MAbs are shown below the IN map with the clone numbers. The
lower part of the figure shows a linear map of HIVIN spanning from aa
160 to 288. Five MAbs recognized epitopes within this region. The
numbers above the map indicate amino acid positions. Individual MAbs
are shown by clone numbers below the IN map, with arrows indicating
epitope regions. The three asterisks in the map indicate predicted
-strands, which may form an interface of homodimerization in a
structure of triple-stranded antiparallel -sheets (14,
29).
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These MAbs displayed two isotypes (Table
1): immunoglobulin G2b (IgG2b) (clones
7-19, 2-19, and 6-19) and IgG1 (clones 8-6, 8-22, and 4-20).
Semiquantitative ELISA (3) (Table 1) demonstrated that the
titers of MBP-HIVIN varied about 150-fold: the minimal antibody
concentration required for detection of IN with MAb 7-19 was the
highest, whereas that with MAb 6-19 was the lowest.
Epitope mapping.
The epitopes for the purified MAbs were
determined by reactivity to HIV/Rous sarcoma virus (RSV) chimera INs
and HIVIN deletion mutants by immunoblot analysis and ELISA (summarized
in Fig. 1; also see Table 2 and Fig. 2 and 3). RSV (strain CS8) IN
fused to MBP was described previously (25). Similarly,
mutants of HIVIN with deletions from aa 271 to 288 (HIVIN ending at aa
270 [HIVIN270]), HIVIN261, HIVIN252, HIVIN236, HIVIN227, HIVIN210, and HIVIN185 were expressed and purified as MBP fusion proteins. HIV/RSV chimera INs {HIVIN aa 1 to 236 with RSV aa 235 to 286 [H(1-236)R(235-286)], H(1-159)R(160-286), and R(1-36)H(40-288)} were also obtained as MBP fusion proteins.
The results of ELISA (Table
2) and
immunoblot analysis (Fig.
2) showed that
MAbs 7-16 and 8-6 recognized an epitope within
the central catalytic
domain of HIVIN and the region from aa 211
to 227, respectively.
Furthermore, we inferred that the epitopes
for MAbs 2-19, 8-22, 4-20, and 6-19 are likely to be contained
in a simple linear sequence in
tandem within the MDBD (Fig.
1 and Table
2). None of these MAbs
recognized RSV IN (Table
2)
or HIV-2 IN (data not shown). The epitopes
for MAbs 2-19 (from
aa 228 to 236) and 6-19 (from aa 261 to 270) are
likely to protrude
outwards, whereas those for MAbs 8-22 (from aa 237 to 252) and
4-20 (from aa 253 to 261) form a homodimer interface
(
14,
29).
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FIG. 2.
Effect of amino acid change on affinity of MAb 2-19. (A)
Immunoblot analysis of various INs with MAb 2-19. Recombinant INs
expressed in E. coli were separated in an SDS-10%
polyacrylamide gel and blotted onto a nitrocellulose membrane. The
proteins were probed with MAb 2-19. Lane 1, MBP-LacZ ; lane 2, MBP-IN
(LAI); lane 3, MBP-IN (NL4-3); lane 4, MBP-IN (BH10). The arrow shows
the position of MBP-HIVIN. (B) Amino acid sequences from aa 228 to 236 of HIVIN strains LAI, NL4-3, and BH10 are shown. Dashes indicate that
the amino acid residues are the same as the corresponding residues of
the IN of the LAI strain. There are no amino acid changes in the region
from aa 237 to 270 among those strains. A panel of MBP-IN proteins were
separated by SDS-PAGE and blotted onto a nitrocellulose membrane. The
blotted proteins were analyzed with MAb 2-19 (C) or MAb 8-22 (D). Lanes
1, MBP-IN (wild-type LAI); lanes 2, MBP-IN (W235A); lanes 3, MBP-IN
(W235AKGA); lanes 4, MBP-IN (W235E); lanes 5, MBP-IN (W235EKGE). The
positions of size markers are shown to the right of the panels.
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Effects of amino acid changes on capability of MAbs to bind mutant
HIVINs.
To test whether the MAbs recognize changes in the amino
acid sequence of HIVIN we prepared several IN mutants. The unique BsmFI-BseRI region of the IN open reading frame
of pLAI was replaced with synthetic double-stranded oligonucleotides;
the wild-type sequence (5'-CACTTTGGAAAGGAC-3') was changed
to 5'-CACTTGCCAAAGGAC-3', 5'-CACTTGAAAAAGGAC-3',
5'-CACTTGCCAAAGGAGCCAAAGGAC-3', or
5'-CACTTGAAAAAGGAGAAAAAGGAC-3' to generate W235A, W235E,
W235AKGA, or W235EKGE, respectively. All the mutant INs as well as
the wild-type INs of NL4-3 (1) and BH10 (35) were
produced as MBP fusion proteins as was HIV INLAI and
subjected to ELISA and immunoblot analysis. First, we found that MAb
2-19 as well as the other MAbs to MDBD (8-22, 4-20, and 6-19)
interacted to a similar extent not only with HIVINLAI but
also with HIVINNL4-3 and HIVINBH10, both in
ELISA (Table 2) and in an immunoblot analysis (Fig. 2A). Apparently,
the amino acid changes within the region from aa 228 to 236 did not
affect the binding capability of MAb 2-19 much (Fig. 2B). Secondly, we observed that MAb 2-19 was capable of interacting with W235A and W235E
in an immunoblot analysis (Fig. 2C) but not in an ELISA (Table 2). In
contrast, MAb 2-19 did not interact with W235AKGA or W235KGE in either
assay (Fig. 2C and Table 2). The data imply that W235A and W235E show a
subtle, yet distinct structural change and agree with the fact that
they retain in vitro integration activity but lack in vivo infectivity
(9, 27). The data suggest that the structural change in W235
IN is probably in the epitope for the MAb.
Effects of MAb binding on in vitro activities of IN.
To test
whether the anti-MDBD MAbs interfere with the in vitro activities of
HIVIN, we assayed TC, ST, and disintegration activities (12,
15) in the presence of each MAb. Unlike ELISA or immunization,
these assays utilized MBP-free HIVIN prepared by cleavage of MBP-HIVIN
with factor Xa (NEB) to minimize the unexpected effects, if any, of the
N-terminal tag. Briefly, each purified MAb was preincubated with 7.5 pmol of purified HIVIN at various MAb/IN molar ratios on ice for 60 min
in 14.5 µl of solution containing 20 mM sodium 3-N-morpholino
propanesulfonate (MOPS-Na, pH 7.0), 10 mM MnCl2, 45 mM
NaCl, 0.1% IGEPAL-CA630 (Sigma), and 1 mM dithiothreitol (DTT).
Reactions were initiated by adding 32P-labeled substrates
(0.2 pmol; specific activity, ~106 cpm/pmol) in 0.5 µl
of solution containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 100 mM
NaCl followed by incubation at 37°C for 20 min. The integration
products were separated by denaturing polyacrylamide gel
electrophoresis (PAGE) and analyzed by autoradiography with an image
analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan).
The effects of MAb binding on the TC, ST, and disintegration activities
of IN are shown in Fig.
3. Both TC and ST
activities
were inhibited by all the four MAbs reactive to the MDBD,
whereas
the disintegration activity was much less affected. This is
compatible
with the results of genetic analyses of HIVIN (
31,
40) and
HIV-2 IN (
31). Moreover, this agrees with the
reports that MAb
(
33) and Fab (
2) reactive to the
region from aa 262 to 273
of HIVIN significantly inhibited both TC and
ST activities and
showed little inhibitory effect on disintegration
activity. We
concluded that the MDBD is essential to TC and ST
activities but
not to disintegration activity.
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FIG. 3.
Effect of MAb binding on in vitro activities of HIVIN.
(A) TC (open square), ST (solid square), and disintegration (open
circle) activities were assayed with the blunt-ended, precleaved, and
Y-mer oligonucleotide substrates, respectively. Each MAb tested is
indicated in each panel. The effect of MAb binding on HIVIN activities
was measured relative to the result obtained in a reaction with MAb
reactive to the hexahistidine tag (100% activity). Results are based
on the average of at least three independent assays (error bar,
standard error). (B) Typical gel image obtained in TC assay. IN was
incubated on ice prior to addition of radiolabeled oligonucleotide
substrates with MAb 2-19 at MAb/IN molar ratios of 2 (lane 2), 1 (lane
3), 0.5 (lane 4), 0.25 (lane 5), and 0.125 (lane 6). Lane 1, without
IN; lane 7, without MAb 2-19 but with anti-hexahistidine tag antibody
as an unrelated antibody. S, substrates; TCP, TC products. (C) Typical
gel image obtained in ST assay. The lane arrangements are the same as
in panel B. S, substrates; STP, ST products.
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Effects of MAb binding on interaction between IN and RT.
Hoping to demonstrate the usefulness of the MAbs in studying IN
function, we performed a pull-down experiment with MAb 2-19 and found
that it inhibited RT-IN interaction. Briefly, MBP-HIVIN (5 µg; wild
type or mutant) immobilized on amylose resin (5 µl; NEB) in buffer B
(20 mM HEPES-Na [pH 7.2], 0.1 M NaCl, 5 mM DTT, 5 mM
MgCl2, 1 mM EDTA, 1% bovine serum albumin) was
incubated with endonuclease (Benzonase; 50 U/ml; Sigma) at 37°C for
10 min followed by an extensive wash with buffer B. The immobilized
MBP-HIVIN was incubated with MAb 2-19 or MAb 8-16 on ice for an hour
and thereafter with 50 ng of RT (F. Hoffman-La Roche Ltd., Basel, Switzerland) on ice for an additional hour. The resin was washed extensively with a solution containing 50 mM HEPES-Na (pH 7.2), 50 mM NaCl, 5 mM DTT, 1 mM EDTA, 1% bovine serum albumin and 0.25% IGEPAL-CA630. The bound proteins were separated by sodium dodecyl sulfate (SDS)-PAGE and analyzed by Western blot analysis with an
anti-RT (p66/p51) antibody (mouse MAb; Advanced Biotechnologies). MAb
2-19 as well as 8-22 inhibited RT-IN interaction (Fig.
4A, lanes 1, 6, and 7). Furthermore,
HIVINs with an amino acid substitution or insertion at W235 lost the
ability to find RT (Fig. 4A, lanes 2 to 5). The results suggest that
the region containing the epitope of MAb 2-19 is responsible for RT-IN
interaction and that W235 mutants lack some structure required for that
interaction.
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FIG. 4.
Interaction of IN with RT. MBP-HIVINs of W235 mutants as
well as wild-type LAI were immobilized on amylose resin in the presence
or absence of MAb 2-19 or 8-22 and were incubated with RT. The bound
proteins were separated by SDS-PAGE and blotted onto a nitrocellulose
membrane. The blotted proteins were probed with an anti-RT (A) or an
anti-MBP (B) antibody. Incubated with 0.5 µg of RT were HIVINs of the
wild-type LAI (lanes 1, 6, and 7), W235A (lanes 2), W235E (lanes 3),
W235 AKGA (lanes 4), and W235EKGE (lanes 5). For assaying the effect of
MAb binding, MBP-HIVIN was incubated for an hour on ice with MAb 2-19 (lanes 6) or MAb 8-22 (lanes 7) prior to incubation with RT. Lanes 8, RT alone as a positive control. (A) Bars show the positions of p66 and
p51 subunits of RT.
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Our current working hypothesis is that MAb 2-19 recognizes a
conformational motif in the MDBD which is conserved across different
primary amino acid sequences, because MAb 2-19 interacted with
INs of
HIV-1
LAI, HIV-1
NL4-3, and HIV-1
BH10
to a comparable extent
(Fig.
2A) although their amino acid sequences
(aa 228 to 236)
are different (Fig.
2B). This seems to be consistent
with a report
that mouse MAbs reactive to epitopes within the carboxyl
region
of HIVIN cross-reacted with HIV-2 IN (
33) and with an
observation
on an anti-RT intracellular antibody (
20). We
speculate that
W235A and W235E mutants contain such a subtle and local
structural
change in the above-mentioned conformational motif that MAb
2-19
detected those W235 mutants in the immunoblot analysis (Fig.
2C).
This is compatible with the fact that W235A and W235E retain in
vitro
integration activity (
9,
10,
27,
28). But we also
infer that
the local conformational change around W235 is so definite
that IN with
a W235E or W235A substitution could not be detected
by MAb 2-19 in
ELISA (Table
2) nor could these mutants interact
with RT (Fig.
4). This
inference agrees with the report that pooled
sera from HIV-1-positive
patients cannot recognize mutants carrying
a substitution at residue
W235 of the HIVIN (
10). This probable
definite, yet subtle
structural change may account for the incompetence
of W235A and W235E
mutants in replication (
9,
10,
27,
28).
In summary, we have described a collection of MAbs with sequential
epitopes on the MDBD of HIVIN demonstrating that the MDBD
is essential
for TC and ST activities but dispensable for disintegration
and have
shown that the MDBD seems to contain a structural motif
common to
various strains of HIV-1. These MAbs should prove useful
for further
studies of the structure and function of HIVIN and
the molecular design
of inhibitors to
HIVIN.
| |
ACKNOWLEDGMENTS |
We thank Keith Peden for pLAI. We thank Tadahito Kanda, Kunito
Yoshiike, and Hiroshi Yoshikura for critical reading of the manuscript.
This work was supported by a grant for AIDS Research from the Ministry
of Health and Welfare of Japan and the Program to Develop Countermeasures for Health Management and Decreasing Immunity in
Relation to Public Hygiene from the Japan Health Sciences Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Molecular Genetics, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan. Phone: 81-425-61-0771, ext. 370. Fax: 81-425-67-5632. E-mail: yochan{at}nih.go.jp.
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Journal of Virology, May 1999, p. 4475-4480, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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