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Journal of Virology, April 2001, p. 3988-3992, Vol. 75, No. 8
Laboratory of Molecular Retrovirology,
Clinical Services Program, Science Applications International
Corporation
Received 26 October 2000/Accepted 19 January 2001
The potential roles of an amino acid deletion at codon 67 ( Current analyses of genotypic drug
resistance patterns have revealed the presence of stable
rearrangements, including amino acid insertions and deletions in the
We have demonstrated that high-level resistance to AZT (up to
1,810-fold) was seen in the setting of the A series of HIV-1 variants were created in a cloned proviral DNA pNL4.3
(1) backbone using a Quickchange site mutagenesis kit
(Stratagene, La Jolla, Calif.) (8). Drug resistance assays were carried out using MT-2 cells (4, 5) following
previously described methods (8). Sensitivities to each
drug were reported as the concentrations of the drugs that inhibited
p24 production by 50% (IC50s) in tissue culture systems
(8, 20). Significant differences between HIV-1 variants in
drug sensitivity were calculated by using the unpaired t
test of the StatView program (Abacus Concepts, Berkeley, Calif.).
Constructs containing either the
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.8.3988-3992.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Amino Acid Deletion at Codon 67 and Thr-to-Gly
Change at Codon 69 of Human Immunodeficiency Virus Type 1 Reverse
Transcriptase Confer Novel Drug Resistance Profiles
Frederick, National Cancer InstituteFrederick,
Frederick,1 and Laboratory of
Immunoregulation, National Institute of Allergy and Infectious
Diseases, Bethesda,2 Maryland
ABSTRACT
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67)
and a Thr-to-Gly change at codon 69 (T69G) in the reverse transcriptase
of human immunodeficiency virus (HIV) type 1 in drug sensitivity and
relative replication fitness were studied. Our results suggest that the
67 and T69G changes can be categorized as mutations associated with
multidrug resistance. The combination of both mutations with an L74I
change (67+T69G/L74I) leads to a novel 3'-azido-3'-deoxythymidine
resistance motif and compensates for impaired HIV replication.
TEXT
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3-4 hairpin loop of the finger domain of the reverse
transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1)
(3, 18a, 19). The emergence of these stable rearrangements
is associated with failures of combination multiple antiretroviral
therapy (3, 7, 11, 13, 16, 18, 18a, 19). A two-amino-acid
insertion between codons 69 and 70 in the RT has been shown to confer
resistance to multiple nucleoside RT inhibitors (NRTIs) (11, 13,
18). We have recently identified an amino acid deletion at codon
67 (67) and a Thr-to-Gly change at codon 69 (T69G) in the RT of
HIV-1 from a patient who had failed combination therapy with
3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI)
(7). The emergence of this same combination of mutations
in the RT has also recently been reported under the selection pressure
of 2',3'-didehydro-3'-deoxythymidine (D4T)-(
)-L-2',3'-dideoxy-3'-thiacytidine
(3TC)-indinavir (14). Therefore, it appears that the
emergence of the 67 deletion and the T69G change is not the result
of any single treatment regimen but rather may be due to an unusual set
of host and/or viral characteristics.
67 and T69G changes in
association with the AZT resistance mutations K70R, T215F, and K219Q
and the non-NRTI (NNRTI) resistance mutations L74I and K103N
(7). Further study has revealed that the emergence of the
T69G mutation confers drug resistance at the expense of fitness (8). Subsequently, the development of the 67 deletion
led to a virus with improved replication and high-level AZT resistance (8). The purpose of the present study was to determine the overall profiles of resistance to licensed NRTIs and NNRTIs in viruses
containing the 67 deletion and the T69G mutation.
67 or the T69G change demonstrated
increases in the IC50s of 3'-dideoxycytidine (ddCi Sigma, St. Louis, Mo.) (P < 0.01), ddI (Sigma) (P < 0.01), D4T (P < 0.05 for 67, P < 0.01 for T69G) (Sigma), 3TC (P < 0.01), and
abacavir (P < 0.01) (Table
1). They were sensitive to AZT (Sigma),
nevirapine, and delavirdine. Interestingly, the 67 or T69G change
increased sensitivity to efavirenz (P < 0.01). A
mutant containing both the 67 and T69G changes (67+T69G) was
sensitive to AZT, ddI, nevirapine, delavirdine, and efavirenz. The
67 and T69G changes increased the ICs50 of ddC
(P < 0.05), 3TC (P < 0.01), D4T
(P < 0.05), and abacavir (P < 0.05)
(Table 1). Ross et al. recently reported that the 67 mutation in the
HXB2 backbone did not lead to high-level resistance to NRTIs
(14). Differences in assay methodology or subtle
difference in the HIV genotypic backbone may explain this difference
between our results and theirs.
TABLE 1.
Susceptibilities of recombinant viruses to RT inhibitors
Since the 67 or T69G change increased sensitivity to efavirenz,
further phenotypic studies were performed to assess the impact of these
mutations on NNRTI-resistant variants of HIV-1. As described by other
groups, the variant containing the K103N change showed resistance to
nevirapine, delavirdine, and efavirenz (15). Addition of
either the 67 or T69G mutation to the K103N mutant diminished resistance to efavirenz. In contrast, the presence of both the 67
and T69G changes in the K103N mutant partially decreased resistance to
all NNRTIs (Table 1).
We have previously reported that the combination of an L74I change, an
NNRTI resistance mutation with the 67 deletion, and a T69G change
(67+T69G/L74I) in the RT resulted in AZT resistance. The
IC50 of AZT for the 67+T69G/L74I mutant was fivefold
higher than that for the wild type (7). The further
addition of a K103N change to the 67+T69G/L74I motif led to a
further increase (16-fold) in the IC50 of AZT
(7).
To define the combined effects of the 67+T69G/L74I motif and other
NNRTI resistance mutations on AZT resistance, a series of constructs
containing the well-described NNRTI resistance mutations were created
by site-directed mutagenesis (8). Addition of an A98G
change to the 67+T69G/L74I motif also led to an increase in the
IC50 (520 ± 70 nM; P < 0.01) of AZT.
No significant changes in AZT resistance were seen upon addition of
V106A (92 ± 20 nM), Y108A (100 ± 20 nM), Y188C (164 ± 48 nM), or G190S (205 ± 53 nM; P < 0.1). The
Y181C and L100I changes can lead to suppression of AZT resistance
(2, 10). Addition of the Y181C or L100I change diminished
the IC50 of AZT for the 67+T69G/L74I motif-containing mutant to 10 ± 1.7 or 12 ± 8 nM, respectively (Fig.
1). An M184V change in the RT confers
resistance to 3TC and suppresses AZT resistance (15).
Addition of an M184V mutation also downregulated AZT resistance in the
67+T69G/L74I construct (24 ± 4.5 nM).
|
Mutations M41L, D67N, K70R, T215F/Y, and K219Q/E are associated with
well-described AZT resistance (15). Codons 41, 67, and 70 are present in the 3-4 loop region of the finger domain of the RT
and separated by approximately 25 Å from codons 215 and 219 in the 11a-11b loop. Meanwhile, codons 98 and 103 are present in
the 5-6 loop of the NNRTI binding site and separated by
approximately 40 Å from the finger domain (17).
Despite this distance, the combination of the 67 deletion and the
T69G and L74I changes with A98G or K103N led to AZT resistance (Fig. 1). The crystal structure of a covalently trapped catalytic complex of
HIV-1 RT has been reported (6). Binding of the
template-primer and a deoxynucleoside triphosphate to the DNA-binding
cleft between the finger and palm domains of the RT has been shown to
induce the outer part of the finger domain, the fingertip, to bend
inward toward the palm domain (6). Recently, Winters et
al. have reported that the 67 deletion in the 3-4 loop and the
T69G mutation in HIV-1 are associated with nucleoside analogs and have
proposed a model to explain their findings (18a). In this
model, these mutations lead to changes in the three-dimensional
structure of the 3-4 loop and alpha helices C and E in both
RT-DNA open (17) and RT-DNA-dTTP closed structures
(6). These changes lead to the loss of hydrogen bonds
between the RT and dTTP (18a). Therefore, the changes in
these regions may lead to an inefficient RT and then allow a novel
interaction between the RT and the template-primer-deoxynucleoside triphosphate, subsequently leading to a novel drug resistance.
In the absence of L74I, AZT resistance was not seen in the
67+T69G/K103N construct (data not shown). Therefore, the L74I mutation or the combination of 67+T69G/L74I changes in the finger domain might facilitate long-distance "communication" between the
finger and palm domains through the template-primer, as first reported
by Kleim et al. (9).
We have previously reported that the T69G mutation led to ddI
resistance in an AZT-resistant backbone and was associated with impaired HIV replication (8). Emergence of the 67
deletion and the L74I change in the RT compensated for this decrease in HIV replication, even though the 67 deletion alone led to impaired HIV replication (8). To characterize the effect of the
67+T69G/L74I motif on the replication capability of HIV containing
NNRTI resistance mutations, relative replication fitness assays were
performed in the presence or absence of 1 µM AZT. Infected MT-2 cells
were cultured at 0.1 × 106/ml for 7 days, and p24
levels in day 7 supernatants were measured by a p24 antigen capture kit
(8) (Fig. 2). In the absence
of AZT (Fig. 2A), a K103N, V108I, Y181C, Y188C, or G190S change in the
RT did not lead to a significant difference in growth compared with the
wild-type (WT) virus (P > 0.05). In contrast, a A98G, L100I, or V106A change, decreased virus replication to 28 ± 7.0, 66 ± 6.5, or 40 ± 4.8% of that of the WT, respectively.
The impaired HIV replication induced by the A98G or V106A change was
compensated for by the addition of the 67+T69G/L74I motif to the
mutant. Meanwhile, addition of the 67+T69G/L74I motif to the L100I,
Y181C, or Y188C change significantly decreased HIV replication to
37 ± 4.4, 46 ± 21, or 10 ± 2.0% of that of the WT,
respectively (P < 0.01). In the presence of 1 µM AZT
(Fig. 2B), all variants without the 67+T69G/L74I motif were
sensitive to AZT. Addition of the motif to the WT or A98G or K103N
mutant resulted in partial restoration of replication to 28 ± 11, 38 ± 11, and 65 ± 6.3%, respectively. Archer et al.
demonstrated that NNRTI resistance mutations could alter the rate of
RNase H cleavage, which is correlated with HIV replication
(1a). Since addition of the 67+T69G/L74I motif to
variants containing mutations associated with resistance to NNRTI
affected their replication capability in this study (Fig. 2A), the
motif might also influence RNase H cleavage.
|
To define the novel AZT resistance mechanism, a virion-associated RT
inhibition assay was also attempted using serial twofold dilutions of
AZT-TTP (DuPont NEN, Boston, Mass.) and a commercially available RT
assay kit (Roche Molecular Biochemical, Indianapolis, Ind.). The
virion-associated RT activity of the 67+T69G/L74I motif did not show
resistance to AZT-TTP (data not shown). Further biochemical study of
this 67+T69G/L74I motif is necessary to understand the precise
mechanism(s) of the novel AZT resistance motif, as well as the
compensated replication fitness. Recently, Meyer et al. demonstrated a
novel mechanism of AZT resistance due to an increase in
primer-unblocking activity (12). It is hoped that such
knowledge will lead to the development of better therapies and salvage therapies.
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ACKNOWLEDGMENTS |
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We thank Hiroaki Mitsuya at the NCI for kindly providing 3TC and abacavir. Nevirapine, delavirdine, and efavirenz were provided by Boehringer Ingelheim (Ridgefield, Conn.), Pharmacia & Upjohn (Kalamazoo, Mich.), and DuPont Pharmaceutical Company (Wilmington, Del.), respectively. MT-2 cells and pNL4.3 were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.
This study was supported by the National Institute of Allergy and
Infectious Diseases under contract N01-CO-56000 with SAICFrederick.
| |
FOOTNOTES |
|---|
*
Corresponding author. Mailing address: Laboratory of
Molecular Retrovirology, Clinical Services Program, SAICFrederick,
NCIFrederick, Building 550, Room 126, P.O. Box B, Frederick, MD
21702-1201. Phone: (301) 846-5450. Fax: (301) 846-6762. E-mail:
timamichi{at}nih.gov.
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Journal of Virology, April 2001, p. 3988-3992, Vol. 75, No. 8
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.8.3988-3992.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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