Next Article 
Journal of Virology, October 2000, p. 9339-9346, Vol. 74, No. 20
0022-538X/00/$04.00+0
Evidence of a Role for the Q151L Mutation and the Viral
Background in Development of Multiple Dideoxynucleoside-Resistant Human
Immunodeficiency Virus Type 1
J. Gerardo
García-Lerma,1
Philip J.
Gerrish,2
Anthony C.
Wright,1
Shoukat H.
Qari,1 and
Walid
Heneine1,*
HIV/AIDS and Retrovirology Branch, Division of AIDS, STD,
and TB Laboratory Research, National Center for Infectious Diseases,
Centers for Disease Control and Prevention, Atlanta,
Georgia,1 and Theoretical Biology and
Biophysics Group, Los Alamos National Laboratory, Los Alamos, New
Mexico2
Received 12 April 2000/Accepted 18 July 2000
 |
ABSTRACT |
The majority of human immunodeficiency virus type 1 (HIV-1)-infected patients treated with zidovudine (AZT) plus
zalcitabine (ddC) and didanosine (ddI) develop AZT resistance mediated
by mutations such as T215Y and M41L. Only a small proportion of
patients develop multiple dideoxynucleoside resistance (MDNR) mediated by the Q151M mutation. To gain insight into the factors responsible for
the low frequency of selection of Q151M, we evaluated the replication
capabilities of recombinant viruses carrying two possible intermediates
(151L or 151K) of the Q151M mutation generated in different reverse
transcriptase (RT) genetic backgrounds. The 151L and 151K mutations
were introduced by site-directed mutagenesis in RTs from two
patient-derived HIV-1 isolates that had either wild type (WT) Q or the
Q151M (posttreatment isolate) mutation. For comparison, both mutations
were also introduced in a laboratory-adapted HIV-1 strain
(HIV-1HXB2). Analysis of replication capabilities showed
that both 151L and 151K were lethal in RT genetic backgrounds of the WT
isolate and in HIV-1HXB2. In contrast, 151L but not 151K
allowed virus replication in RT backgrounds of the posttreatment isolate. Three mutations (V35I, S68G, and I178M) were present in the RT
background of the posttreatment isolate but not in the WT isolate.
Introduction of S68G in the RT of both the WT isolate and
HIV-1HXB2 partially restored replication capacity of
recombinants carrying the 151L mutation. The S68G mutation alone did
not confer a significant replicative disadvantage in WT viruses. Like
HIV-1151M, HIV-1151L RT was found to have six-
to eightfold resistance to AZT-triphosphate (TP), ddA-TP, and ddC-TP,
indicating an MDNR phenotype. However, HIV-1151L was found
to be less fit than HIV-1151M, which may explain the
preferential selection of HIV-1151M observed in vivo. The
demonstrated ability of HIV-1151L/68G to replicate and the
associated MDNR suggest that 151L is a potential intermediate of Q151M.
The dependence of HIV-1151L on other mutations, such as
S68G, for replication may explain the low frequency of the Q151M-mediated pathway of resistance.
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INTRODUCTION |
The nucleoside reverse transcriptase
(RT) inhibitors zidovudine (AZT), didanosine (ddI), zalcitabine (ddC),
stavudine (d4T), lamivudine (3TC), and abacavir are widely used to
treat persons infected with human immunodeficiency virus type 1 (HIV-1)
(3). However, specific patterns of mutations in the HIV-1
pol gene usually occur in these patients following treatment
and have been associated with resistance and cross-resistance to
each of these compounds. For instance, the Met184Val (M184V) mutation
is associated with resistance to 3TC, the L74V mutation is associated
with resistance to ddI and ddC, the T69D mutation is associated with
resistance to ddC, and the T215Y/F, M41L, and K70R mutations are
associated with resistance to AZT (21). The presence of
these mutations generally results in the loss of the antiretroviral and
clinical benefit of these drugs.
The T215Y mutation is the first mutation identified in the majority of
patients receiving combination antiretroviral therapy with AZT and
ddC-ddI (2). However, in the past few years several reports
have shown that HIV-1 carrying a different pattern of mutations is
selected in 3 to 16% of patients treated with AZT and ddC-ddI and
sporadically in patients receiving other ddI-containing regimens
(12, 22-26). These mutations occur at positions 62 (A62V), 75 (V75I), 77 (F77L), 116 (F116Y), and 151 (Q151M) and confer resistance to all currently approved nucleoside analogs including AZT,
ddI, ddC, d4T, 3TC, and abacavir (6, 26-28; K. Van
Laethem, M. Witvrouw, J. Balzarini, J.-C. Schmit, S. Sprecher, P. Hermans, M. Leal, T. Harrer, L. Ruiz, B. Clotet, M. Van Ranst, J. Desmyter, E. De Clercq, and A.-M. Vandamme, Letter, AIDS
14:469-471, 2000). The emergence of these multiple
dideoxynucleoside resistance (MDNR) mutations may
compromise the clinical efficacy of this entire class of compounds,
thus severely limiting treatment options.
Among the five MDNR mutations, the Q151M mutation plays a pivotal role
in the acquisition of the MDNR phenotype. Q151M is the first mutation
identified in the majority of patients who develop MDNR and by itself
confers low-level resistance to AZT, ddI, ddC, and d4T (11, 12,
22, 27). In contrast, the A62V, V75I, F77L, and F116Y mutations
do not affect drug susceptibility by themselves, but their
co-occurrence with Q151M results in high-level resistance to AZT, ddI,
ddC, and d4T and low cross-resistance to 3TC (6, 11).
Patients treated with AZT plus ddI-ddC develop AZT resistance through
acquisition of either the T215Y or Q151M mutation but very rarely
through both mutations (12, 22). The lack of coexistence of
these two mutations observed in vivo indicates the presence of two
different pathways for HIV-1 to develop AZT resistance. Although the
viral determinants that may influence selection of either Q151M or
T215Y remain undefined, it is known that coexistence of T215Y and Q151M
is not constrained since introduction of both mutations in the same
virus does not significantly affect replication or enzymatic activity
of the RT (15, 28).
The factors responsible for the low frequency of selection of Q151M
compared to T215Y are not known. The low frequency of Q151M cannot be
explained by the number of mutations required for each amino acid
change, since both Q151M (CAG
ATG) and T215Y (ACCTAC) require two-base transversions and
therefore, may preexist at a similar low frequency in the absence of
drug pressure. In addition, the replicative fitness of
HIV-1Q151M has been found to be higher than that of
HIV-1T215Y both in the presence and in the absence of
selective pressure with AZT (13, 15). Thus, decreased
fitness of HIV-1Q151M does not explain the low frequency of
selection of the Q151M mutation compared to T215Y. Therefore, additional factors such as drug susceptibility and replication capabilities of putative intermediates of Q151M may influence selection
of the Q151M pathway of resistance.
The possible intermediates that require a single base substitution from
the wild type (WT) Q (CAG) to M (ATG) at codon 151 are leucine (L;
CTG) or lysine (K; AAG). Little is known about the replication capability and drug susceptibility of
HIV-1151L or HIV-1151K. A recent report
indicated that these intermediates were deleterious when present in a
laboratory-adapted (HIV-1HXB2) genetic background
(13). Based on these results, Kosalaraksa et al. postulated
that for Q151M to develop, two base-pair transversions are needed
concurrently or within a short period of time, which may be a
relatively infrequent event (13). However, studies using
HIV-1HXB2 may be limited in their ability to assess how polymorphisms present in the RT genetic background of patient-derived HIV-1 isolates may influence or compensate the deleterious effect of
the 151L or 151K mutation. Therefore, to fully evaluate the role of the
151L and 151K mutations in the acquisition of Q151M, additional
analysis using patient-derived isolates may be necessary.
In the present study, we investigated whether replication of
HIV-1151L or HIV-1151K may limit acquisition of
the Q151M mutation. We introduced the 151L and 151K mutation in HIV-1
RT backgrounds obtained from a patient who developed the Q151M mutation
during antiretroviral therapy. We compared the effect of these
mutations in the patient-derived isolates with that seen in a
laboratory-adapted HIV-1 strain and found that only the genetic
background associated with Q151M allows replication of
HIV-1151L. We also identified a mutation at codon 68 (S68G)
that alone can partially restore replication of HIV-1Q151L
in WT viruses. Our results suggest that Q151M may potentially be
acquired through a 151L intermediate. The dependence of 151L on other
mutations such as S68G may explain the low frequency of selection of
the Q151M-mediated pathway of resistance.
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MATERIALS AND METHODS |
Virus isolates.
HIV-1 isolates L1S and L2S were obtained
from an HIV-1-infected patient who developed MDNR after sequential
treatment with AZT, ddC, and ddI. They were kindly provided by
Anne-Mieke Vandamme (Rega Institute for Medical Research and University
Hospital, Katholieke Universiteit, Leuven, Belgium). Detailed
information about the patient antiretroviral treatments, as well as the
two isolates used, has been previously described (23).
Isolate L1S has WT Q at codon 151, while isolate L2S has the Q151M
mutation. No other MDNR mutations were present in isolate L2S. L1S and
L2S were isolated from samples collected 14 months apart
(23).
Cloning of full-length HIV-1 RT from isolates L1S and L2S.
Full-length RT from isolates L1S and L2S was amplified by RT-nested PCR
and cloned using the TA cloning kit (Invitrogen; Promega). Briefly, RNA
was extracted from culture supernatants using the QIAmp Viral RNA kit
(Qiagen). The RT reaction was done for 1 h at 42°C using primer
RT2 as described previously (29). After a first round of PCR
amplification using primers AV150 and RT2, 4 µl was subjected to a
second round of amplification using primers IN3 and IN5
(29). A 1,703-bp PCR product comprising the complete HIV-1
RT was gel purified (QIAquick Gel Extraction; Qiagen) and ligated into
the pCR2.1 vector. OneShot competent cells (TOP10F') were then
transformed, and single colonies were screened for the insert by PCR
amplification using primers IN3 and IN5 (29). Plasmids
containing the whole HIV-1 RT from L1S or L2S (referred to as pL1S or
pL2S, respectively) were purified from individual colonies (Qiagen
Plasmid Purification Kit) and were used for site-directed mutagenesis.
Primers used for site-directed mutagenesis at codons 151 and 68 of HIV-1 RT.
The following primers were used to generate the 151L
or 151K mutation (mutations are underlined in the primer sequences): 5'-CAG TAC AAT GTG CTT CCA CTG GGA TGG AAA GGA TCA CC-3'
(151LF1, sense) and 5'-GG TGA TCC TTT CCA TCC CAG TGG AAG
CAC ATT GTA CTG-3' (151LR1, antisense) for the 151L mutation; and
5'-CAG TAC AAT GTG CTT CCA AAG GGA TGG AAA GGA TCA CC-3'
(151KF1, sense) and 5'-GG TGA TCC TTT CCA TCC CTT TGG AAG
CAC ATT GTA CTG-3' (151KR1, antisense) for the 151K mutation. The
primers used to generate mutants at codon 68 (68G) of HIV-1 RT were
5'-GCC ATA AAG AAA AAA GAC GGT ACT AAG TGG AG-3' (68GF1,
sense) and 5'-CT CCA CTT AGT ACC GTC TTT TTT CTT TAT GGC-3'
(68GR1, antisense).
Site-directed mutagenesis.
Mutations at positions 151 and/or
68 were introduced in the pHXB2RIP7-based infectious clone pSUM9
(kindly provided by H. Mitsuya) or in plasmid preparations
containing full-length L1S- or L2S-derived RT sequences (plasmids
pL1S.18Q151, pL1S.28Q151, pL2S.4M151, and pL2S.16M151). The
following 13 plasmids with mutant RTs were made:
pL1S.18Q151L, pL1S.18Q151K,
pL1S.28Q151L, pL1S.28Q151K, pL1S.28Q151L/S68G, pL1S.28S68G,
pL2S.4M151L, pL2S.4M151K,
pL2S.16M151L, pL2S.16M151K,
pHXB2Q151L, pHXB2Q151K, and
pHXB2Q151L/S68G.
Site-directed mutagenesis was done using the QuikChange Site-Directed
Mutagenesis Kit (Stratagene). To generate mutants at codon 151 in
patient-derived RTs, 25 ng of purified plasmids
pL1S.18Q151, pL1S.28Q151,
pL2S.4M151, or pL2S.16M151 was added to a
cocktail containing 125 ng of forward and reverse mutagenic primers
(151LF1-151LR1 or 151KF1-151KR1), 2.5 U of PfuTurbo DNA
polymerase, and deoxynucleoside triphosphates (dNTPs). Cycling
conditions were 95°C for 30 s and then 12 cycles of 95°C for
30 s, 55°C for 1 min, and 68°C for 12 min. After digestion of
the parenteral (nonmutated) DNA template by the DpnI
restriction enzyme, Epicurian Coli XL1-Blue supercompetent cells were
transformed, and individual colonies were screened for the presence of
the 151L or 151K mutation by sequence analysis using primers AV36 and
NE(1)35. Plasmid preparations pL1S.28Q151L/S68G and
pL1S.28Q151/S68G were generated from
pL1S.28Q151L and pL1S.28Q151, respectively,
using the cycling conditions described above and the mutagenic primers
68GF1 and 68GR1. All plasmids containing the desired mutations were
purified, and their RT sequence inserts were used to generate
recombinant viruses. Mutant RT inserts were all sequenced following
mutagenesis and cloning to verify absence of unexpected changes.
The pSUM9 infectious clone was used to generate pHXB2
Q151L
and pHXB2
Q151K. Conditions for site-directed mutagenesis
were as
described above, except the extension at 68°C was increased
to
32 min. Following mutagenesis, the whole RT was amplified by PCR
using primers IN3 and IN5 and cloned using the TA cloning system.
Individual colonies were then screened for the 151L or 151K mutation
by
sequence analysis. Mutagenic primers 68GF1 and 68GR1 were used
to
generate pHXB2
Q151L/S68G from pHXB2
Q151L as
described
above.
Generation of recombinant viruses with cloned RT sequences from
isolates L1S, L2S, and HIV-1HXB2.
Both mutated or
nonmutated RT sequences were used to generate recombinant viruses with
the RT-deleted HXB2-based proviral molecular clone pHIV
RTBstEII.
Recombinants were generated in MT-4 cells as previously described
(10, 29). Briefly, the full-length RT was amplified by PCR
from plasmid preparations using primers IN3 and IN5 and purified with
the QIAquick PCR purification kit (Qiagen). MT-4 cells (2.5 × 106) were then cotransfected with 1 or 2 µg of purified
PCR product and 10 µg of BstEII-linearized pHIVRTBstEII
(10). Culture supernatants were collected at different time
points or when the full cytopathic effect (CPE) was observed, and then
they were aliquoted and stored at 
70°C. Levels of p24 antigen
(Coulter HIV-1 p24 Antigen Assay) and RT activity were quantitated in
cell-free culture supernatants. Amino acids 7 to 246 of HIV-1 RT were
sequenced in all virus stocks to exclude possible spontaneous reversion
of relevant mutations or accumulation of additional mutations.
The following recombinant viruses were obtained using the RT from
HIV-1
HXB2: HXB2
Q151 (nonmutated),
HXB2
Q151L, HXB2
Q151K,
and
HXB2
Q151L/S68G. The recombinant viruses generated
using the
RT from isolate L1S were L1S.28
Q151 (nonmutated),
L1S.28
Q151L,
L1S.28
Q151K,
L1S.28
Q151L/S68G, L1S.28
S68G,
L1S.18
Q151L, and L1S.18
Q151K.
The recombinant
viruses generated using the RT from isolate L2S
were
L2S.4
M151 (nonmutated), L2S.4
M151L,
L2S.4
M151K, L2S.16
M151L,
and
L2S.16
M151K.
Titration of virus stocks.
To determine the infectious virus
titers of each stock, MT-4 cells (30,000 cells/well in six replicates)
were added to 96-well flat-bottom microtiter culture plates (Costar)
and exposed to serial dilutions of each infectious virus. Cells were
examined for CPE at day 5 of culture, and the 50% cell culture
infectious dose (CCID50) was determined by the method of
Reed and Muench (19).
Analysis of replication kinetics in MT-4 cells.
Inocula of
450 CCID50 was used to infect 4.5 × 105
MT-4 cells (multiplicity of infection [MOI] = 0.001). After
incubation for 2 h at 37°C, cells were washed twice with
phosphate-buffered saline and resuspended in complete medium at
7.5 × 104 cells/ml. Two-milliliter cultures were done
in triplicate using 24-well tissue culture plates (Costar).
Supernatants (200 µl) from each culture were collected at different
days and then an equal volume of culture media was added. Levels of p24
antigen were quantitated in cell-free culture supernatants and were
used to monitor replication kinetics.
Sequence analysis of HIV-1 RT.
Sequence analysis of HIV-1 RT
(from nucleotides 2529 to 3333 of HXB2; amino acids 7 to 246) was done
in an ABI 373 automated sequencer using primers AV36, AV44, A35, and
NE(1)35 (23). The DNAsis program was used to analyze the
data and to determine deduced amino acid sequences.
Amino acid frequencies in HIV-1 sequence databases were analyzed using
the RT and Protease Sequence Analysis Program developed
by Robert
Shafer, Duane Jung, and Brad Betts (HRP-ASAP v1.1, Stanford
University;
http://hivdb.stanford.edu/hiv/).
Analysis of relative replicative fitness in virus mixtures.
Relative replicative fitness was analyzed in growth competition assays
as previously described (8). Viruses were adjusted according
to their CCID50 values before mixtures were prepared. Briefly, a 300-µl inoculum of the two competing variants mixed at
different ratios was used to infect 3.0 × 105 MT-4
cells at an MOI of 0.001 as described above. After 5 to 6 days in
culture, 200 µl of the supernatant from 2-ml cultures were used to
reinfect a fresh aliquot of 3.0 × 105 MT-4 cells. The
relative proportion of the two competing variants was determined both
at baseline and in each passage based on the ratios of the specific
mutations. Ratios were estimated based on the relative peak heights in
electropherograms obtained by automated DNA sequencing as previously
described (13). The ratios of leucine and methionine in
mixtures of L2S.4M151L and L2S.4M151 were
determined based on the relative proportions of cytosine and adenosine
at the first nucleotide position at codon 151, respectively. Similarly,
the ratios of serine and glycine in mixtures of L1S.28S68 and L1S.28S68G were determined based on the relative
proportions of thymidine and cytosine at the second nucleotide position
at codon 68, respectively. To control for the possible spontaneous reversion of mutated codons, HIV-1 RT from cultures done with each
virus separately was also sequenced after each viral passage.
Quantitation of RT activity in culture supernatants.
Levels
of RT activity were quantitated in 10 µl of cell-free culture
supernatants using the ultrasensitive PCR-based Amp-RT assay
(9). Quantitation of Amp-RT signals was done by an
enzyme-linked immunosorbent assay-based, nonradioactive oligoprobing
system using a standard curve of known units of RT activity/milliliter as previously described (5).
RT drug susceptibility testing in recombinant viruses.
The
susceptibility to AZT-triphosphate (AZT-TP), ddA-TP (the active form of
ddI), and ddC-TP of RTs from recombinant viruses was determined
enzymatically by measuring IC50 values using the Amp-RT
assay as previously described (6). Testing conditions included duplicate Amp-RT reactions done in the absence or presence of
several concentrations of the TP form of the nucleoside analog (6). The concentration of each dNTP in the RT step of Amp-RT was variable depending on the nucleoside analog tested. A concentration of 15 µM dTTP was used for reactions containing AZT-TP, while 5 µM
dCTP and 5 µM dATP were used for reactions done with ddC-TP and
ddA-TP, respectively. The other three dNTPs were used at 20 µM each.
The RT-generated cDNA was detected by PCR amplification as described
previously after increasing the dNTP concentrations to 200 µM
(6). AZT-TP was obtained from Moravek Biochemicals, Inc.
(Brea, Calif.), and ddC-TP and ddA-TP were from Sigma Chemicals (St.
Louis, Mo.).
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RESULTS |
Generation of recombinant viruses carrying the 151L or 151K
mutation in a patient-derived WT genetic background and in
HIV-1HXB2.
We first investigated the effect of the
151L or 151K mutations in the WT RT background of isolate L1S. We
introduced these mutations in two randomly selected RT clones from this
isolate (clones L1S.28 and L1S.18) and determined virus production
following cotransfection of MT-4 cells with an RT-deleted proviral
molecular clone. The 151L and 151K mutations were also introduced in
the RT from HIV-1HXB2 as a control genetic background.
Table
1 shows the levels of p24 antigen
and RT activity observed after 12 days of culture. The infectivity of
recombinant
viruses measured as CCID
50 values per
milliliter is also shown.
Transfections done with the WT RT clone
L1S.28
Q151 resulted in
high levels of both RT activity and
p24 antigen. A high titer
of infectious virus was also seen, indicating
that this RT sequence
did not compromise replication capability of
HIV-1 (Table
1).
In contrast, little or no RT activity or p24 antigen
was observed
in transfections done with either L1S.28
Q151L
or L1S.28
Q151K.
The low RT levels seen in
L1S.28
Q151L and L1S.28
Q151K were not
associated
with detectable infectious virus in culture supernatants,
suggesting
that the 151L or 151K mutations profoundly affected
the replication
capabilities of these viruses. Comparison of the
RT sequence from
plasmid pL1S.28
Q151 with the sequences of
pL1S.28
Q151L or pL1S.28
Q151K showed no
mutations other than 151L or 151K (not
shown), confirming that the
observed lack of productive infection
is due to a deleterious effect of
the 151L or 151K mutation. Similar
results were seen in two other
mutants that were generated with
an additional RT clone from isolate
L1S (clone L1S.18) (Table
1), further supporting the deleterious effect
of both 151L and
151K on virus replication. The two RT clones used
(L1S.18 and
L1S.28) were not identical and differed at four amino acids
at
codon positions 82 (K or E), 113 (D or N), 202 (I or L), and 220
(K
or I). These amino acid changes had no effect in replication
of
HIV-1
151L or HIV-1
151K, and they may likely
represent natural
polymorphisms.
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TABLE 1.
Levels of p24 antigen, RT activity, and infectious virus
titers (CCID50) in culture supernatants obtained following
cotransfection of MT-4 cells with an RT-deleted clone and RTs from
HIV-1HXB2 or isolate L1S (clones L1S.28 and L1S.18)
having Q, L, or K at codon 151a
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The effect of the 151L and 151K mutation was also determined using the
RT genetic background from HIV-1
HXB2. High titers of
p24
antigen, RT activity, and infectious virus were seen with
a recombinant
generated using the WT RT sequence from HIV-1
HXB2 (Table
1). In contrast, low or undetectable levels of p24 antigen
and RT
activity were seen in HXB2
Q151L and HXB2
Q151K,
respectively.
Both sequences were not associated with detectable
infectious
virus (Table
1), demonstrating that HXB2
Q151L
and HXB2
Q151K replicate
much less efficiently compared with
HXB2
Q151. Taken together,
our results indicate that the
151L or 151K mutation profoundly
decreases the replication capability
of HIV-1, suggesting that
acquisition of Q151M may be limited by
impaired replication of
a 151L or 151K
intermediate.
The RT genetic background of an isolate carrying the Q151M mutation
supports the replication of HIV-1151L but not
HIV-1151K.
To determine whether the genetic background
associated with Q151M could support replication of either
HIV-1151L or HIV-1151K, we mutated M151 to L or
K in two different RT clones from isolate L2S (clones L2S.4 and
L2S.16). Table 2 shows that the levels of
p24 antigen and RT activity observed in recombinants carrying the 151L
mutation (recombinants L2S.4M151L and
L2S.16M151L) 12 days after cotransfection were similar to
those observed in a parenteral virus having the Q151M mutation
(recombinant L2S.4M151), indicating that the genetic
background associated with Q151M could support the 151L mutation
without compromising replication capabilities. Both recombinant viruses
L2S.4M151L and L2S.16M151L were able to
reinfect MT-4 cells with kinetics of p24 antigen production similar to
those of WT viruses or viruses carrying the Q151M mutation, indicating
that these two viruses can replicate efficiently (Fig. 1). These findings also indicate that the
amino acid changes observed between the two RT clones L2S.4 and L2S.16
(R or K at codon 83, and W or R at codon 153) have no effect on
replication capacities and may likely represent natural polymorphisms.
In contrast to the 151L mutation, cotransfections done with two
different RT clones carrying the 151K mutation (L2S.4M151K
and L2S.16M151K) resulted in little or no RT activity or
p24 antigen, indicating that the 151K mutation was deleterious in these
RT backgrounds (Table 2).
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TABLE 2.
Levels of p24 antigen and RT activity in culture
supernatants obtained following cotransfection of MT-4 cells with
an RT-deleted molecular clone and two RT clones from isolate L2S
(L2S.4 and L2S.16) having M, L, or K at
codon 151a
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FIG. 1.
Replication kinetics of WT HIV-1 (HXB2Q151
and L1S.28Q151) and mutants carrying the 151L
(L2S.4M151L and L2S.16M151L) or 151M
(L2S.4M151) mutation. Virus production was monitored
overtime by measuring p24 antigen in supernatants. Mean p24 values from
triplicate cultures are shown. Mock, uninfected cells.
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HIV-1151L has a significant replication disadvantage
compared to HIV-1151M.
We next compared replicative
fitness of HIV-1151L and HIV-1151M to determine
whether a low replication capability of HIV-1151L could
explain why viruses carrying the 151M mutation but not 151L are
selected in vivo. Replicative fitness was determined by using a
competitive HIV-1 replication assay of HIV-1151L and
HIV-1151M and by measuring virus infectivity/total HIV-1
virion particle ratios. Total HIV-1 virion particles in culture
supernatants were expressed as levels of p24 antigen or RT activity.
Virus infectivity/virion particle ratios measured in
L2S.4
M151L and L2S.16
M151L were found to
be 20- to 73-fold and 125- to
200-fold lower than the mean ratios seen
in WT viruses, respectively,
suggesting that the 151L mutation
decreases replication capabilities
in HIV-1 (Table
3). In contrast, no clear evidence of
reduced
replication capabilities was seen when HIV-1
151L
was compared
to HIV-1
151M by analysis of virus
infectivity/virion particle
ratios. Ratios measured in
L2S.4
M151L and L2S.16
M151L were either
similar
to or 3- to 10-fold lower than those of L2S.4
M151. The
decreased replication capability of HIV-1
151L compared to
that
of HIV-1
151M was more evident in a competitive HIV-1
replication
assay of L2S.4
M151L and L2S.4
M151
(Fig.
2): L2S.4
M151
completely
outgrew L2S.4
M151L after 12 days in culture,
even in experiments
started with 85% L2S.4
M151L and 15%
L2S.4
M151. Cultures done with
L2S.4
M151L or
L2S.4
M151 alone showed no mutations at codon 151
after 12 days of cultivation, indicating stability of the 151L
and 151M mutation
(Fig.
2). These results demonstrate that replicative
fitness of
HIV-1
151L is lower than that of HIV-1
151M and
may explain
why only viruses carrying the Q151M mutation are observed
in vivo
(
12,
22).
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Virus infectivity (CCID50)/virion particle
ratiosa in recombinant viruses generated with
wild-type RTs (HXB2Q151 and L1S.28Q151) or RTs
carrying the 151M (L2S.4M151) or 151L
(L2S.4M151L L2S.16M151L) mutation
|
|
View larger version (12K):
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|
FIG. 2.
Competitive replication assay of HIV-1151M
(L2S.4M151) and HIV-1151L
(L2S.4M151L). Two experiments, each initiated at different
proportions of the two viruses (85% L2S.4M151L-15%
L2S.4M151 or 55% L2S.4M151L-45%
L2S.4M151) are shown. The proportion of L at codon 151 is
plotted over time. Day 0 represents proportions in the initial virus
mixtures. The results of infections done with L2S.4M151L
and L2S.4M151 separately are also shown.
|
|
The S68G mutation restores replication of HIV-1151L in
both HXB2 and a patient-derived WT genetic background.
We next
compared RT sequences of isolates L1S and L2S to identify the genetic
changes in isolate L2S that may have allowed replication of
HIV-1151L. Figure 3 shows all
mutations present in L1S.18, L1S.28, L2S.4, and L2S.16. A consensus
sequence for L1S and L2S is also shown. Each consensus sequence
represents the amino acid most frequently observed in seven RT clones
from isolates L1S and L2S. Three mutations at codon positions 35 (ValIle), 68 (SerGly), and 178 (IleMet) were found in L2S.4
and L2S.16 compared to L1S.18 and L1S.28. Analysis of the frequency of
each of these amino acid changes in the data base using the HRP-ASAP v1.1 program indicated that V35I and I178M are common mutations identified in 5 to 10% of both treated and untreated HIV-1-infected patients, suggesting that both mutations may be natural polymorphisms. The S68G mutation, however, has been previously found to be associated with Q151M in approximately 50% of patients who develop MDNR mutations (12, 22), suggesting that this mutation might play an
important role in replication of viruses having mutations at codon 151.
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|
FIG. 3.
Comparison between deduced amino acid sequences of the
RT (amino acids 7 to 246) from two clones from isolate L1S (L1S.28 and
L1S.18) or isolate L2S (L2S.4 and L2S.16). Consensus sequences of
isolate L1S and L2S are also shown. Sequences are compared with the
reference HIV-1HXB2 strain. Asterisks indicate amino acids
changes observed in L2S.4, L2S.16, and consensus L2S, but not in
isolate L1S or HIV-1HXB2.
|
|
To examine the effect of S68G on replication of HIV-1
151L,
we determined whether the deleterious effect of the 151L mutation
seen
in the genetic background of L1S.28 could be reverted by
introducing
the S68G mutation. Figure
4A shows the
mean levels
of p24 antigen obtained in duplicate transfections done
with the
double mutant L1S.28
Q151L/S68G. Results of control
duplicate transfections
done with the nonmutated L1S.28 WT RT
(L1S.28
Q151) or RTs carrying
the Q151L or S68G mutations
alone (L1S.28
Q151L and L1S.28
S68G,
respectively) are also shown. Transfections done with
L1S.28
Q151L did not result in detectable p24 antigen even
after 20 days of
culture. In contrast, transfections done with the
double mutant
Q151L/S68G (recombinant L1S.28
Q151L/S68G)
resulted in detectable
p24 antigen, although the kinetic of virus
production was delayed
compared to that of the control nonmutated WT
virus or the virus
carrying the S68G mutation alone (Fig.
4A). The
effect of S68G
on replication of viruses carrying the 151L mutation was
not limited
to L1S.28. Introduction of S68G in HXB2
Q151L
also enhanced replication
capability of this virus. Figure
4B shows the
higher levels of
p24 antigen following transfections with
HXB2
Q151L/S68G compared
to those observed in
HXB2
Q151L during the entire culture. These
results
demonstrate that the S68G mutation increases replication
of viruses
carrying the 151L mutation and suggest a compensatory
role for Q151L.
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|
FIG. 4.
Effect of S68G on replication of HIV-1151L.
WT RTs from either L1S.28 (A) or HIV-1HXB2 (B) were used to
generate site-directed mutants at codon 151 or 68. MT-4 cells were
transfected with 1 µg of proviral WT or mutant RT and 10 µg of the
RT-deleted proviral molecular clone as indicated in Materials and
Methods. Virus production was monitored by measuring p24 antigen levels
in culture supernatants. The results are the mean values observed in
duplicate transfections.
|
|
The S68G mutation is not deleterious in a WT RT genetic
background.
To determine whether the S68G mutation alone could
affect replicative fitness of WT HIV-1, we compared replicative fitness of L1S.28S68G with that of L1S.28S68 by
measuring replication kinetics and by using a competitive HIV-1
replication assay. Figure 5A shows
similar kinetics of p24 antigen production following reinfection of
MT-4 cells with recombinants L1S.28S68 and
L1S.28S68G, suggesting that the S68G mutation does not
affect replication capability of HIV-1. Similar replicative fitness of
both viruses was evident in competitive assays containing different
mixtures of L1S.28S68 and L1S.28S68G (Fig. 5B).
The proportion of L1S.28S68G remained constant after 24 days in culture in all virus mixtures, thus confirming that S68G does
not significantly affect replication capabilities in HIV-1. Taken
together, these results indicate that the S68G mutation in a WT genetic
background does not confer a significant replicative disadvantage or
loss of fitness to the virus, suggesting that viruses carrying the S68G
mutation would likely preexist in the virus population.
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|
FIG. 5.
Replicative fitness of L1S.28S68 and
L1S.28S68G. (A) Replication kinetics of
L1S.28S68 and L1S.28S68G in MT-4 cells. Virus
production was monitored overtime by measuring p24 antigen in
supernatants. Mean p24 values from triplicate cultures are shown. (B)
Competitive replication assay of L1S.28S68 and
L1S.28S68G. Two experiments, each initiated at different
proportion of the two viruses (80% L1S.28S68G-20%
L1S.28S68, or 40% L1S.28S68G-60%
L1S.28S68) are shown. Day 0 represents proportions in the
initial virus mixtures. Results of infections done with
L1S.28S68 and L1S.28S68G separately are also
shown.
|
|
Susceptibility to nucleoside analogs of RTs from recombinant
viruses carrying the 151L or 68G mutation.
We next sought to
determine whether the 151L or 68G mutations could affect susceptibility
to nucleoside analogs. Table 4 shows that
RT susceptibility to AZT-TP, ddA-TP, and ddC-TP in a recombinant virus
carrying the 151L mutation (L2S.4M151L) was six- to
eightfold lower than that of WT HIV-1 (L1S.18). Interestingly, IC50 values observed in L2S.4M151L were similar
to those of L2S.4M151 for the three drugs tested,
indicating that both 151L and 151M confer similar levels of MDNR.
View this table:
[in this window]
[in a new window]
|
TABLE 4.
Susceptibility to AZT-TP, ddA-TP, and ddC-TP or RT in
recombinant viruses containing WT (L1S.28) or mutant RTs carrying the
S68G (L1S.28S68G), 151M (L2S.4M151), or 151L
(L2S.4M151L) mutation
|
|
We also compared RT susceptibility in L1S.28 and
L1S.28
S68G. Table
4 shows similar IC
50
values for AZT-TP, ddA-TP, and ddC-TP
in L1S.28
S68G and
L1S.28, indicating that the S68G mutation does
not affect the
susceptibility to these
drugs.
| |
DISCUSSION |
We investigated whether decreased replication capabilities of
viruses carrying the two intermediates between Q and M at codon 151 might explain the lower frequency of selection of MDNR compared to the
classical AZT resistance pathway mediated by the T215Y mutation. We
have focused on the 151L and 151K intermediates since preexistence of
HIV-1151L and/or HIV-1151K could allow
emergence of Q151M via a single-base substitution. We also wanted to
investigate the effect that the RT genetic background has on the
replication of both HIV-1151L and HIV-1151K to
identify possible viral determinants that might influence selection of
the Q151M mutation.
Several reports have shown that viruses carrying intermediates of the
T215Y mutation, such as T215S and T215N, replicate efficiently and that
these mutations confer little or no fitness cost in the absence of AZT,
thus facilitating the development of AZT resistance through this
pathway (7, 13, 14). However, in contrast to T215S or T215N,
our results indicate that both the 151L and 151K intermediates are
lethal in all WT RT genetic backgrounds tested, including those
obtained from a WT patient-derived isolate or from
HIV-1HXB2. The decreased replication capabilities of these two intermediates may imply that two base transversions are needed to
evolve from the WT Q to M at codon 151, which may be a relatively infrequent event. Similar findings and conclusions were also reported by Kosalaraksa et al. who analyzed the effect of 151L and 151K in the
HIV-1HXB2 genetic background only (13). However,
our findings on the effect of the 151L mutation in the genetic
background associated with Q151M provide new information that suggests
a role for Q151L as a potential intermediate of Q151M. Several
observations suggest that Q151L could be a viable intermediate. First,
the observed reversion of the deleterious effect of 151L in the
151M-derived RT background indicated that replication of
HIV-1151L is possible. Second, we found that the 151L
mutation confers MDNR, which can favor the selection of viruses with
this mutation in patients treated with AZT and ddC or ddI. Third, our
results showed that the replicative fitness of HIV-1151L is
lower than that of HIV-1151M, further supporting a role of
151L as a transient intermediate. The higher replicative fitness of
HIV-1Q151M may explain the frequent observation in vivo of
Q151M but not Q151L (12, 22-26).
Our data also indicate that replicative fitness of
HIV-1151L depends on the presence of at least one
compensatory mutation. We demonstrated the compensatory role of S68G by
showing that introduction of this mutation partially restores
replication capacity of HIV-1151L in WT genetic
backgrounds. The S68G mutation might therefore provide a "bridge"
across the adaptive "valley" caused by the 151L mutation. These
findings indicate a dependence of 151L on other mutations such as S68G
for replication which may explain the low frequency of the
Q151M-mediated pathway of resistance.
Several observations suggest that S68G may represent a preexisting
polymorphism. First, the S68G mutation only requires a single base
substitution (AGUGGU), which is a relatively
frequent event (4, 17). Second, fitness assays showed that
S68G does not confer a significant replicative disadvantage to the
virus, indicating that this mutation is near neutrality. Third,
HIV-1S68G has WT susceptibility to AZT, ddC, and ddI, as
expected from a natural polymorphism. Fourth, S68G is present in ~3%
of untreated patients, as indicated by our analysis of HIV-1 sequence databases.
The compensatory role of S68G in HIV-1151L demonstrated in
our study in both a patient-derived virus and in HIV-1HXB2
may provide a mechanism by which other patients acquire Q151M. The frequency of S68G in treated individuals is significantly higher among
patients who carry the Q151M mutation than in those having the
classical AZT resistance mutations (12, 22). About 50% of
patients with Q151M also have S68G, supporting a role of this mutation
in the acquisition of Q151M, possibly through 151L. However, the fact
that many patients with MDNR phenotypes do not show evidence of a S68G
mutation may suggest either a transient role for S68G or the
involvement of alternative compensatory mutations (12, 22).
Our findings on the effect of the S68G mutation in replication of
HIV-1151L emphasize the importance of the genetic
background in determining the fitness of mutations. The role of the
genetic background in replication of viruses carrying different
mutations associated with resistance to protease and RT inhibitors has
been previously documented. For instance, an isoleucine at codon 10 of
the protease has been found to be critical for allowing replication of
viruses carrying several protease resistance mutations, and a proline
at codon 63 compensates for the deleterious effect of a L90M mutation
(16, 20). Similar compensatory effects on virus replication
have been observed in the RT for the G190E and L74V mutations or for
the Y115W and M230I mutations (1, 18).
Selection of the Q151M or T215Y pathways of resistance may also be
influenced by other factors such as virus population size. The higher
fitness of HIV-1Q151M compared to HIV-1T215Y
observed in several studies (13, 15) implies that if just
one viable Q151M mutant appears before fixation of T215Y, then Q151M
should displace T215Y, thus leading to MDNR. As population size
increases, the time required for fixation of T215Y should also
increase. A large population size might also result in an increased
number of replication-competent HIV-1151L. Thus, the
probability of appearance of Q151M may increase as the effective viral
population size increases.
In conclusion, we show evidence that suggest that 151L but not 151K is
a potential intermediate of Q151M. We demonstrate that replication
capabilities of HIV-1151L are dependent on the presence of
other polymorphisms such as S68G, which may explain the low frequency
of selection of MDNR mediated by Q151M.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: HIV/AIDS and
Retrovirology Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd., N.E., MS G-19, Atlanta, GA 30333. Phone: (404) 639-0218. Fax: (404) 639-1174. E-mail: wmh2{at}cdc.gov.
| |
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Abstract
Introduction
