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Current Drug Targets – Infectious Disorders 2003, 3, 355-371
355
Fitness Variations and their Impact on the Evolution of Antiretroviral Drug
Resistance
Luis Menéndez-Arias 1,*, Miguel
A. Martínez2, Miguel E. Quiñones-Mateu3,
2
and Javier Martinez-Picado
1
Centro de Biología Molecular “Severo Ochoa”, Consejo Superior de Investigaciones
Científicas – Universidad Autónoma de Madrid, 28049 Madrid, Spain; 2Laboratorio de
Retrovirología, Fundación irsiCaixa, Hospital Universitario Germans Trias i Pujol,
Badalona, Barcelona, Spain; 3Department of Virology, Lerner Research Institute,
Cleveland Clinic Foundation, Cleveland, Ohio, USA.
Abstract: The human immunodeficiency virus (HIV) exhibits extensive heterogeneity
due to its rapid turnover, high mutation rate, and high frequency of recombination. Its
remarkable genetic diversity plays a key role in virus adaptation, including
development of drug resistance. The increasing complexity of antiretroviral regimens
has favored selection of HIV variants harboring multiple drug resistance mutations. Evolution of drug resistance is
characterized by severe fitness losses, which can be partially overcome by compensatory mutations or other
adaptive changes that restore virus replication capacity. Recent reports have addressed the impact of drugresistance mutations on viral fitness. Methods include in vitro estimates based on the determination of viral
replication kinetics, viral infectivity in single-cycle assays and growth competition experiments; as well as
estimates of the relative fitness of viral populations in vivo calculated from standard population genetics theory.
This review focuses on the effects in viral fitness of mutations arising during treatment with reverse transcriptase
and protease inhibitors, and the molecular mechanisms (including compensatory mutations) that improve the viral
fitness of drug-resistant variants.
Key Words: HIV, fitness, drug resistance, antiretroviral drugs, protease, reverse transcriptase, evolution.
INTRODUCTION
In a human immunodeficiency virus (HIV)-infected
individual, the rapid turnover, the high mutation rate and the
high frequency of recombination result in a diverse
population. The HIV-1 reverse transcriptase (RT) is errorprone, and its error rate has been estimated at between 10 -4 to
10-5 mutations per nucleotide and cycle of replication (for
recent reviews see ref. [1, 2]). If one assumes that 109-1010
virions are produced each day in an infected person, then
they must be the product of at least 107-108 replication
cycles. Given the length of the HIV-1 genome
(approximately 10,000 nucleotides), it is likely that every
single possible point mutation (and probably many double
mutations) will occur at least once each day, in an infected
individual. Although specific combinations of multiple
mutations may be rare, it is clear that the degree of potential
genetic change drives the diversification of HIV-1 in
response to the selective pressure of host immune responses
or antiretroviral therapy.
The introduction of potent antiretroviral therapies has
been an important achievement towards the control of HIV
infection and AIDS. However, the development of resistance
*Address correspondence to this author at Centro de Biología Molecular
“Severo Ochoa”, CSIC – Universidad Autónoma de Madrid, Cantoblanco,
28049 Madrid, Spain; Tel: 34-913978477; Fax: 34-913974799; E-mail:
[email protected]
1568-0053/03 $41.00+.00
constitutes a major hurdle towards long-term efficacy of
current antiretroviral therapies [3]. The evolutionary
pathways leading to resistance have been widely studied for
many antiretroviral drugs. In general, drug resistance
mutations emerge at the expense of a loss in viral fitness.
Fitness is a complex parameter aimed at describing the
replicative adaptability of an organism to its environment.
For HIV (and other RNA viruses), an experimentally useful
approach to fitness is its relative ability to produce stable
infectious progeny in a given environment (i.e. cell culture,
blood stream, etc.) (for reviews, see refs. [4, 5]). Evolution
of resistance to antiretroviral drugs is characterized by
significant fitness loss and subsequent repair strategies that
include compensatory mutations in the targeted gene, as well
as other molecular mechanisms that improve the replication
capacity of the virus. The recognition of virological and
clinical correlates of viral fitness is becoming more and more
important in the clinical setting, and in this review we will
discuss on the relevant techniques used to measure fitness,
the effect of drug resistance mutations on viral fitness and
their implications in the management of HIV infection.
TECHNIQUES USED TO ESTIMATE VIRAL
FITNESS WITH DRUG-RESISTANT HIV VARIANTS
Viral fitness refers to the ability of a virus to reproduce in
a defined environment. In vivo fitness depends on multiple
viral and host factors. First, all events involved in the virus
© 2003 Bentham Science Publishers Ltd.
356 Current Drug Targets – Infectious Disorders 2003, Vol. 3, No. 4
life cycle could have an impact on the virus replication
capacity: (i) target cell entry, (ii) reverse transcription, (iii)
integration, (iv) gene expression, (v) assembly, (vi) egress,
and (vii) maturation. In addition, there are selective factors
that impose certain constraints on viral replication. These
factors include the state of the host cell for optimal
replication and virion production, the host immune system,
the density of target cells at the site of infection, the number
of transmitted virions, and the complex distribution of
closely related but distinguishable genomes that constitute
the so-called viral ‘quasispecies’ [5].
Emergence of mutations during treatment with current
antiretroviral drugs [mostly, RT and protease (PR) inhibitors]
is likely to affect the catalytic efficiency of those viral
enzymes, having a detrimental effect on viral replication.
Enzymatic assays provide good estimates of the effect of
drug-resistance mutations on the catalytic efficiency of the
viral enzymes. PR activity assays monitoring cleavage of
model oligopeptide substrates or precursor polyproteins have
been used to determine their proteolytic efficiency. Resistance
mutations are often associated with a significant loss of
catalytic efficiency (reduced kcat /Km values), or with low
vitality values [6, 7]. In a similar way, the effect of
mutations in the RT are clearly revealed through monitoring
the polymerase or RNase H activities of the enzyme, or its
processivity. Amino acid substitutions that diminish the
catalytic efficiency of the viral enzymes are expected to have
a detrimental effect on the replication capacity of the virus,
rendering viruses which are less fit that their wildtype counterpart.
For this purpose, replication kinetics assays that quantify
the efficiency of HIV replication in parallel cultures, and
competitive culture assays in which the proportions of the
competing viruses are carefully monitored over time using
various techniques (sequencing, differential hybridization,
heteroduplex mobility assays, TaqMan real-time polymerase
chain reaction, etc.) are a much better choice (reviewed in
[8]). Several studies have extended the definition of relative
fitness by comparing (i) the virus turnover in HIV-1-infected
individuals, (ii) HIV-1 production in cultures infected with
different viruses, (iii) virion (infectious)/virus particle ratios,
and (iv) HIV infectivity in single-cycle infectious assay.
The simplest method to obtain a relative fitness value is
by comparing the replication curves for each virus after
infection of cell cultures, and then measuring the amount of
p24 antigen or RT activity in the culture supernatants [9-13].
These assays are usually done with MT-2, MT-4 or
phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMC). This method is suitable to show
significant differences in replication kinetics between viable
viral variants, but is less sensitive to subtle fitness
differences between viruses.
In growth competition experiments, two viral variants are
mixed to produce an infection, and after successive passages,
the amount of each virus is measured by using a distinctive
genetic trait. For example, an infection with wild-type and
drug resistant variants, at 1:1 or a different ratio is carried
out, and then followed in cell culture after several passages
(i.e. in MT-2 cells, or in phytohemagglutinin-stimulated
Menéndez-Arias et al.
PMBCs), by measuring the relative amount of the drug
resistance mutation [12-16].
Mathematical interpretation of the growth competition
experiments may result in the determination of a relative
fitness value from the equation p/q = [p(0)/q(0)] x (fitness)T ,
where p is the proportion of less fit virus, q is the proportion
of more fit virus, 0 indicates time 0, and T is the time in
viral generations [17, 18]. This approach may not be
adequate to study HIV fitness variation, since the model
does not take into account changes in the multiplicity of
infection over time. More complex methods that can be used
not only for steady-state viral populations, but also for
decreasing or expanding viral populations have also been
described [19, 20]. Growth competition experiments are more
sensitive than replication kinetics in order to detect fitness
differences between viral variants. Another advantage of this
method is that the replicative abilities of the two competing
viral strains are determined under identical conditions.
However, these assays can take weeks to perform and are
labor intensive, limiting their use in the clinical setting.
Relative estimates of viral infectivity have been determined
in experiments carried out independently with each virus. In
these assays, a fixed amount of virus (equivalent to 5-10 ng
of HIV-1 p24), grown in the absence or in the presence
of antiretroviral drugs, is used to infect indicator P4 cells in
a single cycle. Upon infection, P4 cells produce βgalactosidase, whose activity can be detected through the
cleavage of chlorophenolred-β-D-galactopyranoside. The ratio
of mutant to wild-type infectivity is calculated to obtain a
fitness-related value [21]. A similar method uses GHOST
cells that are stably transduced with the green fluorescent
protein linked to the HIV-1 long terminal repeat. Then, the
expression of the green fluorescent protein is induced with
the viral protein Tat [22]. The first commercially available
assay uses luciferase expression as reporter of single cycle
infection, and a virus containing a recombinant pol gene [23,
24]. This method is a modification of the single-round
recombination virus assay used to determine viral drug
susceptibility (marketed as PhenoSense, ViroLogic Inc., San
Francisco, California, USA) [25 ].
An important issue to be considered when obtaining
relative fitness values refers to the viral isolates or strains
used in the assays. Reported fitness determinations have
been obtained with reference viral strains (usually, HXB2 or
NL4-3) in which mutations were introduced by site-directed
mutagenesis, with recombinant variants obtained by
introducing the PR or the RT coding-region within a
reference clone lacking that particular gene, or alternatively
with natural isolates obtained from HIV-infected patients
after coculture in PBMCs. It is clear that the genetic background in each case adds complexity to the interpretation of
the results. Thus, it is easier to draw conclusions from
fitness experiments carried out with viruses containing one
or two mutations introduced by site-directed mutagenesis,
than from experiments involving clinical isolates. A recent
study has shown that molecular defects in the RT, in the
PR, or in both may contribute to the infectivity and
replicative capacity phenotype of a clone [22]. For example,
the increase or diminution of fitness observed when a PR
variant is introduced in a recombinant virus may not be the
Viral Fitness and Antiretroviral Drug Resistance
same if the recombined fragment includes the PR alone or
both the PR- and the RT-coding regions. In addition, these
defects can be expressed differently depending on the cellular
system used for testing [26].
Analyzing Fitness In Vivo
The relative fitness of viral populations in vivo can be
estimated from their relative rates of outgrowth which can be
calculated from standard population genetics theory. Thus, if
time is given in generations, the relative frequency over time
is given by the equation:
ln[p2(t)/p1(t)] = ln[p 2(0)/p1(0)] + (s2-s1)t
where p2 and p 1 represent the relative frequency of two viral
populations, with constant fitness of 1+ s1 and 1 + s2,
respectively (s2 > s 1, and the fitness of wild-type virus is 1).
Hence, if ln(p2/p1) is plotted against time, the intercept gives
an estimate of the relative frequency of the mutants prior to
therapy and the slope gives an estimate of the relative fitness
of the mutants, which can be obtained by fitting linear
models. For HIV, the generation time is usually assumed to
be 2.6 ± 0.8 days [27]. Examples illustrating the
applications of these methods to HIV drug resistance have
been described [17,28,29].
As in growth competition experiments, DNA sequencing
[20, 28], differential hybridization [30] or primer-guided
nucleotide incorporation assays [29] can be used to detect the
distinct genetic trait characterizing the competing viral
populations (i.e. the relative amounts of the wild-type ATG,
and resistant mutants ATA or GTG at codon 184 of HIV-1
RT, during treatment with lamivudine [29]). Although in
vivo fitness studies are useful to follow the emergence of
drug resistant mutants, and provide the best estimate of
fitness, they cannot make an accurate determination of the
impact of specific substitutions on the viral replicative
capacity. The dominance of specific quasispecies (i.e. drugresistant variants) in the blood, which could be less fit in
other compartments (i.e. lung or central nervous system), or
the differences in host genetics and immune response are
important issues that hamper the analysis of fitness in vivo.
FITNESS AND DEVELOPMENT OF RESISTANCE
TO PR INHIBITORS
The proviral DNA is transcribed and translated by cellular
enzymes to produce large polypeptide chains that are referred
to as polyproteins. The HIV-1 PR cleaves two of these
polyproteins (Gag and Gag-Pol) into smaller functional
proteins, thereby allowing the virion to mature. The HIV-1
PR is a homodimeric enzyme composed of two polypeptide
chains of 99 residues. The side chains of Arg-8, Leu-23,
Asp-25, Gly-27, Ala-28, Asp-29, Asp-30, Val-32, Ile-47,
Gly-48, Gly-49, Ile-50, Phe-53, Leu-76, Thr-80, Pro-81,
Val-82 and Ile-84 form the substrate binding pocket and can
interact with specific inhibitors [31], such as those used in
clinical treatment of AIDS.
Currently approved PR inhibitors include saquinavir,
ritonavir, indinavir, nelfinavir, amprenavir, lopinavir and
atazanavir. They all share relatively similar chemical
structures and cross-resistance is commonly observed in the
clinical setting. Primary resistance mutations usually involve
Current Drug Targets – Infectious Disorder 2003, Vol. 3, No. 4 357
amino acid substitutions at positions located at the
substrate/inhibitor binding site. Examples are D30N, G48V,
I50V, V82A, or I84V, among others. Often, these amino
acid changes have a deleterious effect on the replication
kinetics of HIV-1 [9, 10, 12, 13, 32] (Table 1). The effects
caused by drug resistance mutations can be rescued by other
amino acid replacements. For example, multidrug-resistant
virus arising during prolonged therapy with indinavir
contained the substitutions M46I, L63P, V82T and I84V in
the PR-coding region [12, 50]. Crystallographic studies of
the mutant enzyme revealed that the substitutions at codons
82 and 84 were critical for the acquisition of resistance,
while the amino acid changes at codons 46 and 63, which are
away from the inhibitor binding site, appear as compensatory
mutations [51, 52] [Fig. (1)]. In a similar way, saquinavir
resistance implies the acquisition of substitutions G48V and
L90M [46], where G48V exerts the major influence on
resistance, and L90M, which is located away from the
substrate/inhibitor binding site, contributes to the stability
of the HIV PR.
The analyses of viral fitness of clones containing PR
inhibitor resistance mutations are broadly consistent with the
hypothesis suggesting that the acquisition of resistance
implies a significant cost in terms of viral replicative capacity
(Table 1). Primary mutations D30N, G48V and V82T, that
arise after treatment with nelfinavir, saquinavir or ritonavir,
respectively, have been shown to produce a severe impact on
viral fitness [12, 30, 32, 35, 37, 41]. In all cases, these
substitutions affect residues that are important to stabilize
the substrate in its binding pocket. Accumulation of two or
more mutations in addition to the one producing the largest
decrease in viral fitness produce a significant recovery of the
viral replication capacity or infectivity (i.e. L10I/G48V/
L90M, M46I/G48V/L63P/L90M). High-level resistance to
PR inhibitors results from the stepwise accumulation of
amino acid substitutions in the PR. The evolutionary
pathways leading to increased levels of resistance are largely
dependent on viral fitness [21, 45]. The selective advantage
conferred by resistance mutations may depend upon several
parameters: the impact of the mutation on virus infectivity in
the absence or in the presence of the inhibitor, the nature of
the antiretroviral drug, and its local concentration.
Fitness profiles determined by measuring the infectivity
of a mutant virus relative to the wild-type in the presence of
different concentrations of PR inhibitors were consistent
with patterns of selection of drug resistant mutants [21]. For
example, in the case of ritonavir, at a low drug concentration
(0.025 µM), single mutations (M36I, M46I, I54V, A71V or
V82A) do not confer a replicative advantage over the wildtype, while a double or a triple mutant (A71V/V82A or
I54V/A71V/V82A) confers a small replicative advantage. At
higher concentrations (i.e. 0.625 µM), the double mutant has
no replicative advantage relative to the wild-type virus, but
I54V/A71V/V82A shows higher infectivity. In these
conditions, the highest fitness was displayed by the
quadruple mutant M46I/I54V/A71V/V82A. Interestingly,
this mutant shows similar infectivity to the wild-type virus
in the absence of inhibitor, thereby indicating that in some
cases, high-level resistance can be achieved without a
significant loss of fitness.
358 Current Drug Targets – Infectious Disorders 2003, Vol. 3, No. 4
Table 1:
Menéndez-Arias et al.
Effects of PR Inhibitor-resistance Mutations on Viral Fitness.
Amino acid substitutions
Viral growth
kinetics
Single-cycle
infection assays
Growth competition
experiments
References c
R8K
++++
n.d.
n.d.
[9]
R8Q
+
n.d.
n.d.
[9]
L10I
n.d.
++++
n.d.
[21]
L10F
++++
n.d.
+++
[13, 33]
D30N
++
++++
+
[12, 34, 35]
V32I
n.d.
n.d.
+++
[36]
M36I
n.d.
++++
n.d.
[21]
M46I
++++
++++
++++
[9, 21, 37]
G48V
+
++
n.d.
[32]
I50V
n.d.
+
n.d.
[38]
I54V
n.d.
++++
n.d.
[21]
L63P
++++
n.d.
++++
[10, 37]
A71V
++++
++++
n.d.
[10, 21]
V82A
++++
++++
n.d.
[21, 32, 39, 40]
V82F
++++
n.d.
n.d.
[10]
V82T
n.d.
n.d.
++
[37, 41]
I84V
++++
n.d.
+++
[10, 36, 41, 42]
N88D
+++
++++
n.d.
[33, 35]
N88S
+++
++
+
[33, 40, 43]
L90M
++++
++++
+++
[12, 21, 34, 35]
++++
+++
n.d.
[38, 44]
p1/p6(P5')
n.d.
++++
n.d.
[38]
p1/p6(P1') + p7/p1b
+++
n.d.
n.d.
[44]
I50V + p1/p6(P1')
n.d.
+
n.d.
[38]
I50V + p1/p6(P5')
n.d.
+
n.d.
[38]
I50V + p1/p6(P1') + p1/p6(P5')
n.d.
+
n.d.
[38]
R8Q/M46I
++++
n.d.
n.d.
[9]
L10F/I84V
+++
n.d.
++
[13]
L10F/N88S
+++
n.d.
n.d.
[33]
L10I/G48V
n.d.
+++
n.d.
[21]
L10I/L90M
n.d.
++++
n.d.
[21]
D30N/L63P
+++
n.d.
++
[12]
D30N/N88D
+++
n.d.
n.d.
[34]
D30N/L90M
+
+
n.d.
[34, 35]
p1/p6(P1')b
b
Viral Fitness and Antiretroviral Drug Resistance
Current Drug Targets – Infectious Disorder 2003, Vol. 3, No. 4 359
Table 1. Contd….
Amino acid substitutions
Viral growth
kinetics
Single-cycle
infection assays
Growth competition
experiments
References c
V32I/A71V
++++
n.d.
n.d.
[36]
M36I/I54V
n.d.
n.d.
++
[41]
M46I/I50V
n.d.
+
n.d.
[38]
M46I/V82A
n.d.
++++
n.d.
[21]
M46L/V82A
+++
n.d.
n.d.
[39, 45]
G48V/V82A
n.d.
+
n.d.
[21]
G48V/L90M
+
+
n.d.
[21, 46]
I54V/V82A
n.d.
++
n.d.
[21]
V62I/V77I
n.d.
n.d.
++++
[41]
L63P/L90M
++++
n.d.
++++
[12]
A71V/V82A
n.d.
++++
n.d.
[21]
V82A/L90M
n.d.
+
n.d.
[21]
V82A/L97V
++++
n.d.
n.d.
[39]
V82F/I84V
++
n.d.
n.d.
[10, 39]
L10F/I84V + p1/p6(P1’)
++
n.d.
++
[13]
M46I/I50V + p1/p6(P1’)
n.d.
++
n.d.
[38]
M46I/I50V + p1/p6(P5’)
n.d.
+
n.d.
[38]
M46I/I50V + p1/p6(P1’) + p1/p6(P5’)
n.d.
++
n.d.
[38]
M46L/V82A + p7/p1
+++
n.d.
n.d.
[45]
R8Q/M46I/A71T
+
n.d.
n.d.
[47]
L10F/M46I/I50V
n.d.
n.d.
++
[13]
L10I/G48V/V82A
n.d.
+++
n.d.
[21]
L10I/G48V/L90M
n.d.
+++
n.d.
[21]
L10I/V82A/L90M
n.d.
+++
n.d.
[21]
D30N/N88D/L90M
+
+++
n.d.
[34, 35]
M36I/I50V/L63P
+
n.d.
n.d.
[48]
M36I/I54V/V82T
n.d.
n.d.
++
[41]
M46I/I54V/V82A
n.d.
++
n.d.
[21]
M46L/V82A/L97V
++++
n.d.
n.d.
[39]
G48V/A71V/V82A
++
n.d.
n.d.
[47]
G48V/V82A/L90M
+
n.d.
n.d.
[47]
I54V/A71V/V82A
n.d.
++++
n.d.
[21]
L63P/V77I/N88S
++++
+++
+++
[43]
L63P/V82F/I84V
+++
n.d.
n.d.
[10]
360 Current Drug Targets – Infectious Disorders 2003, Vol. 3, No. 4
Menéndez-Arias et al.
Table 1. Contd….
Amino acid substitutions
Viral growth
kinetics
Single-cycle
infection assays
Growth competition
experiments
References c
L10F/M46I/I50V + p1/p6(P1’)
+
n.d.
+
[13]
M36I/I50V/L63P + p1/p6(P1’)
+++
n.d.
n.d.
[48]
M46L/I54V/V82A + p7/p1
++
n.d.
n.d.
[45]
M46L/I54V/V82A + p1/p6(P1’) + p7/p1
++++
n.d.
n.d.
[45]
L10F/M46I/I47V/I50V
n.d.
n.d.
+
[13]
K20R/M36I/I54V/V82A
+++
n.d.
n.d.
[49]
K20R/M36I/L63P/V82S
+++
n.d.
n.d.
[49]
V32I/M46I/A71V/I84A
++
n.d.
n.d.
[44]
M36I/I54V/A71V/V82T
n.d.
n.d.
++++
[41]
M46I/G48V/L63P/L90M
+++
n.d.
n.d.
[49]
M46I/I54V/A71V/V82A
n.d.
++++
n.d.
[21]
M46I/L63P/V82T/I84V
++++
n.d.
++++
[12]
M46L/L63P/V77I/N88S
+++
+++
++
[43]
G48V/A71T/V82A/L90M
+
n.d.
n.d.
[47]
L10I/L23I/M46I/I84V + p1/p6(P1’)
+++
n.d.
+++
[36]
V32I/M46I/A71V/I84A + p1/p6(P1’)
+++
n.d.
n.d.
[36, 44]
V32I/M46I/A71V/I84V + p1/p6(P1’)
+++
n.d.
n.d.
[44]
L10R/M46I/L63P/V82T/I84V
++++
n.d.
++++
[12]
K20R/M36I/I54V/A71V/V82T
n.d.
n.d.
++++
[41]
I54V/L63P/A71T/I72E/V82A/I85V
++
n.d.
n.d.
[48]
I54V/L63P/A71T/I72E/V82A/I85V + p7/p1
++++
n.d.
n.d.
[48]
L23I/V32I/M46I/I47V/I54M/A71V/I84V
++
n.d.
n.d.
[44]
L23I/V32I/M46I/I47V/I54M/A71V/I84V + p1/p6(P1’) + p7/p1
+++
n.d.
+++
[36, 44]
L10I/K20R/M36I/M46I/F53L/L63P/A71V/V82A
+++
n.d.
n.d.
[49]
a
Reported data were obtained using HIV-1 strains NL4-3 [9, 10, 12, 13, 21, 36 – 38, 43 – 45, 47, 49], HXB2 [32 – 35, 40, 41, 48], RF [39, 42] and GB8 [46] as reference
virus. Mutations were obtained by passaging the virus in cell culture [9, 39, 42], through site-directed mutagenesis [10, 12, 21, 32 – 38, 40, 43, 44, 46], or by
recombination using protease sequences derived from clinical isolates [13, 41, 44, 45, 47 – 49]. Viral replication, or growth competition experiments were carried out with
different cell types: MT-2 [12, 13, 32, 33, 37, 39, 42, 44, 45], MT-4 [9, 10, 38, 40, 47 – 49], H9 [43], CEM [43, 46], C8166 [36, 44], HUT [49], or PBMCs [12, 34, 36,
41]. 293 cultures, and HeLa-CD4+ and P4 cells were used in infectivity assays [21, 35, 43].
Symbols are: ++++, fitness similar or slightly higher than the wild-type virus; +++ and ++, lower than the wild-type virus; +, very low fitness compared with the wild-type
HIV; and n.d., not determined.
b
p1/p6(P1’) indicates a Leu to Phe amino acid substitution at the P1’ position of the Gag cleavage site between p1 and p6 (RPGNF/FQSRP instead of RPGNF/LQSRP),
p1/p6(P5’) indicates the presence of Leu instead of Pro at the P5’ position of the Gag cleavage site between p1 and p6 (RPGNF/LQSRL instead of RPGNF/LQSRP), and
p7/p1 represents the replacement of the wild-type sequence -Gln-Ala- by -Arg-Val-, at positions P3 and P2 of the Gag cleavage site between p7(NC) and p1 (ERRVN/FLGKI
instead of ERQAN/FLGKI).
c
References 9, 10, 12, 13, 21, 32, 35, 37, 38, 41, 42, 44, 45, 46, 47 and 48 are also cited in the text.
PR Inhibitor-resistance Mutations and their Impact on
Viral Fitness In Vivo
Viral replication kinetics, single-cycle infectivity assays
and/or growth competition experiments have consistently
shown that mutations D30N, G48V and V82T have a
deleterious effect on viral fitness. These data were also
consistent with experiments using a library of HIV-1
mutants containing random combinations of amino acid
substitutions in the PR coding region, that conferred
resistance to ritonavir and other PR inhibitors [53]. Authors
Viral Fitness and Antiretroviral Drug Resistance
Current Drug Targets – Infectious Disorder 2003, Vol. 3, No. 4 361
Fig. (1). Structural location of PR residues involved in drug resistance. Crystallographic structure of HIV-1 PR complexed with an
inhibitor, showing the location of amino acid residues involved in primary (A) or secondary (B) resistance mutations. (C) shows the
HIV-1 Gag and Gag-Pol precursor processing sites, indicating the sequence around the cleavage sites p7(NC)/p1 and p1/p6.
Underlined residues are involved in mutations appearing during the antiretroviral treatment. Viral protein abbreviations are: MA,
matrix protein; CA, capsid protein; NC, nucleocapsid protein; PR, protease; RT, reverse transcriptase; IN, integrase.
analyzed the stability of mutations in viral populations
selected with ritonavir. L10I, M36I, I54V, L63P, A71V,
V82A and I84V were maintained in the population,
indicating that they did not impair virus replication.
However, D30N, N88D, V82T and L90M showed lower
fitness in the absence of specific PR inhibitors [53].
The fitness in vivo conferred by the amino acid
substitution V82A, which appeared during treatment with
ritonavir, was estimated to be 96 – 98 % of the wild type
virus, as obtained from a quantitative analysis of the
replacement of the mutant population with the wild-type
virus in the absence of drugs [30]. In this case, acquisition of
additional mutations at codons 54, 63 and 84 was associated
with increases in plasma RNA levels, indicating a
compensatory effect on viral fitness. In another study
involving eleven patients, the effects on fitness were
determined for various mutations after therapy was stopped
[54]. The results of these analyses revealed a substantial
reduction of viral fitness associated with primary mutations
D30N (12.4 % loss), M46I/L (21 % loss) and V82A (21 %
loss). Other mutations such as I54V, G73S, I84V and N88D
were associated with moderate reductions of fitness (ranging
from 8 to 12 %), while L10I/V, K20R, M36I, L63P,
A71V/T, V77I and L90M had little effect on viral fitness
and persisted in most patients after therapy was stopped.
Several mutations detected at virological failure after
second or third line antiretroviral therapy have been shown to
revert to wild-type, when PR inhibitors were withdrawn,
suggesting a negative influence on viral fitness. Examples
are D30N [55], M46I/L [56], or V82A/F/S/T [56, 57].
Despite these findings, mutations that affect fitness (i.e.
V82A, N88D or L90M) may persist for months in some
patients, after the discontinuation of treatment [56, 58]. It
should be noted that the viral genetic background and
accompanying mutations found in clinical isolates may
differ from patient to patient and could have an influence
on the selective advantage of each particular amino
acid substitution.
Molecular Mechanisms Leading to Fitness Recovery
During PR Inhibitor Treatments
In addition to compensatory mutations within the PRcoding region (Table 1; [10, 30, 41, 47]), there are genetic
alterations that affect other loci within the viral genome that
also contribute to improve the HIV replication capacity and
fitness [Fig. (1)].
During HIV maturation, the viral PR hydrolyzes a series
of peptide bonds within the precursor polyproteins Gag and
Gag-Pol, rendering the structural proteins of the mature
capsid, plus the viral enzymes PR, RT and integrase.
Compensatory mutations within the PR-coding region
increase the catalytic efficiency of the enzyme. However, an
alternative mechanism to improve viral maturation involves
amino acid substitutions at the polyprotein cleavage sites,
making them better substrates for the altered viral PR. This
was observed with virus selected after culture in the presence
of palinavir derivatives [44], and in viral isolates from
patients treated with indinavir [45] or amprenavir [38]. These
mutations appear at the cleavage sites that flank the p1 spacer
peptide [p7(NC)/p1 and p1/p6]. Cleavage at these sites is the
rate limiting steps for polyprotein processing and viral
maturation [44, 48]. Enzymatic studies reveal that resistance
mutations at the p7(NC)/p1 and p1/p6 cleavage sites
improve the catalytic efficiency of mutant PRs carrying the
drug resistance mutations M46L, V82S, V82A, I84V or
362 Current Drug Targets – Infectious Disorders 2003, Vol. 3, No. 4
L90M, an effect that is not always observed with the wildtype PR [59]. This observation explains why cleavage site
mutations appear late during development of high-level
resistance. A number of examples shown in Table 1 reveal
the beneficial effect of mutations at p1/p6 or p7(NC)/p1 on
the viral fitness of PRs carrying two or more mutations
conferring antiretroviral drug resistance. Mutations at other
Gag cleavage sites (i.e. MA/CA and CA/p2) could also
enhance the rate of cleavage by the viral PR [60], although
these sites are less prone to change. Nevertheless, an amino
acid substitution at the CA/p2 cleavage site (ARVL/AEAM
to ARIL/AEAM) has been found in a nelfinavir-resistant
variant selected in vitro under subinhibitory concentrations
of the drug [61].
A third mechanism to improve viral maturation efficiency
involves mutations that produce an increased level of GagPol frameshifting. Frameshifting is a translational event that
occurs at a low efficiency (approximately 5 %) during Gag
synthesis. It is facilitated by specific sequence and structure
motifs in the viral RNA, which are found near the end of the
Gag open reading frame. Mutations of the frameshift signal
have been found in PR inhibitor-resistant variants. Thus,
Doyon et al. [62] have shown that the substitution of Leu by
Phe at the P1’ position of the p1/p6 cleavage site, which
improves fitness of palinavir-resistant viruses, originates
from a C-to-T transition of the first base of the leucine
codon. This substitution also affects the viral RNA
frameshift signal, leading to a 3- to 11-fold increase in the
expression of pol [62].
Recently reported data suggest that mutations at noncleavage sites in the Gag polyprotein may also recover the
reduced replicative fitness of HIV-1 containing resistance
mutations in the PR-coding region. Highly-resistant HIV-1
variants selected against amprenavir, and other PR inhibitors,
were found to contain additional mutations in the matrix
protein (MA) (L75R, within the sequence context
…GSEELRSLY…), the capsid protein (CA) cyclophillin
binding loop (H219Q, in the sequence context
…HPVHAGPI…), the nucleocapsid protein (NC) (V390D or
V390A and R409K, within the sequence …RKTVKCF…
RAP R KKG…) and at p6 (E468K, within …FGEETT…).
These mutations were indispensable for HIV-1 replication in
the presence of PR inhibitors. Although the molecular basis
of resistance mediated by these substitutions is unknown, it
is suggested that the identified Gag mutations should render
the polyprotein cleavage sites more accessible to the PR,
thereby improving the efficiency of the maturation process
[63]. Further evidence on the influence of non-cleavage sites
in Gag has been obtained from viral isolates harboring a
nelfinavir-resistant PR containing mutations L10I/M36I/
G48V/I54V/I62V/I72V/T74S/V82A, which acquired three
additional amino acid substitutions in the PR (E35D, N37S
and K43T), a single amino acid change in the CA/p2
cleavage site (ARVL/AEAM to ARIL/AEAM), and 7
substitutions in the MA protein (E55Q, G62R, V82I, S109N,
Q117E, N129D, and D130N), during antiretroviral treatment.
When tested in vitro, this variant showed increased
replication capacity in the presence of nelfinavir [61].
Menéndez-Arias et al.
FITNESS AND DEVELOPMENT OF RESISTANCE
TO RT INHIBITORS
It is widely accepted that amino acid changes conferring
resistance to RT inhibitors do not reduce fitness to the same
extent as PR inhibitor-resistance mutations. This may be due
to several reasons: (i) the RT could be more flexible than the
PR in absorbing resistance mutations, (ii) mutations
conferring resistance to RT inhibitors are often relatively
distant from the dNTP binding site (as occurs with
nonnucleoside inhibitors, or zidovudine and stavudine), and
(iii) the degree of cross-resistance among RT inhibitorspecific mutations is significantly lower than the one
observed with PR inhibitor-specific mutations. In contrast
to PR inhibitors, evolutionary pathways leading to resistance
to RT inhibitors can be rather different depending on the
drug.
Impact of Mutations Conferring Resistance to
Nonnucleoside RT Inhibitors
Nonnucleoside RT inhibitors in clinical use include
nevirapine, delavirdine and efavirenz. These drugs bind to a
hydrophobic cavity located 8 –10 Å away from the
polymerase active site [Fig. (2)]. Mutations in the binding
pocket are rapidly selected during nonnucleoside RT
inhibitor-therapy. Available data indicate that the drugresistance mutations L100I [42], K103N [65 – 68], V179D
[69], Y181C [57, 67, 68, 70], Y181I [67], Y188C [67],
Y188H [67], and Y188L [67] have a relatively small effect
on viral fitness, despite having a large influence on
resistance. In contrast, other mutations confer reduced
replication capacity. Examples are V106A [69, 70], P225H
[67], M230L [67], P236L [65, 67, 71], and several mutants
at codons 138 [72] and 190 [24, 70, 73].
Fig. (2). Nonnucleoside RT inhibitors binding site, showing the
location of relevant residues involved in drug resistance, whose
effects on viral fitness have been determined experimentally.
The crystallographic structure used was taken from ref. [64] and
shows the HIV-1 RT complexed with delavirdine.
Viral Fitness and Antiretroviral Drug Resistance
Replication defects produced by mutations V106A,
G190E and P236L are likely to result from impaired RNase
H activity [65, 69, 74]. Mutations at position 138 may affect
RT stability, since this residue contributes to electrostatic
interactions between the RT subunits p66 and p51. The
fitness ranking for these mutants is: E138D > E138G >
E138K > E138A > E138Q = E138Y > E138F [72].
Introducing large hydrophobic residues at this position
produced a large diminishing effect on viral fitness. Another
residue that could influence RT stability is Gly-190. Several
mutations affecting this position confer reduced
susceptibility to nevirapine and efavirenz (i.e. G190A,
G190C, G190Q, G190S, G190V, G190E, or G190T). The
replication capacity of five of them (G190C, G190Q,
G190V, G190E, and G190T) was severely impaired and
correlated with reduced virion-associated RT activity and
incomplete PR processing of the viral Gag polyprotein [24].
However, recombinant patient-derived viruses harboring the
substitutions G190V or G190E displayed higher or similar
replication capacity than the wild-type virus. Viral fitness
recovery shown by these viruses was attributed to the
presence of Val instead of Leu-74, which probably increases
protein stability [24]. The background RT sequence could
also explain why clinical isolates containing the P236L
mutation display a similar replication capacity than the wildtype virus, even though a compensatory mutation has yet to
be identified [71].
Impact of Mutations Conferring Resistance to Nucleoside
RT Inhibitors
In contrast to nonnucleoside RT inhibitors, high-level
resistance to zidovudine is achieved through the acquisition
of several mutations including M41L, D67N, K70R,
L210W, T215F/Y and K219Q/E [Fig. (3)]. Different levels
of resistance are observed depending on the particular
combination of resistance-related mutations found in one
viral clone. The first substitution arising during zidovudine
treatment is usually K70R, followed by T215Y. The K70R
mutation appears frequently, since it requires only one
nucleotide change, and does not have a major impact on viral
Current Drug Targets – Infectious Disorder 2003, Vol. 3, No. 4 363
fitness [75]. However, its effect on viral resistance is
relatively small [76]. Fitness determinations based on
growth competition experiments carried out in MT-2 or MT4 cells, revealed the following ranking on the effect of
zidovudine resistance mutations: Wild-type > K70R (97 %)
> T215Y (85 %) = M41L/T215Y (85 %) > M41L (80 %)
>> L210W (21 %) [14, 15, 75]. This fitness ranking
indicates that L210W has a strong deleterious effect on viral
fitness. However, M41L and T215Y show rather similar
fitness, as well as the double mutant M41L/T215Y. Unlike
M41L/T215Y and K70R/T215Y, the replication capacity of
the double mutant M41L/K70R in SupT1 cells was very
poor [77]. These observations are consistent with the
sequential appearance of mutations in zidovudine-treated
patients [78]. Both mutations M41L and K70R are rarely
found together in the same viral clone, although in the
presence of other amino acid substitutions (i.e. S163N), viral
replication capacity is restored [77].
The substitution of Tyr-215 by Cys, Asp or Ser has been
observed in vivo in the absence of zidovudine [28, 79].
Growth competition experiments carried out in MT-2 cells
show that both T215S and T215D display a small but
significant advantage over the wild-type virus in the absence
of inhibitor [20]. In another study, competition experiments
showed that virus harboring mutations T215C or T215D
were outgrown by variants carrying the substitution T215S
[80]. However, the replicative advantage conferred by mutation
T215S was lost in the presence of zidovudine-resistance
mutations such as M41L or L210W [80]. These results were
consistent with the observed switching to T215C and T215D
found in untreated individuals that were infected with
T215F/Y-containing variants [79]. These findings are
important with regard to transmission of zidovudine-resistant
strains. Zidovudine-susceptible strains containing Cys, or
Asp at position 215 are as fit as the wild-type virus in the
absence of drug, but retain the potential for rapid emergence
of high-level zidovudine resistance, since just a single
nucleotide substitution at codon 215 is sufficient to render a
resistant isolate (for further discussion, see [81]).
Fig. (3). Evolution of zidovudine resistance. Accumulation of resistance mutations during the antiretroviral treatment implies
increasing phenotypic drug resistance (the fold-increase of the IC50 for the inhibitor is shown between parenthesis). Highly-resistant
viruses contain 4 or more drug-resistance mutations.
364 Current Drug Targets – Infectious Disorders 2003, Vol. 3, No. 4
A number of mutations related to resistance to nucleoside
analogues will decrease viral fitness. The most extensively
studied are M184V and M184I that confer high-level
resistance to lamivudine. Mutant viruses carrying any
of those mutations showed impaired replication in
phytohemagglutinin-stimulated PBMCs [82-84]. This effect
was also observed with virus having different sequence
contexts, as for example, in the presence of mutations
V75I/F77L/F116Y/ Q151M [85].
The low replication efficiency of M184V- and M184Icontaining isolates is attributed to their diminishing effect
on RT processivity [82], which was accentuated in PBMCs
due to the low dNTP intracellular pools found in those cells
[83]. When introduced in a wild-type HIV-1 RT, the
mutation M184I had a larger diminishing effect on
processivity than M184V, in agreement with the results of
growth competition experiments carried out with mutant
viruses [86, 87]. Furthermore, estimates obtained from
population dynamics in vivo revealed that in the presence of
lamivudine, virus having the mutation M184V showed only
10 % of the fitness value calculated for wild-type virus in the
absence of drug, but only in individuals with high CD4+
cell counts [29, 54, 57, 88]. This fitness reduction could be
smaller (less than 50%) depending on the viral genetic
background [89]. M184V was around 23 % more fit than
M184I during treatment [29]. In contrast to thymidine
analogue-related mutations, M184V reverts to wild-type
when lamivudine is eliminated from second or third line
antiretroviral regimens [56], although reversion may be
accelerated by zidovudine pressure [90]. Otherwise, the low
fitness conferred by M184V impairs the potential for
compensatory mutations and reversion in HIV-1 [91, 92].
Other nucleoside analogue resistance mutations (i.e. the
didanosine-resistance mutation L74V, the tenofovirresistance mutation K65R, or the adefovir-resistance
Menéndez-Arias et al.
mutation K70E) also have a significant impact on viral
fitness, which correlates with an RT processivity defect [15,
93 – 95]. The presence of K65R together with M184V or
L74V has a strong deleterious effect on viral fitness, due to
the low processivity of the double mutant K65R/M184V
[94], or to a poor ability of K65R/L74V to use natural
nucleotides relative to wild-type RT [96]. These observations
are consistent with the low prevalence of the K65R mutation
among isolates from antiretroviral-drug experience patients,
and give rational support to the benefit in combining
mutations that impair virus replication.
DEVELOPMENT OF RESISTANCE TO MULTIPLE
NUCLEOSIDE ANALOGUES
Resistance to multiple nucleoside inhibitors of RT has
been associated with an amino acid substitution at the
nucleoside binding site of the enzyme (i.e. Q151M) and with
insertions or deletions around positions 67-70 of the RT.
The acquisition of resistance through the Q151M pathway
was first observed in virus isolated from patients receiving
zidovudine and didanosine [97]. In this situation, the first
amino acid change that appears in the population is Q151M
[Fig. (4)]. Viral clones harboring this amino acid
substitution display moderate resistance to zidovudine and
zalcitabine, and low resistance to other nucleoside analogues
[97, 98]. However, further acquisition of additional
mutations, such as A62V, V75I, F77L, or F116Y lead to the
appearance of highly-resistant virus.
Fitness assays involving the determination of replication
kinetics or growth competition experiments show that the
mutations at codons 62, 75, 77 and 116 improve the
replication capacity of the resistant virus [11, 16]. Emergence
of Q151M requires two nucleotide changes (from CAG to
AUG). Therefore, the first step in the evolutionary pathway
towards multidrug resistance would be either the substitution
Fig. (4). Evolution towards high-level resistance to multiple nucleoside inhibitors through the Q151M pathway. The order of
accumulation of mutations is shown on the left, indicating between parenthesis the fold-increase of the IC50 for zidovudine
(AZT), didanosine (DDI) and zalcitabine (DDC) [11]. The structural location of the amino acids involved in this resistance
pathway relative to the dNTP binding site of HIV-1 RT is shown on the right.
Viral Fitness and Antiretroviral Drug Resistance
Current Drug Targets – Infectious Disorder 2003, Vol. 3, No. 4 365
Q151K (CAG to AAG) or Q151L (CAG to CUG). Viral
clones having any of these mutations were lethal for viral
replication [16]. However, in the presence of compensatory
mutations (i.e. S68G, or M230I), mutant viruses having the
Q151L mutation in the RT retained significant replicative
capacity [99, 100]. Both S68G or M230I could facilitate the
emergence of multidrug-resistance through the Q151M
pathway.
in replication kinetics experiments [107]. In the presence of
enfuvirtide, the fitness ranking determined using growth
competition experiments was SIM > DIM > DTV > wildtype [108]. These effects can be modulated by modifications
in gp120, since mutations in its V3 loop can impact
coreceptor binding constants and interactions between gp120
and gp41; exerting a significant influence on the viral
sensitivity to enfuvirtide [109].
Another group of multidrug-resistant viruses are those
having an insertion between codons 69 and 70 of its RT.
Viruses with a dipeptide insertion (usually Ser-Ser, Ser-Gly
or Ser-Ala) and additional mutations such as M41L, A62V,
K70R, M184V, T215Y and others, display high-level
resistance to zidovudine and other nucleoside analogues
[101]. Dual infection/competition experiments revealed that
in the presence of low concentrations of zidovudine, removal
of the two serine residues in the multidrug resistant isolate
does not cause a detrimental effect on the replication capacity
of the virus [102]. Furthermore, in the absence of drug, RT
insertions improve the fitness of viruses carrying a number
of accompanying mutations associated with resistance to
multiple nucleoside analogues (i.e. M41L, L210W, T215Y,
etc.). This observation is consistent with the strong
association found between the dipeptide insertion and T215F/
Y, which appear together in >95% of isolates having a
dipeptide insertion in the RT [101]. Viral isolates harboring
the dipeptide insertion were replaced by wild-type variants
following cessation of therapy, indicating a competitive
advantage of the wild-type over the insertion mutant in the
absence of selective drug pressure [103, 104]. However, these
multidrug-resistant mutants were able to maintain high viral
loads in the presence of antiretroviral therapy.
HIV-1 requires chemokine receptors such as CCR5 or
CXCR4 as entry cofactors in combination with CD4 to
infect lymphocytes, macrophages and other cell types. HIV-1
strains with a syncytium-inducing phenotype that use
CXCR4 as cofactor (X4 strains) have been associated with
faster disease progression and AIDS. Sequence variation in
env has a measurable impact on fitness. This is demonstrated
by the good correlation found between fitness determinations
obtained with wild-type HIV-1 isolates and those obtained
with env recombinant viruses [110]. Resistance to entry
inhibitors targeting coreceptors CCR5 or CXCR4 can be
achieved through switching coreceptor use. Alternatively,
alterations in the gp120 envelope glycoprotein can also
confer resistance to the inhibitor. Both strategies are
manifested with the CXCR4 antagonist, AMD3100. In all
cases, fitness of the resistant viruses was reduced in
comparison with the wild-type strain [111]. AMD3100resistant variants accumulate a relatively high number of
mutations plus a 5-amino acid deletion, but its potential for
fitness optimization has not been explored so far.
The deletion of codon 67 has also been associated with
multidrug-resistance, conferring up to 1,810-fold resistance
to zidovudine in the presence of mutations T69G, K70R,
L74I, K103N, T215F and K219Q. The emergence of the
T69G mutation confers drug resistance at the expense of
fitness [66]. The subsequent appearance of the deletion led to
a virus with improved replication and high-level resistance to
zidovudine [66, 105]. The deleterious effect on fitness
produced by the substitution of Thr-69 is also documented
in vivo. In the absence of antiretroviral therapy, mutations
T69D and T69N produce a moderate loss of fitness (approx.
7.8 %) [54].
THE ACQUISITION OF RESISTANCE TO NOVEL
ANTIRETROVIRAL
DRUGS:
PRELIMINARY
FITNESS ASSESSMENTS
The development of antiretroviral drugs targeting entry,
integration or any other step in the virus life cycle
constitutes an important challenge in HIV research. This
year, enfuvirtide, a synthetic peptide which blocks viral
infection by impairing virus-host cell membrane fusion, has
obtained approval for clinical use. Resistance to enfuvirtide
is mediated by amino acid substitutions at codons 36-38 of
the envelope glycoprotein gp41. The sequences found in
drug-sensitive viruses (DIV, SIV, GIV or GIM) are replaced
by SIM, DIM or DTV in the drug-resistant clones [106]. As
observed with RT and PR inhibitors, resistance mutations
cause a fitness loss, which was estimated to be around 10 %
Integrase inhibitors are also promising targets in
antiretroviral therapy. Resistance to various diketoacids has
been studied. Viruses displaying up to 70-fold resistance to
derivatives of L-708,906 and L-731,988, and containing
mutations S153Y and N155S in the integrase active site
displayed good replication capacity [112]. In contrast,
resistance to the novel compound S-1360 (which is acquired
through mutations T66I, L74M and S230R in the integrasecoding region) is attained with a concomitant loss of fitness
[113]. In any case, these assessments are all preliminary and
further studies will be necessary to evaluate their impact on
HIV fitness in vivo.
VIRAL FITNESS OF ANTIRETROVIRAL DRUGRESISTANT MUTANTS: CLINICAL ASPECTS
The selection of drug resistant HIV variants during
antiretroviral therapy is the result of complex interactions
involving both the influence of acquired mutations on drug
susceptibility and their effect on viral fitness. From a clinical
standpoint, assessing the HIV-1 replication capacity would
be extremely useful if it could be used as an in vitro correlate
of viral pathogenesis. However, there are currently many
problems that hamper our understanding on how fitness and
replication capacity of drug-resistant HIV-1 would influence
disease progression and transmission. First, the influence of
drug-resistant genotypes on viral fitness is not clear in many
cases. For example, the pre-existence of secondary mutations
in the PR-coding region is likely to have implications for
HIV-1 fitness evolution [114, 115]. However, prediction of
viral fitness based on PR or RT genotypes is not reliable.
Second, as discussed earlier in this review, there are many
different methods to estimate viral fitness ex vivo.
366 Current Drug Targets – Infectious Disorders 2003, Vol. 3, No. 4
Specifically, we should emphasize the observed differences
between data obtained using recombinant viruses in contrast
to clinical viral isolates. Fitness data originating from
studies with resistant variants of laboratory HIV-1 strains
may not be relevant to clinical resistance selection due to the
influence of their specific genetic background.
Although some reports have shown coincidence of results
comparing different methodologies [13, 116], the association
between viral pathogenesis and viral fitness is usually a hard
task due to technical variations in the assays. Third, in vivo
fitness of HIV-1 depends on the interaction with host factors
and genetic background [117, 118], immune control [119 –
121], and target cell availability [29]. These host factors are
not taken into account in ex vivo replication capacity
experiments. Finally, antiretroviral drug concentrations are
also important determinants of viral fitness [13, 21].
Impact of Viral Fitness on Disease Progression
So far, the implications of viral fitness for the clinical
management of HIV-1-infected patients have only been
partially addressed. In a study designed to examine the
impact of HIV-1 fitness on disease progression, the ex vivo
replication capacity was measured in 12 samples from
patients that were classified either as long-term nonprogressors or as progressors. The authors found an
association between plasma HIV-1 RNA and replication
capacity of viral isolates measured by dual competition/
heteroduplex tracking assays, using a series of HIV-1 strains
as reference virus. The results showed that progressors had
higher plasma viremia and viral isolates collected from these
individuals showed higher replication capacities than those
from long-term non-progressors [122].
Studies involving structured treatment interruptions show
that in patients failing antiretroviral therapy, and infected
with viruses having multiple drug resistance mutations,
discontinuation of treatment produced significant increases of
viral load as well as concomitant reductions in the number of
CD4 cell counts. These changes were temporally associated
with reemergence of a wild-type virus population displaying
higher replication capacity [123]. These data suggest that
reduced viral fitness is an important factor contributing to
persistent partial suppression of viral replication during longterm virological failure.
Conversely, in those patients that remain on long-term
antiretroviral therapy despite incomplete suppression, plasma
HIV RNA levels often remained stable or increased slowly,
while phenotypic resistance increased and virus replicative
capacity decreased slowly [124]. The emergence of primary
genotypic mutations in the PR was temporally associated
with increased phenotypic resistance and decreased replicative
capacity, while the emergence of secondary mutations within
the PR-coding region was associated with more gradual
changes in both phenotypic resistance and replication
capacity [124]. These data suggest that HIV-1 may be
constrained in its ability to become both highly resistant and
fit and that this may contribute to the continued partial
suppression of plasma HIV-1 RNA levels that is observed in
some patients infected with drug-resistant viruses.
Menéndez-Arias et al.
The relationship between impaired viral fitness, as
measured by a replication capacity assay, and viral load
reduction in response to a new therapeutic regimen is
unknown. The impact of the replication capacity on viral
load reduction may be difficult to assess in patients that
display an undetectable viral load. Obviously, in the absence
or with very low levels of viral replication, an impaired
replication is difficult to detect. Therefore, evaluation of the
correlation between replication capacity and viral load
reduction is likely to be more informative in patients who
receive a new regimen but show continued viral replication.
In a recent study, replication capacity was measured with a
single cycle recombinant virus assay in a study performed in
97 patients failing antiretroviral therapy. A replication
capacity value lower than 35%, measured at the time of
initiation of a new salvage regimen, was a significant
predictor of reduced plasma viremia 6 months later for
patients that did not achieve undetectable HIV-1 RNA levels
[125]. Similarly, D30N, a nelfinavir resistance-associated
mutation that induces a severe reduction in viral replication
capacity [12] was a predictor of short term virologic response
to salvage therapy [126, 127]
The influence of viral fitness in patients with discordant
responses to antiretroviral treatment has also been addressed
in various studies. In a significant number of cases, anti-HIV
therapy maintains persistent immunological benefits despite
virological failure. In this regard, the viral replication
capacity could be a marker of continued immunological
benefit of the antiretroviral regimen when measured at the
time of virological failure of an ongoing regimen [Fig. (5)].
Patients with lower viral replication capacity have
significantly greater CD4 increases from nadir than those
with less impaired viruses. This effect is independent of
other common clinical markers including reduced phenotypic
susceptibility [125].
Generally, greater reductions in relative replication
capacity have been observed in viruses harboring PR
inhibitor resistance mutations than in viruses containing RT
inhibitor resistance mutations. This observation, and the fact
that PR inhibitor-selected mutant viruses could replicate in
PBMCs or lymphoid tissues but not in human thymus [26,
128, 129], prompted the hypothesis that PR inhibitorcontaining regimens could have a clinical benefit because
PR-resistant viruses had reduced replication capacity.
However, a recent pilot study designed to test such
hypothesis, is producing controversial results. In this study
involving patients infected with multidrug-resistant HIV,
administration of either PR or RT inhibitors was
discontinued. Interrupting RT inhibitor therapy was
associated with increased viral load and decreased CD4 cell
counts. In contrast, interrupting PR inhibitor therapy was
associated with stable viremia, immunological benefit,
prevented overgrowth of wild-type viruses and delayed viral
evolution [130]. Although several mechanisms were
proposed to explain this phenomenon, two seem more
relevant. First, archived RT inhibitor-resistant and PR
inhibitor-susceptible virus likely contained fewer RT
inhibitor-associated mutations than more recent variants,
making the archived virus relatively more susceptible to RT
inhibitors. Second, viral evolution in presence of PR
Viral Fitness and Antiretroviral Drug Resistance
Current Drug Targets – Infectious Disorder 2003, Vol. 3, No. 4 367
Fig. (5). Schematic diagrams of the relationship between viral fitness and clinical parameters in HIV-infected individuals. (A)
Infection with a wild-type HIV-1 strain, in the absence of antiretroviral therapy is followed by a gradual increase in viral load (plasma
HIV-1 RNA) and subsequent decrease in CD4+ T-cell count. Viral fitness increases as the initial virus inoculum adapts to the new
environment. (B) Antiretroviral treatment causes a decrease in viral load since the wild-type virus is not fit to replicate in the presence
of drug. Therefore, both viral load and viral fitness decrease while CD4+ T-cell counts recover. Selection of drug-resistant HIV-1
variants would eventually lead to an increase in viral fitness and viral load, similar to that shown in panel A. (C) However, in some
instances, in the presence of multidrug-resistant viruses with impaired viral fitness, maintaining antiretroviral therapy in patients
with persistently detectable viral load may still have some immunological benefits for the patient.
inhibitor therapy is associated with the accumulation of
compensatory mutations that increase viral fitness. Back
mutations may require significant structural alterations of the
targeted proteins and may produce an early decrease in
relative fitness; thereby preventing reversion [130]. In
conclusion, available data suggest that a decreased replication
capacity associated with PR inhibitor resistance will persist
even in the absence of inhibitor.
Impact of Viral Fitness on HIV-1 Transmission
There is increasing evidence of transmission of resistant
HIV strains in North America and Europe due to the
difficulty of completely suppressing viral replication [131 –
135]. It is clear that replication capacity is an important
determinant, not only of HIV-1 pathogenicity, but also of
transmissibility. Therefore, it has been suggested that
antiretroviral drug-resistant viruses with relatively good
replication capacity rates would be more easily transmitted.
However, it is estimated that the frequency of transmission
of resistant strains is only about 20% of the expected value,
probably due to the potentially reduced fitness of the drugresistant viruses [136]. Although HIV-1 transmission
represents a selective evolutionary bottleneck, detection of
drug-resistant variants in patients with acute primary
infection implies that these viruses possess replication
characteristics that allow them to become the dominant viral
population in a drug-free environment [137]. The long-term
virological and clinical implications of such phenomenon are
of concern because the transmission of drug resistance to
newly infected persons can result in treatment failure and
clinical progression.
There are many reports showing that antiretroviral
resistant viruses have diminished replication capacity and
should be outgrown by drug-sensitive wild-type viruses.
Different studies that explored structured antiretroviral
treatment interruptions in chronic infection have shown rapid
reappearance of drug-sensitive viruses when therapy is
withdrawn [123, 138, 139]. These observations led authors
to propose that drug-resistant viruses acquired during
primary infection would not persist over time in the absence
of antiretroviral pressure. Thus, preliminary studies have
evaluated the differential transmission of HIV-1 strains
harboring mutations in RT in newly infected persons
compared with prevalence in the potential transmitter
population. Interestingly, the prevalence of the lamivudineresistance selected mutation M184V was only 3%, much
lower than thymidine analogue associated mutations (6.7%)
or non-nucleoside analogue associated mutations (7.4%)
suggesting that reductions in viral fitness of M184Vcontaining HIV-1 strains may significantly impact on rates
of viral transmission [140].
Estimates of HIV-1 replication capacity in newly infected
individuals exhibited a broad spectrum, ranging from 1 to
113% of wild type control, when measured with the
ViroLogic single-cycle recombinant virus assay. Although,
low replication capacity was associated most strongly with
PR inhibitor resistance, the wide variation is probably
conferred by genetic interactions, and mutations not
associated with drug resistance. Thus,
impaired
transmissibility of PR inhibitor resistant viruses could
explain the relative scarcity of PR inhibitor resistance among
recently infected persons [141]. In addition, CD4 T-cell
368 Current Drug Targets – Infectious Disorders 2003, Vol. 3, No. 4
counts were higher in those subjects infected with drugresistant viruses with lower replication capacity, especially if
PR inhibitor resistance was evident [142]. Nevertheless, and
despite their impaired replicative competence, transmitted
multidrug-resistant viruses can establish persistent (up to 5
years) infections [88]. The durable persistence of transmitted
drug-resistant virus is consistent with the establishment of
widespread infection with a pure population of resistant
clone(s), in contrast to the rapid reversion observed in vivo in
chronically infected subjects who discontinue therapy after
virological failure [143]. Reversion of resistance is gradual
and usually incomplete, resulting in the persistence of
mixtures of wild-type and resistant variants in plasma HIV
RNA. This persistence of certain transmitted drug-resistant
mutations (i.e. K103N) among subjects with primary HIV
infection deferring antiretroviral therapy is consistent with
the modest fitness effect of these amino acid changes as
determined in assays carried out in vitro [143].
Recent data indicate that recombination of the PR and
RT regions of drug-resistant viral isolates into an isogenic
HIV-1NL4-3-based molecular backbone resulted in chimeras
that displayed a significant reduction in replication capacity
[137, 144]. Similarly, a recent study that used complete viral
isolates has suggested that the total number of PR
mutations, together with individual polymorphisms, could
not be associated with viral fitness [114]. This could be
explained assuming that drug-resistant HIV-1 isolates could
bear adaptive mutations that allow replication recovery, and
thereby overcome the potential defects associated with
genetic changes necessary for drug resistance [45, 63, 137,
145, 146]. The consequence is that the viral fitness obtained
from assays that use recombinant viruses might be
systematically overestimated with respect to that obtained
from complete viral isolates.
Previously we have discussed that increased replicative
capacity of an infecting HIV-1 isolate is associated with
more rapid disease progression [122]. Paradoxically, because
the probability of HIV-1 transmission may be related to
survival time, the more fit viruses infecting rapid progressors
may actually be spread less efficiently in the human
population [147].
Menéndez-Arias et al.
replication competence of drug-resistant mutants by affecting
their ability to accommodate resistance mutations. Moreover,
replication capacity assays are strictly virological methods
that do not consider the influence of important host variables
as the genetic background, the immune control or the target
cell availability.
ACKNOWLEDGEMENTS
Work in the laboratory of L.M.-A. is supported by grants
from Fondo de Investigación Sanitaria (FIS) 01/0067-01,
FIPSE (36200/01 and 36207/01) and an institutional grant
of Fundación Ramón Areces. Work in the laboratory of
M.A.M. is supported by Fundació irsiCaixa and grants from
FIS (01/0067-02), FIPSE (3014/99, 36293/02 and 36207/
01) and Fundació Marató de TV3. Research performed at the
Cleveland Clinic Foundation (M.E.Q-M) is supported by
research grants from the National Heart, Lung, and Blood
Institute, NIH (5-KO1-HL67610-03), and the Center for
AIDS Research (AI36219) at Case Western Reserve
University. Work in the laboratory of J.M.-P. is supported
by grants from FIS (01/1122), FIPSE (36177/01) and
Fundació Marató de TV3. J.M.-P is supported by contract
FIS 99/3132 from the “Fundacio Institut d’Investigacio en
Ciencies de la Salut Germans Trias i Pujol” in collaboration
with the Spanish Health Department. Support from FIS
through the “Red Temática Cooperativa de Investigación en
SIDA (Red G03/173)” is also acknowledged.
ABBREVIATIONS
CA
=
Capsid protein
HIV
=
Human immunodeficiency virus
MA
=
Matrix protein
NC
=
Nucleocapsid protein
PBMC =
Peripheral blood mononuclear cells
PR
=
Protease
RT
=
Reverse transcriptase
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