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Journal of Virology, January 2000, p. 1023-1028, Vol. 74, No. 2
0022-538X/00/$04.00+0
High-Level Resistance to 3'-Azido-3'-Deoxythimidine due to a
Deletion in the Reverse Transcriptase Gene of Human
Immunodeficiency Virus Type 1
Tomozumi
Imamichi,1
Tanima
Sinha,1
Hiromi
Imamichi,1
Yi-Ming
Zhang,1
Julie A.
Metcalf,2
Judith
Falloon,2 and
H.
Clifford
Lane2,*
SAIC Frederick, Frederick Cancer Research and
Development Center, National Cancer Institute,
Frederick,1 and Laboratory of
Immunoregulation, National Institute of Allergy and Infectious
Diseases, Bethesda,2 Maryland
Received 26 April 1999/Accepted 13 October 1999
 |
ABSTRACT |
A variant of human immunodeficiency virus type 1 (HIV-1) possessing
a deletion in the reverse transcriptase (RT) gene at codon 67 was
identified in a patient who had failed combination antiretroviral therapy. This deletion initially emerged under the selective pressure of combination therapy with 3'-azido-3'-deoxythymidine (AZT) plus 2',3'-dideoxyinosine. It has persisted for more than 3 years in association with the accumulation of a variety of other well-described drug resistance mutations and an uncharacterized mutation at RT codon 69 (T69G). Phenotypic studies demonstrated that the codon 67 deletion by itself had little effect on AZT sensitivity. However, in
the context of the T69G mutation and three other mutations known to be
associated with AZT resistance (K70R, T215F, and K219Q), this deletion
led to a increase in AZT resistance from 8.5-fold to 445-fold. A
further increase in resistance (up to 1,813-fold) was observed when two
mutations associated with nonnucleoside RT inhibitor resistance (K103N
and L74I) were added to the deletion T69G K70R T215F K219Q construct.
Hence, these results establish that a deletion at RT codon 67 may
be selected for in the presence of antiretroviral therapy and may lead
to high-level resistance to AZT.
| |
TEXT |
Current studies of the treatment of
patients with human immunodeficiency virus type 1 (HIV-1) infection
with combinations of antiretroviral drugs have demonstrated a dramatic
reduction in AIDS-related morbidity and mortality (11, 20, 36,
39). These therapies, while potent, are not capable of
eradicating HIV-1, and thus, today, the medical management of
patients with HIV-1 infection requires the development of long-term
strategies (6, 14, 38, 51). Treatment failure is a complex
phenomenon (41). It is typically characterized as an
inability to achieve adequate suppression of viral replication and may
be due to the emergence of drug-resistant virus and/or noncompliance
with the prescribed medical regimen (13, 49). Genotypic
analyses have revealed that drug-resistant viruses may acquire
mutations not only in protease (PRT) and reverse transcriptase (RT)
coding regions, but also in the gag-pol cleavage sites
(53). Once multidrug resistance viruses emerge in the
setting of combination therapy, it is often difficult to regain control
of viremia (41, 49). Thus, studies of the precise genetic
features of drug resistance and viral fitness are of value in achieving
a better understanding of these phenomena and in developing better
salvage regimens.
Long-term treatment with 3'-azido-3'-deoxythymidine (AZT)
can result in the development of AZT resistance (27, 34,
43). Amino acid substitutions in the HIV-1 RT at amino acid
codons 41, 67, 70, 210, 215, and 219 have all been well
characterized as conferring AZT resistance (19, 21, 24, 28).
To acquire high-level resistance (50% inhibitory concentrations
[IC50s] increased more than 100-fold) to AZT, the
accumulation of four to six mutations in the RT gene is generally
required (24, 30, 44). In contrast to AZT resistance,
high-level resistance to nonnucleoside RT inhibitors (NNRTIs) is
generally conferred by a single mutation in HIV RT at either codon
103 (N to K) or codon 181 (Y to C) (44).
Patient profile.
In the present study, we have identified a
patient with persistent high levels of viral replication despite
multiple regimens of combination antiviral chemotherapy (Fig.
1). The patient is a 52-year-old man who
was initially enrolled in a monotherapy study of an NNRTI (L697,661)
(16). He was subsequently treated with AZT monotherapy for
10 months, followed by AZT plus didanosine (ddI) combination therapy
for 22 months. He then received AZT plus ddI plus delavirdine
(10) for the next 4 months, followed by a switch to
indinavir (IND) (48) monotherapy and subsequently a series
of combination regimens, including zalcitabine (ddC), lamivudine
(3TC) (46), stavudine (d4T) (32), abacavir
(7), ritonavir (33), saquinavir
(42), amprenavir (E. E. Kim, B. G. Rao, D. D. Deininger, C. T. Baker, M. D. Dwyer, M. A. Navia, T. A. Thomson, and R. D. Tung, Abstr. 10th Int. Conf. AIDS,
abstr. 319A, 1994), efavirenz (52), and hydroxyurea
(3). Over this 7-year period, he also received 28 cycles of intermittent interleukin 2 (IL-2) therapy. A detailed
description of these therapies and the HIV-1 RNA and total
CD4+ cell counts during this time are shown in Fig. 1.
Particle-associated HIV-1 RNA levels in plasma were determined by
the branched-DNA (bDNA) signal amplification assay (Chiron)
(9). The detection limit of the assay before September 1996 (version 1) was 10,000 copies per ml. From September 1996 to August
1998, the detection limit of the assay (version 2) was 500 copies/ml.
After August 1998, the detection limit of the assay (version 3) was 50 copies/ml. As can be seen, this is a patient for whom sustained
virologic control has not been possible.
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FIG. 1.
Virological and immunological responses of the patient
to antiretroviral therapy. The copy number of HIV RNA in plasma was
measured by the bDNA assay. Solid circles show bDNA levels, and open
circles show the number of total CD4+ cells. Arrows
indicate the time points when HIV sequence analyses were performed. The
times at which IL-2 was administered are noted by the solid triangles
at the top of the figure. The drug treatment history is shown on top of
the graph. L, L-697,661; DLV, delavirdine; RIT, ritonavir; SAQ,
saquinavir; APV, amprenavir; ABC, abacavir; EFV, efavirenz; NEL,
nelfinavir; HU, hydroxyurea.
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|
To attempt to elucidate the mechanisms leading to multidrug resistance
in this patient, nucleic acid analysis was performed
with a 1.4-kb
fragment of the
gag, PRT, and RT region of the HIV-1
genome
(nucleotides 2010 to 3492) by using a plasma sample from
October 1997. PCR amplifications were performed as previously
described
(
53). A total of 10 clones were sequenced. Changes
in the
gag, PRT, and RT regions were compared with the HIV-1 clade
B consensus sequence as a reference (
37). Multiple mutations
were observed in RT (Table
1).
Mutations associated with resistance
to AZT (M41L, K70R, L215F,
and K219Q), 3TC (M184V), and NNRTIs
(K103N, A98G, L74I,
Y188C, and P236M) were detected (
44). In
addition, a
deletion was observed at RT codon 67 (
67). Novel
amino acid
substitution patterns were observed at codons 69 (T69G),
75 (V75T),
and 179 (V179I). Other mutations were seen at codons
known to be
associated with ddC (T69D) or NNRTI (V75L/I and V179D/E)
resistance
(
15,
25,
44). No amino acid substitutions were
observed at
the codons associated with multinucleoside analog
resistance
(codons A62, F77, F116, and Q151) (
45). In addition,
position P2 in the
p7
gag-p1
gag cleavage site
was mutated from A to V (
53). The PRT sequence
revealed that
there were nine amino acid substitutions (L10I,
M36I, M46I, I54V, L63P,
A71V, G73S, V82A, and L90M) (
44).
To determine when the codon 67 deletion arose and if it emerged in
a sequential manner along with resistance mutations, analyses
were
performed with longitudinal samples spanning 6 years (Table
1). Due to
the limited availability of plasma samples from the
earlier time
points, proviral DNA was used as the primary source
for sequence
information. In December 1991 (initiation of AZT
therapy), all 10 clones were wild type. By January 1993 (3 months
following the addition
of ddI and the time of maximal virus suppression),
multiple mutations
were noted in the RT gene, including a D67N
substitution. One year
following this, while the patient was still
on AZT plus ddI, the
67
deletion was first seen. This unusual
deletion was found in 8 of 10 clones from 1994 and 10 of 10 clones
from 1997. The T69G substitution
emerged at the same time as
67.
Although the T69G change was seen in
clones in which codon 67
was present, the
67 deletion was only
seen in clones that contained
the T69G mutation (Table
1). A recent
abstract also reported
the emergence of a deletion at codon 67 in
association with a
T69G change during combination therapy with
3TC, D4T, and IND
(L. Ross, M. Johnson, N. Graham, M. Shaefer, M. Griswold, and
M. St. Clair, Abstr. 5th Conf. Retrovir.
Opportunistic Infect.,
abstr. 679, 1998). The emergence of mutant forms
of RT with two
to three amino acid inserts between RT codons
69 and 70 have also
been reported (
8,
50; Ross et
al., Abstr. 5th Conf. Retrovir.
Opportunistic Infect.). These residues
are all part of the finger
domain of RT (
22,
47).
Site-directed mutagenesis studies confirmed
the important role of this
insert in conferring reduced susceptibility
to ddI, ddC, 3TC, and
9-(2-phosphonyl methoxyethyl)adenine (PMEA)
(
40), but
not to AZT or d4T. This two-codon insert plus a T69S
mutation in
the context of AZT resistance mutations decreased
susceptibility to AZT
approximately 200-fold (
50). Thus, a codon
69 substitution seems to be an important accompaniment of insertions
or
deletions in the finger domain of RT. Given the lack of prior
reports
(
21,
31), it is likely that the prevalence of this
67
deletion is very rare. The crystal structure for the HIV RT
was
recently reported by using a combinatorial disulfide cross-linking
strategy (
22). That study revealed that the binding of the
template-primer
hybrid and the deoxynucleoside triphosphate (dNTP)
complexes to
the RT enzyme induces a significant conformational change
in the
enzyme, in which there is a closure of the outer part of
the finger
domain toward the palm domain. Amino acids from codons
67, 69,
and 74 define the ß3-ß4 loop in the finger domain of the
HIV-1
RT (
22,
47), and, therefore, one can speculate
that the
67
deletion may influence this aspect of RT structure. The
importance
of this interaction is supported by the fact that many of
the
key mutations that enhance AZT resistance are located in this
finger domain. Taken together, these observations suggest that
the
emergence of the codon 67 deletion may be dependent upon a
prior
change in the structure or function of
RT.
Drug susceptibility of chimeric HIV.
To further assess the
potential relevance of this deletion at RT codon 67, a series of
phenotypic analyses were carried out with recombinant viruses. A
chimeric HIV was constructed with NL4.3 (1) as the parental
HIV-1 strain and a cloned RT from the patient from December 1994. This
chimera, HIVRT39180, contained the deletion at codon
67; three amino acid substitutions associated with AZT resistance
(K70R, T215F, and K219Q), two mutations associated with NNRTI
resistance (L74I and K103N) (44), a substitution of T69G,
and 11 additional amino acid substitutions of unknown significance
(V35I, S48G, E102K, K122Q, I135M, C162S, G196E, R277K, R284K, V292I,
and Q297A). Phenotypic studies were performed by previously described
methods (2, 53) with MT-2 cell lines (17, 18).
Table
2 compares the drug
susceptibility profiles of wild-type NL4.3 and the chimera
HIV
RT39180 to four RT inhibitors and
one PRT
inhibitor. The PRT inhibitor IND (
48) was used as a
control
inhibitor for these experiments. The chimera, HIV
RT39180,
showed extremely high-level resistance to AZT (1,450-fold) in
the
absence of resistance to IND. Therefore, this decreased
sensitivity
to AZT of HIV
RT39180 was caused by drug
resistance and was not
due to a nonspecific characteristic of this
chimeric virus.
Prior studies have identified high levels of AZT resistance as being in
the range of 100- to 350-fold differences in IC
50 compared
to that of the wild type and in association with amino
acid
substitutions M41L, D67N, K70R, L210W, T215F, and K219Q (
19,
21,
24,
28,
30,
44). To further define the effect of
the deletion at
codon 67 on AZT susceptibility in this chimeric
clone, the
wild-type codon GAC (aspartic acid [D]) was inserted
at RT
codon 67. The resulting virus, HIV
RT39180 + D67,
showed
reduced IC
50s of AZT and nevirapine (
26,
35) and increased
IC
50s of ddI and ddC (Sigma) (Table
2). These results suggest
that the presence of the deletion at RT
codon 67 was an important
component in conferring high-level
resistance to
AZT.
Drug susceptibility of HIV constructs.
To assess the impact of
the deletion alone on susceptibility to RT inhibitors and the potential
interaction of the deletion with other amino acid substitutions,
including known drug resistance mutations, a series of recombinant
viruses were constructed by site-directed mutagenesis (Table
3). Constructs containing only the
deletion at codon 67 () or only T69G (G) showed reduced
susceptibility to ddC and ddI, but not to AZT or nevirapine. A
construct containing the deletion and T69G (+G) showed diminished
sensitivity to only ddC (Table 3). However, the presence of +G in
the context of known AZT resistance mutations K70R, T215F, and K219Q
(+RFQG) led to a 445-fold increase in AZT resistance. Although
addition of either of the single amino acid mutations K103N or L74I in the context of +RFQG showed little effect on AZT susceptibility (data not shown), the presence of both mutations in the context of the
+RFQG motif increased the resistance to AZT to 1,813-fold (Table 3).
A mutant containing K103N alone showed 150-fold resistance to
nevirapine (data not shown). Addition of the
67 deletion to
the
K103N mutant did not result in any significant change in the
sensitivity to nevirapine (data not shown). The addition of the
T69G
change to the K103N mutant (GN) decreased the sensitivity
to nevirapine
from 150-fold to 37-fold (Table
3), suggesting
that the T69G mutation
can influence sensitivity to NNRTIs. The
presence of the
67 deletion
in the context of the T69G and K103N
mutations led to an approximately
threefold increase in sensitivity
to nevirapine (Table
3). While the
67 deletion increased sensitivity
to nevirapine, given the fact that
different NNRTIs possess distinct
binding specificities (
4,
12,
23), the impact of this deletion
on delavidine or efavirenz
sensitivity remains unclear. In order
to elucidate the mechanism for
this marked increase in AZT resistance,
an additional series of HIV
constructs were created (Fig.
2).
A
construct containing the deletion T69G and L74I (
+GI) showed
only a
fivefold increase in the IC
50 of AZT (93.7 ± 11.1 nM).
A construct containing the deletion T69G, L74I, and K103N
(
+GNI)
showed only an 18-fold increase in IC
50 (305 ± 43.9 nM). Thus,
the high-level resistance to AZT observed in
+RFQGNI was due
to a synergistic interaction between the
+GNI and
K70R T215F
K219Q mutations (RFQ). It has been reported that several of
the
mutations associated with NNRTIs, such as Y181C and
L100I, suppress
AZT resistance (
5,
29). Thus far, however,
none of the mutations
associated with NNRTI have been reported to
enhance AZT resistance.
K103N is one of the mutations that is
commonly seen in the context
of NNRTI resistance (
44).
It has been reported that this mutation
has no impact on
preexisting AZT resistance (
5). In the present
study, the
presence of K103N and L74I conferred high-level resistance
to AZT in
the presence of the
67 deletion, and thus, these mutations
can be
categorized as mutations associated with AZT resistance
under certain
circumstances.
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FIG. 2.
Impact of the deletion of an aspartic acid at RT
codon 67 on AZT susceptibility in recombinant HIV-1 variants.
Recombinant viruses were constructed by site-directed mutagenesis.
NL4.3 was used as the wild-type (wt) virus. Variants containing T69G,
L74I, and K103N in the RT gene were designated G, I, and N,
respectively. GI, GN, and NI show the variants containing T69G and
L74I, T69G and K103N, and K103N, and L74I, respectively. A variant
containing T69G, K103N, and L74I was designated GNI. Recombinant
viruses were assessed for AZT susceptibility with the drug resistance
assay described previously (53). Solid bars show variants
without a deletion at RT codon 67, and hatched bars show variants
with the deletion. IC50s were determined from at least
three independent assays.
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|
Further studies are needed to develop new approaches for impeding the
selection and development of these highly-drug-resistant
forms of HIV-1
and to identify a salvage regimen or
regimens.
| |
ACKNOWLEDGMENTS |
We thank Robin Dewar for providing data on HIV RNA levels in plasma
during the course of therapy and critical reading of the manuscript and
Michael Baseler, Randy Stevens, and Laurie Lambert for providing the
data on total CD4 T-cell counts.
This work was supported by the National Institute of Allergy and
Infectious Diseases under contract N01-CO-56000 with SAIC-Frederick.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Immunoregulation, National Institute of Allergy and Infectious
Diseases, 10 Center Dr., Rm. 11S231, Bethesda, MD 20892. Phone: (301)
496-7196. Fax: (301) 480-5560. E-mail:
CLANE{at}niaid.nih.gov.
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