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Journal of Virology, October 1998, p. 8420-8424, Vol. 72, No. 10
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Resistance to the Anti-Human Immunodeficiency
Virus Type 1 Compound L-Chicoric Acid Results from a
Single Mutation at Amino Acid 140 of Integrase
Peter J.
King1 and
W. Edward
Robinson Jr.1,2,*
Departments of Microbiology and Molecular
Genetics1 and
Pathology,2 University of
California, Irvine, California 92697
Received 7 April 1998/Accepted 24 June 1998
 |
ABSTRACT |
L-Chicoric acid is an inhibitor of human
immunodeficiency virus type 1 (HIV-1) integrase in vitro and of HIV-1
replication in tissue culture. Following 3 months of selection in the
presence of increasing concentrations of L-chicoric acid,
HIV-1 was completely resistant to the compound. Introduction of the
mutant integrase containing a single glycine-to-serine amino acid
change at position 140 into the native, L-chicoric
acid-sensitive virus demonstrated that this change was sufficient to
confer resistance to L-chicoric acid. These results confirm
through natural selection previous biochemical studies showing that
L-chicoric acid inhibits integrase and that the drug is
likely to interact at residues near the catalytic triad in the
integrase active site.
| |
TEXT |
With the recent success of
combination therapies targeting human immunodeficiency virus (HIV)
protease and reverse transcriptase (RT) (5, 15, 17), it
seems apparent that multiple drug therapy, rather than monotherapy,
will be required for long-term survival of HIV-infected individuals.
However, due to the rapid turnover of HIV-infected cells and the likely
repetitive rounds of HIV replication within an infected individual
(18, 41), the emergence of resistant organisms is a likely
sequela of drug therapy. Indeed, although it is slower to appear than
with monotherapy, resistance of HIV to combination therapy has been
demonstrated (15). Therefore, to improve upon existing
multidrug therapies, anti-HIV agents that inhibit enzymes other than
HIV protease and RT will likely be required. One such therapeutic
target is HIV integrase.
Integration is absolutely required for the maintenance of stable and
productive HIV infection (8, 21, 37, 38, 40); therefore,
inhibition of integration is likely to profoundly impair HIV
replication. To date, a large number of candidate inhibitors of HIV
integrase have been reported based on the ability to inhibit HIV
integrase enzyme in vitro. However, few have affected HIV replication
in tissue culture (reviewed in reference 33).
Recently, we described a group of compounds, the dicaffeoylquinic and
dicaffeoyltartaric acids, that blocked HIV replication in tissue
culture at nontoxic concentrations (34, 36). In vitro
studies indicated that they were potent and selective inhibitors of HIV
integrase (25, 34, 36). Indeed, these compounds exhibited
10- to 100-fold selectivity against HIV integrase over other HIV
enzymes (25); nevertheless, definitive proof of their
mechanism of anti-HIV activity has been lacking. Indeed, several
findings by other investigators suggested that they might be acting
through mechanisms other than the inhibition of integrase. For example,
other bis-catechols do not inhibit HIV replication (3, 7, 9-11,
20, 24, 27, 42), and several previous reports indicated that
compounds related to the dicaffeoyltartaric acids inhibited gp120
binding to CD4 (22) and RT (29, 30).
Although inhibition of integrase in vitro by small molecules has been
relatively simple to demonstrate, inhibition of HIV integration within
the cell has proven difficult to study. One method by which the
mechanism of an antiviral compound can be deduced is via the isolation
of drug-resistant variants and the demonstration that drug resistance
maps to changes in the amino acid sequence of a viral enzyme. For
example, the resistance of HIV to nucleoside and nonnucleoside RT
inhibitors maps to amino acid changes in HIV RT (12-14,
23). In contrast, a G quartet oligonucleotide currently in
clinical trials (1) was reported to inhibit HIV-1
replication via the inhibition of integrase (31). However, a
recent report indicates that drug resistance maps to the HIV envelope,
not integrase (4), indicating this putative integrase
inhibitor acts via inhibition of virus penetration, not integration.
Therefore, to address whether L-chicoric acid inhibited HIV
replication at the level of integrase, we chose to isolate variants of
HIV resistant to the antiviral effects of L-chicoric acid.
H9 and MT-2 are CD4+ T-lymphoblastoid cell lines that
support replication of tissue culture-adapted and syncytium-inducing, lymphocytotropic clinical isolates of HIV-1. Both were obtained from
the National Institutes of Health AIDS Research and Reference Reagent
Program (Rockville, Md.). HIVNL4-3 plasmid (a gift from P. Krogstad, University of California, Los Angeles) was transfected in
HeLa cells with Lipofectin (Gibco/BRL). Excess DNA was removed by
washing, and the cells were cocultured with H9 cells for 18 h. The
H9 cells were removed and recultured in growth medium. When the culture
was 100% positive for HIV antigens by indirect immunofluorescence
(35), the virus was inoculated onto H9 cells and incubated
at 37°C for several weeks in the presence of 2 µM L-chicoric acid. When this culture was 100% positive, the
virus was isolated and one aliquot was passaged in a similar manner in
4 µM L-chicoric acid. Finally, the virus was cultured in
the presence of 8 µM L-chicoric acid, and the resultant
virus was filter clarified, aliquoted, and stored at 
70°C.
HIVNL4-3, following culture in 8 µM
L-chicoric acid, was tested for resistance to the anti-HIV
activity of the compound by using a cytopathicity-based assay (34,
36) first described by Montefiori et al. (26) (Fig.
1). This assay takes advantage of the
lytic nature of T-cell-tropic clones of HIV, and decreased cell
viability in the assay has been shown to correlate well with HIV
replication (35). The 50% effective dose (ED50)
of L-chicoric acid against HIVNL4-3 control
virus was 400 nM, while HIVNL4-3 passaged in the presence
of 8 µM L-chicoric acid was completely resistant to the
compound (Fig. 1).
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FIG. 1.
HIVNL4-3 passaged in L-chicoric
acid develops drug resistance. HIVNL4-3 passaged in the
presence (squares) or absence (circles) of increasing concentrations of
L-chicoric acid was tested for sensitivity to
L-chicoric acid. Each point is the mean of triplicate
samples; the bars represent 1 standard deviation. HIV is a lytic virus,
and increased levels of virus cause increased death of cells.
Therefore, viability-based assays are good measures of HIV replication
and anti-HIV activity. The viability of the cells is relative to that
of cell controls (eight replicates; 100% viable) and virus controls
(eight replicates; 0% viable) and was measured as first described by
Montefiori et al. (26). Decreased cell viability in this
assay correlates well with levels of HIV RNA, HIV protein expression,
RT release, and numbers of infectious HIV particles (35).
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The overall cloning and sequencing strategy is illustrated in Fig.
2. For cloning and sequencing, HIV from
10 ml of culture was centrifuged at 33,000 × g for
4 h at 4°C. Virions were lysed, and RNA was isolated with
Purescript (Gentra, Frederick, Md.). Primers used to amplify cDNA under
these conditions recognize the 5' and 3' ends of integrase at
nucleotide positions 3580 to 3605 (INS primer;
5'-GGTCTCCGCGGGAATCAGGAAAGTAC-3') and 4497 to 4522 (INX
primer; 5'-GCTTTTCTAGAAATATACATATGGTG-3'), respectively, and
generate a 943-bp product. First-strand synthesis with INX primer and
Superscript II, an avian myeloblastosis virus RT (Gibco/BRL), was
performed at 42°C for 50 min according to the manufacturer's instructions. Thirty-eight-cycle amplification was performed with thermostable Pfu DNA polymerase (Stratagene, La Jolla,
Calif.) according to the manufacturer's instructions. The optimum
Mg2+ concentration for these studies was determined to be 1 mM. The conditions for PCR were 96°C for 1 min, 40°C for 30 s,
and 72°C for 2 min for the first 2 cycles, followed by 96°C for 1 min, 55°C for 1 min, and 72°C for 3 min for 36 cycles. The final
cycle included a 10-min, 70°C elongation step. The resulting RT-PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Appropriately sized products were eluted
from the gel and blunt end ligated into pCR-Script (Stratagene) for
dideoxynucleotide sequencing with Sequenase II (U.S. Biochemical, Cleveland, Ohio) according to the manufacturer's instructions. The
entire integrase sequence was determined through the use of six
oligonucleotide primers: INS, INX, Core 1 (5'-CAGCTGTGATAAATGTCAGCTA-3' [nucleotides {nt} 3721 to
3741]), Core 2 (5'-CCATTTGTACTGCTGTCTTAA-3' [nt 4122 to
4142]), INSPF (5'-GCAATTTCACCAGTACTACAGT-3' [nt 3962 to
3983]), and INSPR (5'-GTAGGGAATGCCAAATTCCTG-3' [nt 4016 to 4036]). Manual sequence analysis was confirmed by automated DNA sequencing.
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FIG. 2.
Cloning strategy for analyzing mutations in integrase.
The diagram illustrates the general cloning scheme designed to insert
integrases from drug-resistant organisms into the wild-type
HIVNL4-3 background. Briefly, RNA was isolated from virions
and subjected to RT-PCR with primers that introduced silent mutations
(SacII and XbaI sites) upstream and downstream of
the integrase gene. These RT-PCR products were ligated into pCR-Script
for sequencing. Clones containing mutant integrases were digested with
SacII and XbaI, and the integrase gene was
ligated into a similarly digested HIVNL4-3 plasmid,
allowing the entire integrase gene, and only the integrase gene, to be
switched into a drug-sensitive HIVNL4-3 background.
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Sequencing the integrase genes from both drug-resistant and control
HIVNL4-3 demonstrated several mutations. The control virus contained two silent mutations at nt 3832 and 4009. These silent mutations are believed to arise from a discrepancy in the published sequences of HIVNL4-3 (2, 39) and were likely
not a result of the passage of HIV in the absence of inhibitor.
Drug-resistant HIVNL4-3 had the same silent mutations as
well as a single G-to-A transition at nucleotide position 4025 (Fig.
3), leading to an amino acid change from
glycine to serine at amino acid 140.
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FIG. 3.
Mutation of integrase at nt 4025 is associated with
drug-resistance. Following the precipitation of virions, RNA was
isolated and subjected to RT-PCR. The RT-PCR products were cloned into
pCR-Script, and multiple clones were sequenced. WT, wild-type
HIVNL4-3 sequence; 7-3, clone 7-3, a control sequence
(HIVNL4-3 passaged in the absence of L-chicoric
acid); 1-D4, clone 1-D4, a drug-resistant virus (HIVNL4-3
passaged in the presence of 8 µM L-chicoric
acid). The arrows indicate the site of the mutation: the native
sequence has a guanine at position 4025, while integrase from
drug-resistant HIVNL4-3 contained an adenine at position
4025.
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To determine whether this amino acid change was responsible for the
observed resistant phenotype, the integrase genes from drug-resistant
HIVNL4-3 were cloned into the native HIVNL4-3
plasmid (pNL4-3). This cloning was accomplished through site-directed mutagenesis, introducing several silent mutations immediately upstream
and downstream of the integrase gene. These mutations generated two
unique restriction sites (2, 39): an upstream SacII site and a downstream XbaI site (Fig. 2).
Introduction of these mutations allowed the entire integrase gene, with
only minimal upstream and downstream nucleotides, to be digested and
"swapped" between drug-resistant and drug-sensitive clones. Two
clones, 7-1 and 7-3, containing control integrase genes and wild type except for silent mutations generating the restriction sites, and clone
1-D4, containing drug-selected integrase with the G140S mutation, were
chosen for further study. Once transfected into HeLa cells and
amplified in H9 cells, the viruses from all three clones maintained the
same sensitivity to zidovudine, a RT inhibitor, as the parental
HIVNL4-3 (Fig. 4A). The three
clones containing wild-type integrase (the control viruses, clones 7-1 and 7-3, and wild-type HIVNL4-3) maintained the
L-chicoric acid-sensitive phenotype (Fig. 4B). Clone 1-D4,
on the other hand, was resistant to the anti-HIV effects of
L-chicoric acid. Although the drug-resistant clone retains
some sensitivity to L-chicoric acid, the compound was
unable to inhibit HIV replication by 50%, a requirement for determining activity (ED50). Therefore, the
ED50 for the drug-resistant clone was >600-fold higher
than that for the drug-sensitive clones (Table
1).
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FIG. 4.
Resistance to L-chicoric acid but not
zidovudine is conferred by G140S mutation in integrase. Wild-type
HIVNL4-3 (circles), HIVNL4-3 control clones 7-1 (squares) and 7-3 (triangles), and clone 1-D4 (inverted triangles) were
tested for sensitivity to zidovudine (A) or L-chicoric acid
(B). The assays were performed as described in the legend to Fig. 1.
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Amino acid 140 of integrase has not been mutated previously by
site-directed mutagenesis. Furthermore, a search of the GenBank database does not indicate any naturally occurring mutations at this
site. This amino acid is also highly conserved in integrases from other
retroviruses, retrotransposons, and transposable elements of bacteria
(19). Our data (Table 2)
indicate that mutation at this site from the highly conserved glycine
to serine has little effect on HIV replication but completely abrogates
the anti-HIV activity of L-chicoric acid.
L-Chicoric acid had been reported previously to have
anti-HIV effects in tissue culture at nontoxic concentrations and to be
a potent and selective inhibitor of HIV integrase in biochemical assays
(25, 34, 36). Although one group has had difficulty demonstrating an anti-HIV effect of L-chicoric acid
(27, 28), that may be due to their use of the tetrazolium
dye, XTT, which is known to be incompatible with studies on
bis-catechols (16), or because they used a different HIV
isolate, HIVRF. Indeed, a previous report on the anti-HIV
activity of a related compound, 3,4-dicaffeoylquinic acid
(22), our reports of the anti-HIV activity of
L-chicoric acid and the dicaffeoylquinic acids, and the
independent confirmation by an outside source (18a) clearly show that these compounds inhibit HIV replication in tissue culture at
nontoxic concentrations. By demonstrating that a single amino acid
substitution at amino acid 140 in integrase confers resistance to
L-chicoric acid, a potent inhibitor of HIV integrase, it is evident that the principal target for the anti-HIV activity of L-chicoric acid is integrase. Thus, there is now very
strong evidence to suggest that small molecules can inhibit integration
within cells and that inhibition of HIV integrase results in impaired HIV replication.
Previous experiments had shown that the ED50 of
L-chicoric acid for the uncloned, tissue culture-adapted
strain of HIV, HIVLAI, was approximately 4 µM
(34). Such a high ED50, coupled with questions
regarding the intracellular (or extracellular) mechanism of action, had
dampened enthusiasm for developing L-chicoric acid as an
individual compound or the dicaffeoylquinic and dicaffeoyltartaric acids as a class of integrase inhibitors worth investigating in detail.
Despite 50% inhibitory concentrations (IC50) of ~200 to 400 nM against recombinant HIV integrase in biochemical assays (34, 36), the compounds lacked potencies suitable for
clinical evaluation. However, the integrase used in biochemical assays was from the HIVNL4-3 molecular clone of HIV, not uncloned
HIVLAI. The results here demonstrate that
L-chicoric acid inhibits HIVNL4-3 replication
(ED50) at a concentration that virtually matches the IC50 against purified HIVNL4-3 recombinant
integrase. Furthermore, small changes in sequence, indeed, a single
amino acid change in integrase, can significantly affect susceptibility
to L-chicoric acid in tissue culture.
These data document that L-chicoric acid inhibits HIV
replication, at least in part, through inhibition of HIV integrase. Furthermore, a single nonconservative amino acid change at a highly conserved amino acid, amino acid 140, is compatible with viral replication (Table 2) and confers resistance to an inhibitor of HIV-1
integrase. It is important to note that the packaging of virus and RT
appears to be unaffected by the G140S mutation, as the RT-to-infectious
particle ratio of the wild-type HIVNL4-3 and that of the
drug-resistant variant were nearly equivalent (Table 2). This mutation
occurs at a potentially important site in the integrase protein. First,
this amino acid, glycine, is highly conserved throughout retroviral
integrases (19). Second, this amino acid forms an anchor
sequence in the integrase crystal structure (6): it was the
last ordered amino acid prior to a disordered loop which contains one
member of the catalytic triad of integrase (DD35E), a glutamine at
residue 151. The location of this mutation in the catalytic core domain
of integrase is indicated in Fig. 5.
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FIG. 5.
HIV integrase catalytic core; diagram of the catalytic
core domain containing the phenylalanine-to-lysine mutation at amino
acid 185 (6). The location of the glycine-to-serine mutation
at amino acid 140 is illustrated, as are the two catalytic residues,
aspartate 64 and aspartate 116. The original figure, courtesy of D. Marcey (Kenyon College, Gambier, Ohio), was downloaded from
http://www.kenyon.edu/depts/bmb/chime/integras/frames/intgrse.htm and
is used with permission. The locations of the catalytic amino acids and
the mutation were manually entered with Microsoft PowerPoint.
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Thus, integrase inhibitors have promise as potential anti-HIV
compounds. Powerful inhibition of integrase can impair virus replication, but any such inhibitors will likely need to be used in
combination with existing anti-HIV agents as resistance, although difficult to achieve, requires only a single nucleotide change and
results in only mild attenuation of viral replication. Now that an
inhibitor of HIV replication that acts on integrase has been
identified, it is possible to determine whether integrase is a suitable
target in combination anti-HIV therapies. Ultimately, this class of
anti-HIV agents may lead to the synthesis of clinically useful
integrase inhibitors which can be used alone or in combination with
existing anti-HIV agents to slow the progression to AIDS.
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ACKNOWLEDGMENTS |
This work was supported in part by grants 1RO1-AI41360 and
5T32-GM-07311 from the Public Health Service.
We thank Suzanne Sandmeyer (University of California, Irvine), Hung Fan
(University of California, Irvine), and Samson A. Chow (University of
California, Los Angeles) for thoughtful suggestions on the manuscript
and Brenda McDougall, Jean Kuan, Tracey Kim, and Keola Beale for their
expert technical assistance. L-Chicoric acid was
synthesized in the laboratory of, and kindly provided by, Manfred G. Reinecke (Texas Christian University, Fort Worth). The diagram of the
integrase catalytic core was obtained from the Kenyon University
website
(http://www.kenyon.edu/depts/bmb/chime/integras/frames/intgrse.htm).
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ADDENDUM IN PROOF |
Recently, the X-ray crystal structure of the core domain of avian
sarcoma virus (ASV) integrase in complex with an inhibitor of both ASV
and HIV integrases was solved (J. Lubkowski, F. Yang, J. Alexandratos,
A. Wlodawer, H. Zhao, T. R. Burke, Jr., N. Neamati, Y. Pommier, G. Merkel, A. M. Skalka, Proc. Natl. Acad. Sci. USA 95:4831-4836, 1998). This structure placed the glycine analogous to G140 of HIV integrase within close proximity to the inhibitor bound within the ASV integrase multimer interface. These findings support the interpretation that G140 is near an
inhibitor-binding site within the HIV integrase core.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pathology, D440 Med Sci I, University of California, Irvine, CA
92697-4800. Phone: (949) 824-3431. Fax: (949) 824-2505. E-mail:
ewrobins{at}uci.edu.
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Journal of Virology, October 1998, p. 8420-8424, Vol. 72, No. 10
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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