HIV-1 Fitness: Implications for Drug Resistance, Disease Progression, and Global Epidemic Evolution
Miguel E. Quiñones-Mateu1 and Eric J. Arts2
1 Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA
2 Department of Medicine, Division of Infectious Diseases, Case Western Reserve University, Cleveland, OH, USA
1 To whom correspondence should be addressed. Tel. (216) 444-2515, Fax (216) 444-2998, E-mail: quinonm@ccf.org
In the past five years, the HIV research field has shown renewed interest in the replicative capacity of human immunodeficiency virus type 1 (HIV-1) due to the potential impact of ex vivo viral fitness on population size (viral load), drug resistance, and disease progression. For example, studies on HIV-1 fitness of drug-resistant mutants under different selective pressures have led to a better understanding of emergence of specific drug resistance mutations during therapy, as well as risk/benefit of these mutations for HIV-infected individuals. In addition, other studies have associated HIV-1 fitness with the rate of transmission and disease progression. HIV-1 continually evolves and migrates through individual hosts, overcoming barriers to transmission, avoiding different immune responses, and resisting various antiretroviral regimens. The "fittest" HIV-1 isolate that survives in growth competition cultures may also lead to increased virulence within a host. However, rapid disease progression may be detrimental to fitness of the same HIV-1 isolate in the human population. Thus, the term "fitness" itself is an enigma requiring a multilayered definition. Rather than attempt to summarize all the studies on HIV-1 replication, this review concentrates on (i) the clinical implications of viral fitness within HIV-infected individuals in the presence or absence of antiretroviral therapy, (ii) changes in viral fitness upon emergence of drug resistance, and (iii) the role of HIV fitness in the AIDS epidemic, including virus transmission and viral/host evolution.
Concepts in viral fitness
Genetic variation is inherent to all RNA viruses but has been best characterized for HIV-1 [94;199]. The extensive heterogeneity observed in the worldwide epidemic originates from the rapid viral turnover (1010 viral particles/day) in an HIV-infected individual [102;226], high rate of incorrect nucleotide substitutions during HIV reverse transcription (10-4/nt) in the absence of proof-reading mechanisms [182], and the pliant conformations/functions of many HIV-1 proteins. In addition to this rapid accumulation of minor genotypic changes, different HIV-1 strains can also recombine at a high rate, generating large genetic alterations [25;106;185;214;221;222]. HIV-1, like other RNA viruses, has a high mutation rate (3 x 10-5 mutations per base pair per cycle [146]), which coupled with selection and rapid turnover [225] results in the generation of swarms of mutants known as viral quasispecies [59;60;71]. Continuous production of viral mutants results in adaptation to most environmental changes [57;60]. It is important to highlight that the quasispecies, and not individual virus genomes, are the subject of selection and evolution [57;60;71]. Thus, HIV-1 quasispecies are evolutionary and clinically relevant since this genetic variability can in a sense respond to selective pressure (e.g., host immune system and antiretroviral therapy).
Fitness is a parameter defining the replicative adaptation of an organism to its environment (reviewed in [57;60]). Survival of the fittest is the concept that drives evolution in a complex population. Within a given viral quasispecies each clone has a fitness, representative of those viral properties (e.g. activity and stability) undergoing selection in that particular environment. During viral replication within a defined microenvironment, different genomes encode virus that replicate at high rates, continually mutate but generally remain under the same selective pressures [57]. Thus, positive (Darwinian) selection implies that one or more members of the quasispecies are better suited to a given environment while negative selection eliminates unfit variants [57;58;60]. In summary, the dynamics of the viral population is a continuous process of growth, competition, and selection that takes place in the sequence space (i.e., all possible sequence permutations for an informational macromolecule). In the case of HIV-1, with a genome size of 10 Kb, the total possible sequence space is 410,000, although only a very small fraction corresponds to functional viruses [57]. The combination of sequence space and fitness (or replicative capacity) constitutes the "fitness landscape" [60;233]. This classical concept, first described seventy years ago [233], suggests that changes in viral fitness can be viewed as a movement of viral genomes in an irregular and adaptive landscape of peaks and valleys. As a result, RNA viruses can find multiple pathways to reach alternative high fitness peaks on the fitness landscape [72].
Multiple studies based on vesicular stomatitis virus (VSV), foot-and-mouth disease virus (FMDV), or HIV-1 quasispecies have assessed different fitness theories related to population size and quasispecies complexity. The Red Queen hypothesis states that viral quasispecies in competition tend to gain fitness with each viral passage [33;58;166]. However, the evolutionary interpretation of the statement implies that population size must continually increase to maintain or increase fitness gains. In contrast, the competitive exclusion principle asserts that in the absence of niche differentiation, one competing species will always eliminate or exclude the other [33;58]. Most experiments designed to test either fitness theory require the continual passage of an increasing virus population in cell culture. Failure to passage a significant proportion of the virus population and/or changes in the tissue culture environment results in bottlenecks and a decreased rate in fitness gain. Repeated bottlenecks will actually result in a net fitness loss [31;58;60;64;72;238]. The Muller's ratchet hypothesis suggests that limiting the population size will result in an accumulation of deleterious mutations which overwhelm the appearance of mutations improving fitness [31;58;64;72]. Extreme examples of Muller's ratchet have been well documented with plaque-to-plaque passages of VSV, FMDV, and bacteriophage f6 [58;64;72]. With the exception of few studies [237;238], HIV-1 has not been utilized as a tool to test fitness theories. This may be due in part to the slower replication rates and production yield of lentiviruses (e.g., HIV-1) as compared to many single-strand RNA viruses (e.g., VSV an FMDV).
Methods to determine HIV-1 fitness
Although differences in viral replicative capacities were described early in the HIV-1 epidemic [82], the role of HIV-1 fitness in drug resistance and pathogenesis have only recently been the focus of detailed investigations [34;164]. Hence, several key conceptual and technical shortcomings remain unresolved. For example, what is the proper assay to measure fitness of HIV-1 isolates or clones? Multiple methods have been employed to measure HIV-1 replication capacity in vitro [34;164] (Table 1). Although viral fitness is best defined by replicative capacity during growth competition experiments [103], many studies have extended the definition of relative fitness by comparing (i) the catalytic activity of HIV-1 enzymes [8;165], (ii) virus turnover on HIV-1 infected individuals [56;90;91], (iii) HIV-1 production in monoinfected cultures [40;62;151;204], (iv) the virion (infectious):virus particle ratios [19], and (v) an HIV-induced event in a single-cycle infection assay [52;143;239]. Use of these assays is generally justified or necessary to test a specific hypothesis. However, there is also little continuity between these studies creating specific ambiguities in the HIV-1 fitness field. Although each study was designed to address specific questions, the use of different assays and viral constructs to define the "fitness" of drug resistant HIV-1 can be very misleading for comparisons.
Table 1. Methods used to estimate HIV-1 fitness
|
Assays |
Methods |
Detection techniques |
References |
|
In vivo |
Viral kinetics in plasma |
Sequencing |
[45;56;90;91;142] |
|
Differential hybridization |
[68;69;97] |
||
|
Primer-guided nucleotide incorporation assay |
[85] |
||
|
Ex vivo |
Protease catalytic activity |
Pr efficiency (Kcat / Km), |
[8;40;62;165;220] |
|
Polyprotein processing/maturation |
[19;24;40;144;148;174;180;180;191;201;239] |
||
|
Genetic complementation |
[135;153] |
||
|
RT catalytic activity |
RT polymerase, Rnase H activity |
[2;8;26;79;88;120;205] |
|
|
Viral growth kinetics |
p24 Antigen / RT activity |
[2;8;16;19;20;26;40;43;62;67;87;88;101;109;110;113;116;122;139;143;144;148;151;177;180;195;197;204;205;210;212;239] |
|
|
Single-cycle infection |
b-galactosidase activity |
[43;143-145;239] |
|
|
GHOST/CCR5-CXCR4 permissibility |
[19] |
||
|
Luciferase activity |
[52;92;149;180;234] |
||
|
Growth competition |
Differential plaque assay |
[155;191] |
|
|
Cloning/sequencing |
[2;40;45;87;96;109;113;122;128;150;152;165;180;204;205;212;236] |
||
|
Heteroduplex mobility assay |
[162;188;192;238] |
||
|
Real-time NASBA |
[45] |
||
|
Recombinant marker virus assay |
[139] |
||
|
Animal model |
SCID mice |
SCID-hu Thy/Liv |
[210] |
|
SCID-hu PBL |
[177] |
In vivo assays
Competitive ability of a virus is the result of many biological processes in its life cycle (i.e., cell entry, genome replication, protein synthesis/processing, particle assembly and release from cells). A human host offers a variety of cell types and microenvironments to the infecting HIV-1 resulting in various selective pressures. To date, most in vivo fitness studies have been performed on blood samples [68;90;91]. Although in vivo assays provide the best estimate of fitness, specific quasispecies (e.g., drug-resistant variants) may be dominant in the blood but less fit in other compartments (e.g., lung or CNS). Host-to-host comparisons of in vivo HIV-1 fitness is very difficult to evaluate due to differences in host genetics and immune response. Therefore, in vivo fitness studies are limited to the emergence of specific quasispecies or drug resistant mutants, and cannot determine the impact of specific substitutions on replicative capacity. In contrast, ex vivo fitness assays of HIV-1 primary isolates remove "variable" host constraints and focus on replication efficiency. Thus, in vitro fitness assays are still indispensable.
In vitro assays
In vitro studies using HIV-1 isolates or recombinant viruses cannot mimic the natural setting within human hosts. However, they are useful models for viral replication in a fixed environment and for the behavior of drug-resistant variants after the initiation of therapy. In the absence of a consensus method for quantifying viral replication capacity, many studies have used different techniques to assess ex vivo HIV-1 fitness. In general, methods to determine HIV-1 fitness in vitro could be grouped in two general techniques: viral growth kinetic assays and growth competition experiments (Fig. 1).
a) Viral growth kinetic assays
Biochemical properties of the protease (PR) and reverse transcriptase (RT) enzymes, as well as the replication kinetics of HIV-1 with particular mutations, have been the subject of extensive investigations. Replicative capacity (viral fitness) of HIV-1 isolates or recombinant infectious clones is examined in monoinfections by determining the amount of virus production over time (e.g., measuring p24 antigen, RT-activity, (b-galactosidase or luciferase activity in reporter cell lines, etc) [20;52;62;144;204] (Table 1). Differences in the replication kinetics of HIV-1 mutants can be compared in parallel infections (Fig. 1A). However, these assays can only discern gross changes in replicative capacity and cannot accurately define the impact of subtle genetic changes on the replication rates of HIV-1 isolates. In general, direct competition between two different viruses is a more accurate and sensitive assay to detect minute fitness differences [103;180] (Fig. 1B).
A novel recombinant virus technique, based on the PhenoSense assay (Virologic) used in drug susceptibility assays [176], has been recently adapted to measure HIV-1 replicative capacity [52;234] (Fig. 1C). Replicative capacity of the recombinant virus (generally PR-RT pseudotyped) is examined in a cell line transfected with a luciferase reporter gene replacing env to monitor a single round of virus replication. A single-cycle assay provides the best estimate of replication efficiency during a mono-infection. However, this assay is also limited by the use of recombinant viruses and assessment of only specific steps in the replication cycle (i.e., only steps leading to viral transcription). This single-cycle infection assay cannot be used effectively in growth competition experiments.
It is important to note that a few studies have analyzed HIV-1 replication of drug-resistant variants using animal models, specifically SCID-hu mice [177;210]. This HIV-1 infection model has been successfully used to assess viral susceptibility to antiretroviral drugs. A recent study has compared the fitness and pathogenesis of protease inhibitor (PI)-resistant HIV-1 strains in SCID-hu mice (see below).
Figure 1. Methods used to determine in vitro HIV-1 fitness. (A) Viral growth kinetics assays involves measuring virus production at various time points using diverse detection methods (e.g., p24 antigen or RT-activity). (B) Growth competition experiments require dual infection with two different HIV-1 isolates (initial proportion of each may vary). Production and quantification of each specific HIV-1 isolate in the mixture can be determined through the use of different methods (Table 1). (C) Single-cycle infection assays. The number of cells infected after a single cycle of replication can be measured using various indicator systems (Table 1) (Figure adapted from [34]).
b) Growth competition experiments
Head on competitions in cell culture between two viral isolates of
the same species provide the internal control lacking in
monoinfections. However, an extreme limitation to growth competition
experiments is developing an assay to accurate detect and quantify
minute genotypic or phenotypic
differences between these viruses. Relative fitness can be directly
compared in dual infections since virus populations in culture
compete until one clone or quasispecies outgrows the other
[60;103]. Following exposure of cells to a viral mixture, the
proportion of viruses after several viral passages is compared with
that in the initial mixture [58;103]. Despite being a
laborious assay, growth competitions provide a more accurate
measurement of relative viral fitness, and are generally reproducible
in the same cell culture environment.
Although the concepts of viral competitions may be similar, different approaches have been developed to measure dual virus production. Most methods rely on laborious point mutation assays or on the sequencing of a large number of clones [40;96;109;150;151;204], whereas new studies utilize more rapid techniques to estimate the frequency of the two viruses in the competition [45;139;162;188;238] (Table 1). Martinez et al. [153] developed a bacteriophage lambda-based genetic screen to characterize the activity and phenotype of HIV proteases. In another study, a "fitness profile assay" was used to determine the relative fitness of drug-resistant variants and wild-type HIV-1 as a function of protease inhibitor concentration (by calculating the ratio of mutant:wt production with each drug concentration, in an assay similar to an IC90 determination) [145]. Heteroduplex tracking assay (HTA) has recently been used (i) to evaluate the production of HIV-1 variants in a competition [188;238] (ii) to detect specific V3-coding regions in env that are associated to NSI/R5 or SI/X4 phenotypes and (iii) to detect drug-resistant vs. wild-type codons in the PR-RT-coding region [162;192]. HTA can accurate detect drug-resistant variants comprising only 3% of the HIV-1 population, and can also be used to estimate in vivo viral fitness.
New technologies such as Real Time PCR have also been applied to dual virus detection. de Ronde [45] was the first to develop a real-time NASBA PCR and molecular beacons technique to quantify a specific AZT RT mutant within the quasispecies of treated patients (specifically, changes of codon 215 of the RT). Lu & Kuritzkes [139] developed a novel recombinant marker virus assay (RMVA) to perform growth competition assays and estimate RT fitness. The nef gene in an RT-deleted HIV-1 clone was replaced by the Salmonella typhimurium histidinol dehydrogenase (hisD) or the human heat-stable placental alkaline phosphatase (PLAP) genes. Relative production of any two RT-pseudotyped HIV-1 isolates was measured by the corresponding marker (hisD or PLAP) using real-time PCR.
Finally, homologous recombination between two HIV-1 variants during competition can present a major obstacle in measuring relative HIV-1 fitness. Recombined HIV-1 isolates did emerge after four passages in a growth competition experiment [128]. This finding suggests that the recombinant was more fit than the two parental infectious clones. A recent study estimated a low frequency of HIV-1 recombination (approximately 3-5% / Kbp in a 5-day infection period) in a high-MOI (0.1 IU/ml) dual HIV-1 infection of PBMC [187]. Reducing the MOI of each isolate provided better estimates of HIV-1 fitness and reduced the frequency of co-infected cells leading to recombination (less than 0.1% for an MOI = 0.01). These results suggest that HIV-1 recombination is minimal in growth competition experiments with low MOI and limited passages.
c) HIV-1 isolates vs. recombinant viruses
To date, the vast majority of in vitro fitness studies are performed with cloned virus pseudotyped with various HIV-1 genes [19;98;119;151;195;206]. The entire gene or even segments of various coding regions (PR, RT, ENV, Gag-Pol protease cleavage sites) are often PCR-amplified directly from patient plasma or cells. One of the main advantages of these recombinant clones is the ease and flexibility of directly comparing fitness and drug sensitivity of various target genes. In HIV-1, protease and reverse transcriptase regions generally harbor the drug resistant mutations. In fact, recombinant viruses, as opposed to HIV-1 primary isolates, can determine the impact of specific sites or regions within a neutral HIV-1 backbone. This pseudotyping technique eliminates the possible fitness effects of polymorphisms or mutations found outside the target sequence in primary HIV-1 isolates [40;109;144;150;151;204;205]. However, comparison between HIV-1 fitness studies are difficult due to the use of a myriad of fitness assays and different HIV-1 clones for pseudotyping. Therefore, the in vivo relevance of fitness studies on pseudotyped virus containing single drug resistance mutations or even the affected gene is still the subject of debate. Other genomic regions external to those containing the drug resistance mutations and not used for pseudotyping could have an equal or greater impact on fitness.
Use of HIV-1 clinical isolates in place of recombinant infectious clones to measure viral fitness will be dependent on the research question. Studies comparing viral fitness with HIV-1 pathogenesis (see below) [16;188] employed primary HIV-1 isolates since the entire genome may affect replicative capacity. Grant et al. [92] analyzed the fitness of PI-resistant and PI-susceptible viruses in vivo (ratio of wild-type:mutant in plasma) and in vitro using the PhenoSense HIV drug susceptibility single-cycle assay (see above). Both methods generated comparable fitness results suggesting that this recombinant/virus single-cycle infection may predict the prevalence of specific HIV-1 drug resistant mutations in the presence or absence of drug selective pressure. Viral fitness studies of amprenavir-resistant viruses also showed a strong correlation between the prevalence of resistant mutations and replication efficiency of PR-RT pseudotyped virus [180]. However, differences in replicative capacity were more apparent using growth competition experiments than in single-cycle mono-infection assays [19;180]. Recently, contributions of PR and/or RT to fitness has been compared in drug resistant HIV-1 primary isolates to fitness of recombinant HIV-1NL4-3 viruses pseudotyped with PR, RT, or PR-RT [19]. All of the HIV-1 clones pseudotyped with the drug-resistant PR and/or RT cassettes were less fit than the wild-type NL4-3 clones. In contrast, two of the three drug-resistant HIV-1 isolates had similar replication rates as the NL4-3 wild-type clone. These results suggest that other genomic regions can compensate the drug resistant substitutions in the PR and RT genes. Continual evolution of HIV-1 regions outside of drug-targeted genes will eventually compensate for defects in fitness. Thus, fitness results on specific viral clone or even primary isolates can only answer defined questions and should not be accepted as absolute in vitro or in vivo fitness.
Fitness of HIV-1 drug-resistant viruses
The ultimate goal of present therapy is to suppress HIV-1 replication for as long as possible. Maintaining low-to-undetectable plasma HIV-RNA levels prevents progression to AIDS and minimizes the possible emergence of drug resistant HIV-1 variants [100]. However, treatments with antiretroviral combinations do not eliminate or even completely suppress HIV-1 replication in all tissue compartments. Each of the sixteen antiretroviral drugs licensed in the United States falls into one of three classes: (i) nucleoside reverse transcriptase inhibitors (NRTI), nonnucleoside reverse transcriptase inhibitors (NNRTI), and protease inhibitors (PI). Aside from the initial pharmacokinetic and dynamic properties of these drugs, duration of treatment success is not measured by drug potency but rather the time required for the dominance of drug resistant isolates in the quasispecies [49;137;157]. In the absence of antiretroviral therapy, strains containing drug-resistance mutations have a reduced fitness compared to the wild-type (wt) quasispecies within the population [37]. Therefore, selective pressure in the form of drug therapy leads to dramatic shifts in the quasispecies distribution and fitness of those mutants with decreased sensitivity to the respective antiretrovirals [37;57;60;61]. During this in vivo selection, several drug-resistant variants will emerge and compete for dominance. These resistant isolates will pass through the drug-induced bottleneck and initiate a new quasispecies distribution that will be governed again by replication efficiency [37;138].
During antiretroviral therapy two types of mutations are associated with drug resistance and respective shifts in fitness (i.e., ex vivo replication efficiency in the absence of drug). An initial decrease in fitness coincides with the appearance of primary substitutions conferring direct drug resistance. Continued drug pressure allows the quasispecies to re-explore the HIV-1 sequence space. Secondary or compensatory mutations are selected to restore the enzymatic activity of the drug-targeted resistant enzyme (PR or RT) leading to a rebound in fitness, similar if not greater than the fitness of the quasispecies prior to treatment [13;34;100;164]. It is important to note that compensatory mutations are not limited to the target/resistant gene unless this resistant enzyme or protein is rendered inactive or severely impaired. In addition, appearance of specific mutations is often highly dependent on the baseline sequence and the sequential selection of "de novo" compensatory mutations that contribute to viral fitness [181;197;224]. These troughs and peaks in the HIV-1 fitness landscape during drug selection were first described in HIV-infected patients treated with lamivudine (3TC) [8]. Subsequently, multiple studies have reported impaired enzyme function and reduced viral fitness of HIV-1 isolates harboring PI and RTI resistant mutations [40;62;96;110;143;144;150;151]. Current treatment guidelines advocate a switch in antiretroviral treatment regimens following the emergence of primary drug resistant mutations and possibly prior to selection of compensatory changes. Several groups have described that an accumulation of drug-resistance mutations would have a debilitating effect on HIV-1 replication efficiency [10;20;62;69;155]. For example, high level resistance to protease inhibitors generally requires a combination of amino acid substitutions within or near the peptide binding/cleavage site [62]. Reduced recognition and cleavage of sites in the HIV-1 precursor proteins was expected but few had anticipated that mutations at the cleavage sites would compensate for changes in enzyme activity.
In the human host, the most fit HIV-1 sequence differs between patients due to variations in host genetics (e.g., co-receptor polymorphisms), immune response, and several viral factors (e.g., replication capacity, mutation rate, and host cell tropism) [34;164]. Several studies have reported that wild-type viruses from treatment-naïve patients have a broad range of replicative capacities (47% to 89%, median 73%) compared to laboratory strains [234]. In addition, pairwise competition experiments with HIV-1 isolates from untreated patients suggest that fitness differences do not necessarily follow a one-dimensional relationship (e.g., since A > B, and B > C, A must be > C), but is much more complex and likely involves competition at multiple points in the replication cycle. Thus, the utility of a molecular HIV-1 clone as a fitness control decreases with the amount of genetic material introduced into that clone for fitness studies. Even the use of primary drug-resistant isolates requires a side-by-side (single-cycle) or head on (competition) comparison with several wild-type HIV-1 strains to approximate ex vivo fitness.
Protease inhibitor (PI)-resistant variants
HIV-1 protease is the enzyme responsible for cleavage of the viral Gag and Gag-Pol polyprotein into mature structural proteins and enzymes found in the infectious virion [157;173]. Six HIV-1 protease inhibitors have been approved to date in the United States, i.e., amprenavir (APV), indinavir (IDV), lopinavir (LPV), nelfinavir (NFV), ritonavir (RTV), and saquinavir (SQV). Numerous primary and/or secondary mutations have now been associated with HIV-1 resistance to these protease inhibitors [157;203]. The protease gene has shown great plasticity, with 49 natural polymorphisms and 20 drug resistance substitutions in the 99 amino acid protein, which is only active as a homodimer [157;203] (Fig. 2). Interestingly, primary drug resistant substitutions rarely dominate the quasispecies in PI-naïve HIV-infected individuals [133], suggesting they confer a selective disadvantage to the virus [129;203]. In fact, multiple mutations appear to be necessary for the development of a replication competent PI-resistant virus [11;228]. For example, viral infectivity was lost when two primary PI-resistance mutations (30N and 90M) were introduced into a HXB2 background, but restored when the entire PR-coding region from a clinical isolate, harboring the same two mutations, was cloned into HXB2 [159]. In patients displaying virological failure to a PI-based regimen, viral evolution leads to a gradual increase in both PI-resistance and replicative capacity (associated with the emergence of secondary mutations) [51;224].
For most of the protease inhibitors, primary PI resistant mutations cluster near the active site of the enzyme (Fig. 2B), reducing both catalytic activity and viral replicative capacity [20;40;144;157]. Secondary mutations within the protease gene (e.g., 10,63,71,77) compensate for the impairment on HIV replication by helping the enzyme to adapt to the primary changes in the active site [20;68;101;144;163;165;197] (Fig. 2). In addition, increases in PI-resistance are often associated with substitutions in the protease cleavage sites (gag and pol genes) [62] (Fig. 3) [34;157;164;240]. Mutations in these regions provide better peptide substrates for the mutated protease and compensate for a potential loss in viral fitness [34;62;144;164;239]. Another compensatory mechanism in RTV+SQV-treated patients may involve insertions in the proline rich region of the p6gag protein, the function of which is still not defined [117]. Although, some PI-resistant viruses display defects in the RT processing [43], mutations in the reverse transcriptase may increase packaging of the Gag-Pol precursor to effectively compensate for this defect and increase the amount of active RT in the virion. AZT-resistance mutations in RT can partially rescue the replicative defect of a PI-resistant virus, which could be relevant to the therapeutic control of HIV-1 infection [43].
Figure 2. (A) Summary of protease mutations associated with resistance to PR inhibitors. Amino acids in red and blue denote primary and secondary/compensatory mutations, respectively. Wild-type amino acids (HIV-1 subtype B) at the codons related to resistance to PI are indicated. Mutations related to loss of sensitivity to PI [99;203] were recently reviewed at http://www.iasusa.org. (B) Structure of the HIV-1 protease (pdb file: 1hxb) [130], indicating amino acid residues associated with PI-resistance. Each numbered circle indicates the codon position and nature of the resistant mutation (primary and secondary mutations in blue and red, respectively).
During the last few years, multiple studies have described a
significant reduction in HIV-1 replicative capacity as a consequence
of PI resistance. A comprehensive list relating ex vivo
fitness to each PI-resistant or secondary mutations is provided in
Table 2. Most of these mutations reduce the replicative capacity but
several amino acid substitutions (usually combinations of primary and
secondary mutations) have been shown to restore or even increase
fitness over a wild-type virus (Table 2). Initial studies reporting
differences in the replicative capacity of viruses resistant to
protease inhibitors introduced a relatively new field to HIV-1
biology and clinical therapy [20]. Transitions in ex
vivo fitness associated with an accumulation of PI-resistant
mutations selected during PI-based regimens (ritonavir, indinavir,
saquinavir, nelfinavir and others) are now well documented in the
literature (reviewed in
[34;164]). Selection of mutant viruses with higher
replication capacity than wild-type virus can be explained by the
Wright's concept of adaptive landscape [233]; "natural
selection drives a population to a local optimum, which is not
necessarily the global optimum". In the absence of PI selection, it
is unlikely that wild-type quasispecies would follow the same
evolutionary pathway to achieve this high level of fitness. During
suboptimal therapy, impaired replication of the intermediate HIV-1
isolate is compensated by the high level of drug resistance.
The latter studies on PI-resistant HIV-1 variants have focused on
ex vivo/in vitro correlations of PI susceptibility and
replication efficiency. Most fitness analyses were performed with
recombinant HIV-1 clones pseudotyped with the PI-resistant protease
or other Gag-Pol regions. Recent studies have compared ex vivo
fitness of PI-resistant variants with an atypical response to
antiretroviral therapy. Particular attention has been focused on
discordant responses in HIV-infected patients treated with PI
(i.e., high viral loads and sustained CD4+ T-cell
counts). PI-resistant HIV-1 isolates and PR-pseudotyped HIV-1 clones
from these patients were used to infect PBMC, human thymic organ
cultures, and SCID-hu Thy/Liv mice [210]. Interestingly,
replication of PI-resistant strains was highly impaired in the
thymus, suggesting a possible preservation of CD4+ T-cell
counts in patients failing PI-based therapy [50].
Figure 3. (A) Organization and processing of HIV-1 Gag and Pol proteins. The nine cleavage sites recognized by the HIV-1 protease in the Gag and Pol proteins are indicated. (B) HIV-1 protease cleavage sites. MA, matrix; CA, capsid; NC, nucleocapsid; TF, transframe; PR, protease; RT, reverse transcriptase; IN, integrase. Scissile bonds are presented as slashes in the amino sequence. Nucleic acid positions are based on the HIV-1HXB2 sequence (GenBank accession number K03455) (Figure adapted from [203]).
Table 2. Effects of protease mutations on HIV&endash;1 replication capacity
|
Amino acid substitutions |
Fitness relative to wildtypea |
Virusb |
Methodc |
References |
|---|---|---|---|---|
|
8K |
|
Sel |
Mono |
[101] |
|
8Q |
|
Sel |
Mono |
[101] |
|
10I |
|
Sel/NL43 |
Mono/Enz |
[145;174] |
|
10I,F |
|
Sel/NL43 |
Mono/Enz |
[145;174] |
|
17R* |
|
HXB2 |
Mono/Comp |
[122] |
|
22V* |
|
HXB2 |
Mono/Comp |
[122] |
|
25E |
|
HXB2 |
Enz |
[135] |
|
25H* |
|
HXB2 |
Mono/Comp |
[122] |
|
30N |
|
NL43 |
Vivo/Mono/Comp |
[56;151] |
|
32I |
|
Sel/NL43/HXB2 |
Mono/Comp/Enz |
[20;40] |
|
35TVLEE* |
|
HXB2 |
Mono/Comp |
[122] |
|
35TD* |
|
HXB2 |
Mono/Comp |
[122] |
|
35TN* |
|
HXB2 |
Mono/Comp |
[122] |
|
36I |
|
NL43 |
Mono |
[145] |
|
36NL* |
|
HXB2 |
Mono/Comp |
[122] |
|
36GL* |
|
HXB2 |
Mono/Comp |
[122] |
|
36DL* |
|
HXB2 |
Mono/Comp |
[122] |
|
37D* |
|
HXB2 |
Mono/Comp |
[122] |
|
37G* |
|
HXB2 |
Mono/Comp |
[122] |
|
37N* |
|
HXB2 |
Mono/Comp |
[122] |
|
46I |
|
Sel/NL43/HXB2 |
Mono/Comp |
[20;101;145;150] |
|
46I,L |
|
n.a. |
Vivo |
[56] |
|
47V |
|
Sel/NL43 |
Mono/Enz |
[174] |
|
48V |
|
NL43 |
Mono |
[145] |
|
50V |
|
Sel/NL43 |
Mono/Enz |
[174] |
|
54V |
|
NL43 |
Mono |
[145] |
|
63P,A |
|
Sel/NL43 |
Vivo/Mono/Comp |
[68;147;150] |
|
71V |
|
Sel/NL43/HXB2 |
Mono |
[20;145] |
|
82A |
|
Sel/NL43/HXB2 |
Mono |
[20;145] |
|
82A,T,F |
|
Prim/Sel/NL43/HXB2 |
Vivo/Mono/Comp/Enz/SCID |
[24;68;147;150;165;177] |
|
84V |
|
Sel |
Mono |
[147] |
|
84A,V |
|
Sel/NL43/HXB2 |
Mono/Comp/Enz |
[40;165;191] |
|
90M |
|
NL43 |
Mono |
[145] |
|
90M |
|
NL43 |
Mono/Comp |
[151] |
|
95TLNFPI* |
|
HXB2 |
Mono/Comp |
[122] |
|
8K/46I |
|
Sel |
Mono |
[101] |
|
10I/48V |
|
NL43 |
Mono |
[145] |
|
10F/50V |
|
Sel/NL43 |
Mono/Enz |
[174] |
|
10F/84V |
|
Sel/NL43 |
Mono/Comp/Enz |
[180] |
|
10I/90M |
|
NL43 |
Mono |
[145] |
|
10F+449F** |
|
Sel/NL43 |
Mono/Comp/Enz |
[180] |
|
30N/63P |
|
NL43/HXB2 |
Mono/Comp |
[151] |
|
30N/88D |
|
HXB2 |
Enz |
[211] |
|
30N/90M |
|
HXB2 |
Enz |
[211] |
|
32I/71V |
|
Sel/NL43/HXB2 |
Mono/Comp/Enz |
[40] |
|
36I/54V |
|
HXB2 |
Comp/Enz |
[165] |
|
46I/63P |
|
HXB2 |
Enz |
[201] |
|
46I/82A |
|
NL43 |
Mono |
[145] |
|
48V/82A |
|
NL43 |
Mono |
[145] |
|
48V/90M |
|
NL43 |
Mono |
[145] |
|
54V/82A |
|
Prim/NL43 |
Mono/SCID |
[145;210] |
|
62I/77I |
|
HXB2 |
Comp/Enz |
[165] |
|
63P/90M |
|
NL43 |
Mono/Comp |
[151] |
|
71V/82A |
|
NL43 |
Mono |
[145] |
|
82T,F/84V |
|
Sel/HXB2 |
Mono/Enz |
[147;201] |
|
82A/90M |
|
NL43 |
Mono |
[145] |
|
10I/48V/82A |
|
NL43 |
Mono |
[145] |
|
10I/48V90M |
|
NL43 |
Mono |
[145] |
|
10I/82A/90M |
|
NL43 |
Mono |
[145] |
|
10F/84V+p1/p6** |
|
Sel/NL43 |
Mono/Comp/Enz |
[150;180] |
|
36I/50V/63P |
|
HXB2 |
Mono |
[195] |
|
36I/54V/82T |
|
HXB2 |
Comp/Enz |
[165] |
|
46I/47V/50V |
|
Sel/NL43 |
Mono/Enz |
[174] |
|
46I/53L/82A |
|
Prim/R8 |
Mono/Enz |
[24] |
|
46I/54V/82A |
|
NL43 |
Mono |
[145] |
|
54V/71V/82A |
|
NL43 |
Mono |
[145] |
|
63P/82F/84V |
|
Sel |
Mono |
[147] |
|
10I/23I/46I/84V |
|
Sel/NL43/HXB2 |
Mono/Comp/Enz |
[40] |
|
10F/46I/50V+p1/p6** |
|
Sel/NL43 |
Mono/Comp/Enz |
[150;180] |
|
10L/46I/82T/84V |
|
NL43 |
Mono/Comp |
[151] |
|
20R/36I/54V/82A |
|
NL43 |
Mono/Enz |
[239] |
|
20R/36I/63P/82S |
|
NL43 |
Mono/Enz |
[239] |
|
20R/63P/82A/90M |
|
NL43 |
Mono/Enz |
[239] |
|
36I/50V/63P+p1/p6** |
|
HXB2 |
Mono |
[195] |
|
36I/54V/71V/82T |
|
HXB2 |
Comp/Enz |
[165] |
|
46I/48V/63P/90M |
|
NL43 |
Mono/Enz |
[239] |
|
46I/54V/71V/82A |
|
NL43 |
Mono |
[145] |
|
46I/63P/82T/84V |
|
NL43 |
Mono/Comp |
[151] |
|
54V/82A+p2/NC+NC/p1 |
|
NL43 |
Mono/Enz |
[144] |
|
10I/23I/46I/84I+p1/p6** |
|
Sel/NL43/HXB2 |
Mono/Comp/Enz |
[40] |
|
10I/36I/48V/84V/90M |
|
Prim/NL43 |
Mono/SCID |
[210] |
|
10F/46I/47V/50V+p1/p6** |
|
Sel/NL43 |
Mono/Comp/Enz |
[150;180] |
|
10L/46I/63P/82T/84V |
|
NL43 |
Mono/Comp |
[151] |
|
20R/36I/54V/71V/82T |
|
HXB2 |
Compt/Enz |
[165] |
|
32I/46I/71V/82A+p1/p6** |
|
Sel/NL43/HXB2 |
Mono/Comp/Enz |
[40;135] |
|
36I/46I/71V/84A+p1/p6** |
|
Sel/NL43/HXB2 |
Mono/Comp/Enz |
[40;62] |
|
10I/46I/63P/77I/84V/90M |
|
Prim/NL43 |
Mono/SCID |
[210] |
|
24I/46I/53L/63P/77I/82A |
|
Prim/R8 |
Mono/Enz |
[24] |
|
54V/63P/71T/72E/82A/85V |
|
HXB2 |
Mono |
[195] |
|
10I/36I/48V/84V/90M+MA/CA+p1/p6** |
|
NL43 |
Mono/Enz |
[144] |
|
10I/54V/63P/71V/77I/82A/90M |
|
Prim/NL43 |
Mono/SCID |
[210] |
|
14V/20R/32I/63P/64V/71V/82A |
|
HXB2 |
Enz |
[153] |
|
10I/20R/36I/46L/48V/71V/82A/90M |
|
Prim/NL43 |
Mono/SCID |
[210] |
|
10I/20R/36I/54V/63P/71V/82T/90M |
|
Prim/NL43 |
Mono/SCID |
[210] |
|
10I/24I/46I/63P/71V/77I/82T/84V |
|
Prim/NL43 |
Mono/SCID |
[210] |
|
10I/36I/46L/48V/63P/71V/82A/90M |
|
Prim/NL43 |
Mono/SCID |
[210] |
|
54V/63P/71T/72E/82A/85V/85V/+p7/p1** |
|
HXB2 |
Mono |
[195] |
|
10I/20R/36I/46I/53L/63P/71V/82A+NCp1?** |
|
NL43 |
Mono/Enz |
[239] |
|
23I/32I/46I/47V/54M/71V/84V+p1/p6+p7/p1** |
|
Sel/NL43/HXB2 |
Mono/Comp/Enz |
[40;62;135] |
|
10I/35D/37D/48V/54V/63P/71V/82A/90M/93L |
|
HXB2 |
Enz |
[153] |
|
10I/14V/33F/36M/37C/54V/63P/67F/71V/72M/73S/77I/82A/84V/90M |
|
HXB2 |
Enz |
[153] |
a Symbols:"&endash;" decreased;"+" increased;"." comparable.
b HIV&endash;1 isolates or pseudotyped viruses used to measure viral fitness (replicative capacity). Prim, HIV&endash;1 primary isolate; Sel, in vitro selection of drug&endash;resistant strains in the presence of the corresponding antiretroviral drug; NL43, HXB2, and R8 correspond to the recombinant infectious clone used as a backbone to introduce drug&endash;resistance associated mutations by site&endash;directed mutagenesis or pseudotyping; n.a., not apply.
c Methods used to determine viral fitness. Vivo, in vivo determination of HIV&endash;1 kinetics in plasma; Mono, ex vivo HIV&endash;1 monoinfections (i.e., viral growth kinetics and single&endash;cycle infection assays); Comp, ex vivo HIV&endash;1 growth competition experiments; Enz, catalytic activities of HIV&endash;1 enzymes; SCID, SCID&endash;hu Thy/Liv or hu&endash;PBL&endash;SCID mice as animal models.
* Insertion mutations at the corresponding codon position.
** Mutations in the protease gene accompanied for mutations in the Gag&endash;processing sites.
Reverse transcriptase inhibitor (RTI)-resistant variants
Multiple studies have explored how RTI-resistant mutations affect HIV-1 replication capacity (Table 3, Fig. 4) (reviewed in [34;164]). Interestingly, amino acid changes conferring RTI-resistance do not appear to reduce viral fitness to the same extent as PI-resistance mutations [13;34]. This may be due in part to a restricted evolution in RT. However, there are other factors/attributes that differ between HIV-1 resistance to PI and to RTI. These include, (i) the appearance of discrete NRTI- or NNRTI-resistant mutations rather than a collection of PI-resistant substitutions during selection, (ii) minimal secondary/compensatory mutations selected during NRTI- or NNRTI-treatment as compared to PI-therapy, (iii) a decrease in relative fitness associated with increasing number of NRTI-resistant mutations (e.g., AZT-resistant mutations) while the opposite is generally true with PI-resistance (i.e., increase in viral fitness due to accumulation of compensatory mutations), and (iv) limited cross-resistance encoded by specific NRTI-resistant mutations as compared to a broad PI cross-resistance conferred by mutations selected during specific PI therapy. It is important to note that these are general observations and do not apply to HIV-1 resistance to all drugs in the NRTI, NNRTI, or PI classes. For example, almost all NNRTI-resistant mutations confer cross-resistance to all other drugs in this class. The next section will review studies on the fitness of RTI-drug resistant HIV-1.
a) Nucleoside reverse transcriptase inhibitors (NRTI)
Nucleoside analogue reverse transcriptase inhibitors (NRTI) were the first class of antiretrovirals to be approved for anti-HIV-1 therapy [137]. These drugs compete for binding to RT with the native deoxynucleoside triphosphates (dNTPs), can be incorporated into elongating HIV-1 DNA, and result in chain termination [6]. To date, six NRTI have been approved for therapy in the United States; i.e., zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), and abacavir (ABC). Tenofovir (TNV) a nucleotide-RT-inhibitor has recently received approval from the FDA. In general, different pairs of NRTI remain the base component of all combination treatment regimens.
It is not surprising that some of the first studies showing the effect of drug-resistance mutations on viral replication fitness were related to AZT [90]. In AZT-experienced patients, the wild-type 215T HIV-1 isolate is more fit in the absence of AZT, can re-emerge from the diverse quasispecies and out-compete the AZT-resistant 215Y strain. This situation appears to be a replacement rather than a true genetic reversion but may be more representative of ex vivo fitness or replicative capacity. In vivo results showing that 215T, 215D, and 215S are equally fit [87] may be due to infection with a homogeneous HIV-1 population, specific mutational pathways in the absence of drug, and the 215T virus as competitor. A stepwise accumulation of AZT resistance mutations (70R, 215Y, and 41L) during in vitro selection was similar to that observed in vivo [96]. Interestingly, ordered accumulation of resistant variants was also predictive of relative fitness (i.e., wt > 70R >> 215Y = 41L/215Y > 41L). Of course, this fitness-drug resistance relationship is inverse to that observed with the accumulation of primary and secondary PI-resistant mutations. However, each new AZT-resistant mutation confers a greater level of AZT-resistance (i.e., 10-fold with 70R to >100-fold with 41L, 67N, 70R, 215Y, 219L) whereas the accumulation of compensatory mutations (e.g., within the PR gene or at the protease cleavage sites) may improve fitness but have limited or no effect on susceptibility to PI.
Unlike other NRTI, AZT selects for mutations outside the RT domains involved in polymerase activity [6]. Residues M41, T215, and K219 reside adjacent but still external to the dNTP or primer binding sites. The role of the 41L mutation in resistance may be more related to enzyme stability [125]. In contrast, the 215Y and 219E substitutions appear to play a role in the reversal of pyrophosphorolysis responsible for removing AZT-MP from the primer terminus. Interestingly, the addition of low AZT concentrations to cell cultures resulted in increased replication of an AZT-resistant 41L/215Y HIV-1 clone, but did not affect growth of the 70R AZT-resistant HIV-1 virus and inhibited the wild-type virus [5]. Similar results were observed with AZT-resistant clinical isolates containing a combination of AZT-resistance mutations [5]. Although 70R is the first AZT-resistant mutation selected during AZT therapy, the less fit 215Y genotype eventually predominates the quasispecies. Thus, the AZT-mediated stimulation of the 215Y as opposed to 70R HIV-1 isolate may play a role in the in vivo selection of 215 mutants.
Table 3. Effects of RT mutations on HIV-1 replication capacity
|
Amino acid substitutions |
|
Virusb |
Methodc |
References |
|
41L |
|
HXB2 |
Comp |
[96] |
|
62V |
|
HXB2 |
Mono |
[143] |
|
67N |
|
n.a. |
Vivo |
[56] |
|
69SS* |
|
HXB2 |
Vivo/Comp |
[142;190] |
|
70R |
|
NL4 |
Mono/Comp |
[204] |
|
70T,R |
|
NL43/HXB2 |
Mono/Comp |
[96;204] |
|
74V |
|
HXB2 |
Mono |
[143] |
|
74V |
|
NL43 |
Mono/Comp/Enz |
[204;205] |
|
75I |
|
HXB2 |
Mono |
[143] |
|
77L |
|
HXB2 |
Mono |
[143] |
|
89K |
|
HXB2 |
Mono/Comp |
[212] |
|
92I |
|
HXB2 |
Mono/Comp |
[212] |
|
98G |
|
NL43 |
Mono |
[110] |
|
100I |
|
NL43/HXB2 |
Mono/Comp |
[110;191] |
|
103N |
|
NL4 |
Vivo/Mono |
[56;110] |
|
103N |
|
NL43 |
Mono/Enz |
[88] |
|
106A |
|
Sel/NL43 |
Mono/Comp/Enz |
[2;108;110] |
|
108I |
|
NL43 |
Mono |
[110] |
|
115L,A,D,W |
|
HXB2 |
Mono/Enz |
[148] |
|
116T |
|
HXB2 |
Mono |
[143] |
|
151M |
|
HXB2 |
Mono |
[143] |
|
151M |
|
HXB2 |
Comp |
[128] |
|
151M,L |
|
HXB2 |
Mono/Comp |
[87] |
|
156A |
|
HXB2 |
Mono/Comp |
[212] |
|
163N |
|
HXB2 |
Mono/Comp |
[113] |
|
179D |
|
NL43 |
Mono/Comp/Enz |
[2] |
|
181C |
|
NL43 |
Mono |
[110] |
|
181C |
|
NL43 |
Mono/Comp/Enz |
[2] |
|
181C |
|
Sel |
Comp |
[108] |
|
184I,V |
|
Sel/NL43/HXB2 |
Vivo/Mono/Comp/Enz/SCID |
[8;56;85;120;139;149;177;205;236] |
|
188C |
|
NL43 |
Mono |
[110] |
|
190S,A |
|
Sel/NL43 |
Mono/Comp |
[108;110] |
|
215Y,S,D |
|
HXB2 |
Mono/Comp |
[45;143] |
|
215Y,F,N,S,D |
|
HXB2 |
Vivo/Mono/Comp |
[45;68;90;91;96;128] |
|
215S |
|
HXB2 |
Comp |
[128] |
|
236L |
|
NL43 |
Mono/Comp/Enz |
[2;67;88] |
|
501W,R |
|
HXB2 |
Mono/Enz |
[3] |
|
41L/70R |
|
HXB2 |
Mono/Comp |
[113] |
|
41L/215Y |
|
HXB2 |
Comp |
[96] |
|
115W/230I |
|
HXB2 |
Mono/Enz |
[169] |
|
151M/215Y |
|
HXB2 |
Comp |
[128] |
|
D67/69G/74I |
|
NL43 |
Mono |
[110] |
|
77L/116Y/151M |
|
HXB2 |
Mono |
[143] |
|
77L/116Y/151M |
|
HXB2 |
Comp |
[128] |
|
67N/70R/ 215Y/219Q |
|
NL43 |
Mono/Enz |
[26] |
|
75I/77L/116Y/151M |
|
HXB2 |
Mono |
[143] |
|
75I/77L/116Y/151M |
|
HXB2 |
Comp |
[128] |
|
62V/75I/77L/116T/151M |
|
HXB2 |
Mono |
[143] |
|
62V/75I/77L/116Y/151M |
|
HXB2 |
Comp |
[128] |
|
69G/70R/74I/103N/215F/219Q |
|
NL43 |
Mono/Comp |
[109] |
|
D67/69G/70R/74I/103N/215F/219Q |
|
NL43 |
Mono/Comp |
[109] |
a Symbols:"&endash;" decreased;"+" increased;"." comparable.
b HIV&endash;1 isolates or pseudotyped viruses used to measure viral fitness (replicative capacity). Prim, HIV&endash;1 primary isolate; Sel, in vitro selection of drug&endash;resistant strains in the presence of the corresponding antiretroviral drug; NL43, HXB2, and R8 correspond to the recombinant infectious clone used as a backbone to introduce drug&endash;resistance associated mutations by site&endash;directed mutagenesis or pseudotyping; n.a., not apply.
c Methods used to determine viral fitness. Vivo, in vivo determination of HIV&endash;1 kinetics in plasma; Mono, ex vivo HIV&endash;1 monoinfections (i.e., viral growth kinetics and single&endash;cycle infection assays); Comp, ex vivo HIV&endash;1 growth competition experiments; Enz, catalytic activities of HIV&endash;1 enzymes; SCID, SCID&endash;hu Thy/Liv or hu&endash;PBL&endash;SCID mice as animal models.
* Mutation at codon 69 (usually 69S), followed by an insertion of 2 or more amino acids (69 insertion complex).
Lamivudine-resistant viruses, harboring the 184V mutation, appear to have increased RT fidelity [79;171], diminished RT processivity and reduced replication capacity [8;132]. Back et al. [8] were among the first to report impaired replicative fitness of viruses carrying 3TC-resistance mutations. Viruses harboring the 184V mutation are more fit and show higher levels of resistance [8;85]. Thus, eventual outgrowth of the 184V 3TC-resistance variant is inevitable. Recent studies suggest that the combination of 184V and AZT-resistant mutations (specifically 215Y) may be incompatible for various RT activities [89]. In general, therapy with multiple nucleoside analogs has exhausted the possible sites for NRTI-resistant mutations in the RT-coding region, all of which reduce fitness. However, most NRTI-resistant mutations have a very modest effect on RT activity. In fact, there is a little evidence of any debilitating effect of AZT-resistant mutations on any HIV-1 RT activity in vitro. Thus, the minor decrease in fitness observed in HIV-1 clones containing NRTI-resistant mutations or pseudotyped with an NRTI-resistant RT-coding region may be overcome by secondary mutations increasing the fitness of other steps in the retroviral life cycle. Few studies have explored this possibility. Interestingly, reversion of most NRTI-resistant mutations is slower than most PI-resistant substitutions in the absence of therapy implying some compensation.
Figure 4. (A) Summary of reverse transcriptase mutations associated with resistance to RT inhibitors. Amino acids in red and blue denote primary and secondary/compensatory mutations, respectively. Wild-type amino acids (HIV-1 subtype B) at the codons related to resistance to RTI are indicated. Mutations related to loss in sensitivity to RTI [99;203] were recently reviewed at http://www.iasusa.org. (B) Structure of the HIV-1 RT (pdb file: 3hvt) [223], and positions of each amino acid residue associated with resistance to RTI. Each numbered circle indicates codon position and the nature of the resistance mutation (primary and secondary mutations in blue and red, respectively).
b) Nonnucleoside reverse transcriptase inhibitors (NNRTI)
Nonnucleoside reverse transcriptase inhibitors (NNRTI) are non- or un-competitive inhibitors that bind to a hydrophobic pocket adjacent to the polymerase active site of RT [49]. This binding inhibits DNA polymerization by an allosteric change of the polymerase active site [73]. Mutations in this hydrophobic binding pocket are rapidly selected during NNRTI-therapy [49;203] (Fig. 4). Three NNRTI are currently approved for antiretroviral therapy in the United States: delavirdine (DLV), efavirenz (EFV) and nevirapine (NVP). Presence of NNRTI resistance mutations results in a slight decrease in RT polymerase activities relative to RNase H activity of the enzyme [2;88]. However, the level of drug resistance in vitro does not always correlate with the likelihood that a drug-resistant variant will emerge in vivo. The viral replicative capacity of NNRTI-resistant viruses has not been extensively studied. Available data suggest that single-point mutations such as 103N or 181C, selected during NNRTI treatment, have limited effects on viral fitness but confer high level resistance and persist in the absence of drug pressure. Unlike other antiretroviral drugs, the genetic barrier to NNRTI resistance is minimal and is found at high frequencies in the HIV-1 quasispecies in the absence of therapy. The NNRTI binding pocket has only a structural role in the catalytic activity of RT in the absence of drug. Thus, changes that maintain the general hydrophobicity and architecture of the pocket will likely confer high level resistance without a negative impact on enzyme function. Interestingly, related lentiviral polymerases (e.g., HIV-1 group O and HIV-2) sharing conserved structural RT domains are resistant to NNRTI [55;186;189]. Most HIV-1 group O isolates are intrinsically resistant to NNRTI due to the presence of three amino acid substitutions (i.e., 98G, 179E, and 181C) in RT [186], which obviously do not affect the wild-type fitness.
Several studies have described a decreased replicative capacity in viruses carrying mutations associated with nevirapine (i.e., 103N and 181C) [97] and efavirenz (i.e., 100I, 103N, and 190S) [215] resistance. However, the high level of delavirdine resistance conferred by the 236L mutation is insufficient to compensate for its decreased fitness compared to other NNRTI resistance mutants (e.g., 103N or 181C). It may also explain why 236L is selected infrequently during DLV therapy [53;65;67;88]. In most cases, the major NNRTI-resistant mutations 103N and 181C have a minimal impact on viral fitness, which could explain for the rapid failure of NNRTI when used in suboptimal treatment regimens. A recent analysis of in vitro selected nevirapine-resistant variants showed that, in the absence of drug, the 181C mutant was more fit than the wild-type virus (fitness gradient: 181C > wt > 106A > 190A) [108]. It is difficult to explain why a drug-resistant substitution that confers increased fitness is not found as the wild-type sequence. Detailed studies on RT activity suggest that several of these mutations may actually increase fidelity thereby slowing the mutation rates. Although a small decrease in mutation frequency will have little impact on intrapatient population size, avoidance of specific selective pressures (e.g., immune response) requires constant evolution (i.e., accumulation of specific mutations).
Multiple dideoxynucleoside resistance (MDR) variants
It is well known that current antiretroviral agents have achieved a remarkable reduction in HIV-related morbidity and mortality. However, a recent study showed that at least 50% of HIV-positive individuals in the United States are infected with drug-resistant variants [194]. Thus, a "second epidemic" of AIDS may involve patients who harbor drug-resistant HIV-1 strains. Prevalence of drug resistant mutations in patients with primary HIV-1 infection has been assessed in several studies conducted in Europe [183] and in the United States [134]. Clinical and virological consequences of primary HIV-1 infection with drug resistant viruses may include suboptimal treatment responses, reduced viral fitness, and the potential for transmission of drug-resistant virus [134]. Moreover, MDR strains acquired during primary HIV infection can persist for more than nine months, despite decreased viral fitness [22].
Accumulation of multiple mutations associated with MDR does not occur at random but follows a sequential order [118]. The 151M mutation is thought to be the first MDR mutation selected in RT, followed by 75I, 77L, 116Y, and then 62V (also know as the MDR-151 complex) [203;207]. As discussed before, HIV-1 can evolve under drug pressure by selecting drug-resistance mutations at the expense of viral fitness, but generally compensate for this defect with secondary mutations. Few studies have documented a replicative advantage of drug-resistant variants over wild-type viruses in the absence of drugs (Tables 2 and 3). Several studies have assessed the in vitro fitness of viruses containing the MDR-151 complex [128;143], but specific combinations of these mutations actually appear more fit than the wt in the absence of drug [128;143]. Garcia-Lerma et al. [87] evaluated the replication capacity of different MDR recombinant HIV-1 clones carrying the 151M mutation along with two intermediate mutations (151L or 151K). They showed that a virus harboring the 151M mutation was more fit than the 151L. Thus, the 151L mutants may be an intermediate of these MDR variants.
Another set of mutations associated with high-level resistance to multiple RT inhibitors centers around the 69G substitution (MDR-69 complex) [99] (Figure 4). An amino acid deletion at codon 67 (D67) improves the replication efficiency of a highly AZT-resistant virus (D67/69G/70R/74I/103N/215F/219Q) to wild-type levels [109]. Recent studies have also reported a rare nucleotide insertion in the reverse transcriptase gene between codons 69 and 70 (e.g., 69SS) that results in high-level resistance to multiple NRTI, including AZT, 3TC, d4T, ddI, and ddC [23;154;229]. Decreased viral fitness in the absence of NRTI is quite evident in HIV-1 clones harboring a dipeptide insertion (69SS) in RT [190]. Finally, Lukashov et al. [142] studied the in vivo evolution of a MDR HIV-1, which contained an insertion of 2 amino acids between positions 68 and 69, and several other mutations within RT. These mutants 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 (16% less fit than the wt virus). However, these MDR mutants were able to maintain high viral loads in the presence of antiretroviral therapy.
Resistance to other HIV-1 inhibitors
Within each drug class targeting PR or RT, there is extensive cross-resistance. For example, 103N in RT confers high-level resistance to the other three NNRTI, 90M in PR results in some degree of resistance to all four available PI, and 151M in RT confers resistance to every NRTI except 3TC. New drugs in development target different steps in the virus life cycle, including host cell entry and viral integration. There are three classes of drugs that interfere with HIV-1 entry into host cells, and are classified as inhibiting: (i) HIV-1 envelope glycoprotein (gp120) interaction with its CD4 receptor [112], (ii) gp120 binding to the chemokine receptors CCR5 or CXCR4 [35;208], or (iii) the fusion of the viral and cellular membranes [121].
Identification of the major HIV-1 co-receptors was facilitated by the initial finding that some (b-chemokines, i.e. RANTES, macrophage inflammatory protein-1a (MIP-1a), and MIP-1b, natural ligands of CCR5 could block infection of nonsyncytium-inducing/CCR5-tropic (NSI/R5) HIV-1 [35]. In contrast, T-cell line tropic isolates, forming cell syncytia during active replication in tumor T cell lines, utilize the CXCR4 co-receptor for entry (SI/X4) [1;54;63;80]. This discovery in the mid 1990 led to rapid development of several drugs that blocked HIV-1 binding or utilization of these co-receptors. The heterogeneous HIV-1 envelope glycoproteins and these RANTES analogs compete for the same CCR5 receptors on the cell surface. Torre et al. examined the sensitivity of 18 primary NSI/R5 HIV-1 isolates to AOP-RANTES inhibition and discovered a 30-fold variation in IC50 values [217]. Although all of these viruses are considered "wild type" quasispecies, there were also significant variations in ex vivo fitness [188]. Considering that the rate of host cell entry appears to control replicative capacity in wild-type viruses [4] (see below), it is quite conceivable that increased viral fitness is also linked to decreased susceptibility to these CCR5 agonists or antagonists.
Several X4 entry inhibitors are now in development (Met-SDF-1b, an a-chemokine analog) or already in clinical trials (AMD3100, a bicyclam compound) [75;235]. Met-SDF-1b inhibits X4 viral entry by a mechanism similar to R5 HIV-1 inhibition by RANTES analogs [75;235]. However, IC50 values required for Met-SDF-1b inhibition of X4 HIV-1 isolates are at least 1,000-fold greater than the concentration required for R5 inhibition by the RANTES analogs [235]. AMD3100 is a low molecular weight bicyclam compound that has an IC50 value in the low nanomolar concentrations (~1.4 nM) and does not induce receptor signaling [42;75]. Resistance to this drug is conferred by a collection of single amino acid substitutions in the env gene or a reduction in the overall positive charge of HIV-1 gp120 V3 loop [42;76]. AMD3100-resistance was not associated with a co-receptor switch (e.g. CXCR4 to CCR5 usage) but did have a negative impact on HIV-1 replication [77].
Another class of entry inhibitors was designed to block virus-cell fusion and target conserved fusion domains in gp41. Included in this class of inhibitors are T-20, T-1249, C34, 5helix, and IQN-17 [30;32;70;84;93;111;161]. T-20 and T-1249 are synthetic peptide analogs that bind to the alpha helix bundle region in the gp41 transmembrane domain and prevent formation of a hairpin structure necessary for membrane fusion. Both drugs are currently in clinical trials [30] and appear to act synergistically with co-receptor antagonists and agonists to inhibit HIV-1 in vitro. Lu & Kuritzkes [140] demonstrated that the introduction of known T-20-resistant mutations (37T, 38M or 36S/38M) into the gp41 coding region of the HIV-1NL4-3 clone reduced replication to levels less than the wild-type control (relative fitness order: wt > 37T > 38M > 36S/38M). T-20-resistant HIV-1 isolates are currently being isolated from patients in ongoing clinical trials and utilized in various fitness assays.
Integrase inhibitors (i.e., diketo acids analogs) comprise the most recent class of drugs shown to effectively inhibit HIV-1 replication [230]. Resistance to these compounds is related to specific mutations in the integrase active site (i.e., 153Y, 66I, and 155S), which also impair enzymatic function in vitro. Increasing levels of resistance to integrase inhibitors are associated with a significant loss of viral fitness [230]. However, the actual appearance or net effect of any resistant mutation on HIV-1 fitness can only be assessed in HIV-infected patients treated with this drug.
A topic that is often overlooked in HIV-1 research and in scientific literature is the impact of fitness on HIV-1 transmission, disease progression, evolution, and prevalence in the human population. This review has thus far focused on the effect of drug resistance on HIV-1 fitness. However, the vast majority of HIV-1 infections worldwide are not being treated with conventional antiretroviral therapies. The rate of new infections continues to increase, people progress to AIDS, and die and yet little is still known about the phenotypic differences between the heterogeneous etiological agent. Recent studies suggest that the nature of the virus itself, and not solely manifestations of host factors and the immune response is contributing to HIV disease progression [188]. Numerous studies have also shown that there are possible differences in interpatient and intersubtype HIV-1 fitness [4;115;188;209]. However, these fitness studies are plagued by the same difficulties and shortcomings described for research on the viral fitness of drug resistant HIV-1 strains. What is appropriate assay to measure ex vivo fitness? Are primary isolates or pseudotyped HIV-1 clones most suitable for accessing differences in fitness? How do we compare ex vivo HIV-1 replicative capacity to viral fitness within a patient or even within the human population? Although this field remains controversial, recent studies aided by new technologies are now making progress in answering some of the questions related to HIV-1 fitness and AIDS [4;115;188;209].
Fitness of the transmitted HIV-1 isolate
A significant genetic bottleneck is quite apparent during transmission by any route of HIV-1 infection (see above). However, individuals may be exposed to varying amounts of virus depending on the mode of transmission. Sexual and vertical transmission generally involves small amounts of virus and infected cells, whereas a bolus dose of virus may be transmitted during direct blood-to-blood contact, e.g. contaminated needles or blood transfusions [170;232;241]. Regardless, the virus population recovered from primary HIV-1 infections is more homogeneous with a narrow genetic distribution of quasispecies as compared to the donor HIV-1 quasispecies. The transmitted variants are likely less fit than the donor quasispecies due to the genetic bottleneck. The selective factors imposing the bottleneck are diverse and likely include: (i) host factors such as innate immune response, (ii) density of target cells at the site of infection, (iii) number of transmitted virions, and (iv) the structure of transmitted viral quasispecies. Quasispecies distribution and size of the transmitted pool may have a significant effect on fitness of the infecting isolate and subsequent disease progression [60;167]. The viral properties selected during initial infection may not be the same attributes necessary for efficient dissemination and rapid turnover during acute disease. A combination of these viral characteristics may only be found in exposures with a significant load of diverse HIV-1 quasispecies. Finally, environmental differences (pH, target cells, mucosal composition) at the site of exposure may not only affect the efficiency of transmission but also fitness of the infecting isolates [18;170]. For example, there appear to be phenotypic and genotypic differences between HIV-1 variants infecting men and women following heterosexual contact [170].
Although there may be an element of chance in the expansion of a particular HIV-1 clone, phenotypic selection does occur in nearly every HIV-1 infection. SI/X4 HIV-1 isolates often dominate the quasispecies late in disease and yet the NSI/R5 variant is typically transmitted to a recipient (reviewed in [83]). Preferential transmission of NSI/R5 over SI/X4 HIV-1 isolates is contradictory to increased turnover of SI/X4 HIV-1 over NSI/R5 isolates in culture (see below) [15;215]. Thus, efficiency or fitness of transmission is likely not related to the HIV-1 replicative capacity measured in normal cell culture. As described below, this fitness dichotomy is not restricted to co-receptor usage but may also be observed in transmission of different NSI/R5 HIV-1 isolates in the human population [17]. Although in vivo findings suggest that NSI/R5 HIV-1 isolates may out-compete the SI/X4 variants at the site of primary infection, one report suggests that the NSI/R5 isolates only predominate after a temporary expansion of SI/X4 HIV-1 isolates is quenched by an activated immune response [38]. However, this observation is difficult to reconcile with the finding that humans who are homozygous for a deletion in the CCR5 gene (i.e. lack CCR5 on any cell surface) are typically resistant to HIV-1 infection [48;168]. To date, the universal factors (i.e. found in almost every human host) involved in the selection of NSI/R5 HIV-1 isolates during transmission and asymptomatic disease are not well defined. Langerhans' cells (LC) are found embedded in mucosa (e.g. vaginal mucosa) and may be the first cell targets for primary heterosexual transmission [18;209]. LC may play a role in NSI/R5 HIV-1 selection since CCR5 and not CXCR4 is preferentially expressed in situ and in the absence of external stimuli [18]. A recent report describes increased replication of an NSI/R5 (HIV-1Bal) over an SI/X4 (HIV-1III-B) isolate in LC embedded in skin-derived explants even though the opposite is true in PBMC cultures or other permissive cell lines [18;209]. This discrepancy only reinforces the definition of fitness, i.e. a measure of an organism's replicative capacity in a given environment.
Impact of HIV-1 fitness on disease progression
Although HIV-1 is the etiological agent of AIDS, it is often assumed that phenotypic characteristics and replication efficiency (ex vivo fitness) of the infecting, wild type HIV-1 isolates has little impact on the rate of disease progression. In general, acute HIV-1 infection is followed by a chronic and progressing disease resulting in immunodeficiency and acquisition of various life-threatening opportunistic infections. Multiple studies have tried to compare both, host and viral factors, with HIV-1 pathogenesis and progression to AIDS. The strongest correlates of HIV-1 disease progression include various host immunological and genetic factors: (i) a strong HIV-specific CD8+ cytotoxic lymphocyte throughout disease, and (ii) retention and proliferation of HIV-specific CD4+ lymphocyte response following acute infection [27;28;66;158;172;179;198], (ii) some polymorphisms in the CCR5, RANTES, and SDF-1 promoter regions or altered expression of the chemokines ([47], reviewed by [12]), and (iii) specific HLA class I genes [44]. However, there are several general observations suggesting that HIV fitness may play an important role in disease progression, i.e., (i) HIV-1 load is the best marker to date for disease progression [156], (ii) treatment with highly active antiretroviral therapy (HAART) may reduce viral loads to undetectable levels and delays progression to AIDS indefinitely, and (iii) emergence of drug resistant isolates and rebound in viral load results in resumption of normal disease progression.
In addition to these general observations, there are also three clear examples that HIV-1 phenotype can affect disease progression. First, the faster replicating SI/X4 HIV-1 isolates are generally isolated during AIDS or in late HIV disease, whereas the slower replicating, NSI/R5 strains generally predominate during asymptomatic stages [7;14;215]. Several studies have now shown that appearance of SI/X4 isolates does coincide with rapid decline in CD4 cells, a burst in viral load, and the onset of AIDS. However, SI/X4 isolates are inconsistently isolated in late stages of disease and are not a prerequisite for progression or AIDS [32;83;202]. Using a mathematical model, Wodarz & Nowak [231] suggested that the evolution of SI/X4 strains depends on the fitness landscape of the HIV-1 quasispecies. Variation in intrapatient HIV-1 evolution and the fitness landscape would explain why SI/X4 viruses only appear in half of HIV-infected individuals. Recently, the dogma that all SI/X4 isolates are more fit in cell culture than NSI/R5 isolates have been challenged by competing several NSI/R5 - SI/X4 pairs in PBMC cultures [188]. Although most SI/X4 were more fit in cell culture, NSI/R5 isolates from rapid progressors could out-compete SI/X4 isolates from long-term survivors or even SI/X4 isolates from patients displaying typical HIV-1 progression [188].
Aside from these changes in co-receptor usage/cell tropism, evidence that HIV-1 genetic alterations could affect disease progression was clearly demonstrated in a few long term nonprogressor patients (LTNP) shown to harbor HIV-1 strains with nef deletions [46;124]. Independently of these observations, Daniel et al. [41] had generated several HIV-1 clones with similar nef deletions, all of which were replication defective in PBMC cultures. Interestingly, several of the LTNP infected with nef-deleted viruses have eventually progressed to AIDS (after >10 years of asymptomatic disease) [21]. Changes in HIV-1 fitness during this time period have not been published but would provide valuable information on the possible accumulation of compensatory mutations. It is important to note that a similar rebound in HIV-1 fitness does occur during the emergence of drug-resistant mutations, albeit on a shorter time scale. In these LTNP now progressing to AIDS, the Red Queen Hypothesis would support the notion that slow but continual replication of HIV-1 would expand the quasispecies diversity and lead to increased fitness. Only genetic bottlenecks due to changes in selective pressure could reverse this trend (i.e. Muller's Ratchet).
As described above, several viral parameters such as infectious dose, route of infection, and viral fitness may be contributing to the clinical course of HIV-1 infection [36;141;216;241]. Subtle differences in viral fitness may also contribute to HIV-1 pathogenesis and even overwhelm an HIV-specific immune response. In an attempt to correlate ex vivo HIV-1 replicative capacity and disease progression, several studies have compared the replication differences of primary HIV-1 isolates using mono-infections of PBMC or a CD4+ tumor cell line [16;82;202;215]. Variations in the micro-environment of tissue culture systems, the lack of an internal control, and the possibility of a high virus titer saturating the target cells all lead to inconsistent results with mono-infections. Moreover, a moderate fitness cannot be accurately measured in mono-infections because of the inherent variability between cultures. This may explain why this assay has only characterized drastic defects in the replication kinetics of HIV-1 isolates from long-term nonprogressor (LTNP). Several studies have examined the replication kinetics of HIV-1 isolated from patients with atypical progression [16;82;202]. Using a mono-infection assay with primary HIV-1 isolates, Blaak et al. [16] showed that some LTNP harbored NSI isolates with slow replication kinetics. A recent study competed HIV-1 isolates from long-term survivors (LTS) and typical progressors (PRO) with four reference HIV-1 strains (i.e. primary isolates) [188]. PRO HIV-1 isolates had out-competed the reference strains in growth competition experiments while the opposite was observed for HIV-1 isolated from long-term survivors. Interestingly, all patient isolates and reference HIV-1 strains had similar replication kinetics in PBMC monoinfections. These results suggest that regardless of the viral phenotype (NSI or SI), HIV-1 isolates from long-term survivors were less fit than strains obtained from progressors [188].
The relative fitness values of each LTS and PRO isolate, derived from competitions with four reference strains, showed strong correlations with viral RNA load. Comparisons of viral load vs. ex vivo fitness indicated that the linear regressions derived from LTS and PRO analyses had similar slopes but did not intersect [188]. This data support earlier findings that the fitness of the infecting HIV-1 isolate may predict the clinical course of disease [141]. In a current model, decreased genetic diversity in donor quasispecies and low infectious dose during transmission result in infection with an HIV-1 clone of reduced fitness. Rosenberg et al. [198] have suggested that rapid depletion of CD4+ cells during acute/early infection results in an irreplaceable loss of HIV-specific T helper cells. In contrast, proliferation and retention of HIV-specific T helper cells during early disease is associated with slower disease progression [198]. Thus, it is quite conceivable that infection with an isolate of poor replicative capacity may lead to limited depletion of CD4+ cells and retention of HIV-specific T helper cells. Slower disease progression may be due to a combination of an enhanced HIV-specific immune response and reduced HIV-1 fitness. However, the model also predicts that continual HIV-1 replication and evolution will eventually increase genetic diversity and the fitness of the quasispecies. This may explain why even LTNP fail to eradicate virus and eventually progress to AIDS. It is important to note that infection with an HIV-1 clone outside of the normal distribution of quasispecies would be rare which could explain the low frequency of atypical progression (e.g. LTS or rapid progressors).
Although the impact of HIV-1 fitness on disease progression is still not well understood, studies on SIV pathogenesis have provided valuable information about the disease process that leads to simian AIDS (reviewed in [227]). Using the SIV model, Kimata et al [123] showed that antigenic and cytopathic properties of the SIV strain and not phenotype (e.g. SI vs. NSI) predict fitness in the host. Emerging SIV variants have increased replicative capacity over the SIV strain used in the initial infection. Furthermore, infectious dose and virulence ("viral fitness") of the initial inoculum did influence viral load during disease progression in SIV-infected macaques [104].
Are there intersubtype HIV-1 variations in viral fitness?
Divergent interpatient HIV-1 evolution coupled with new introductions into susceptible human populations has lead to the current HIV-1 diversification from original zoonotic jumps in central Africa [95;127]. Although a founder effect results in further HIV-1 spread and divergent evolution in different regions in the world, the phylogenies of most non-African HIV-1 strains can be traced to central African isolates [95;127]. At least two to three separate zoonotic jumps from chimpanzees into humans led to the disproportionate spread of HIV-1 groups M (main), O (outlier), and N (non-M/non-O) [86;95;131]. Increased fitness has likely played a key role in the predominance and extreme variation of HIV-1 group M over group N or O isolates. Phylogenetic and recombination analyses further subdivided the HIV-1 group M into nine subtypes (A, B, C, D, F, G, H, J, and K) and fourteen circulating recombinant forms (CRF) (reviewed in [175;185]). These subtypes and CRFs are unequally distributed across the globe, e.g. subtype B in the Americas and Europe whereas A, C, and CRF02-AG are the most prevalent clades in Africa [175;185]. To date, CRF have been identified in nearly every region of the world where two or more subtypes co-circulate and may account for over 10% of new HIV-1 infections [126;175;185].
The proportions of subtypes in defined populations are not stable but are in constant flux due to new introductions of HIV-1 subtypes, changes in human behavior, therapeutic intervention, mode of transmission, and possibly, subtype fitness. Over the past decade, there has been a considerable shift in the epicenter of the HIV-1 epidemic from Sub-Saharan Africa to Southern Africa, India, and Southeast Asia [74;114;136;200]. Subtype C has now emerged as the predominant clade in the world due to these regional pandemics and accounts for at least half of all infections worldwide [105;196]. Although subtype B likely preceded subtype C as a founder clade in India and China, most of the new infections in these countries are attributable to subtype C isolates or intersubtype B/C recombinants [178;196]. Similar trends have been observed in Kenya, Tanzania, and South America (e.g. Brazil and Argentina) [74;105;136;184;219]. This rapid insurgence of subtype C may be due to a founder effect or to intersubtype fitness differences.
Any difference in ex vivo fitness likely reflects genotypic or phenotypic variations between subtypes. For example, most subtype C isolates appear to have an extra or third NFkB element in the LTR as compared to the two sites found in most subtype, which augment transcription in the presence or absence of HIV-1 Tat protein [107;196;218]. A subsequent study on HIV-1 protease activity showed increased cleavage of peptide substrates by HIV-1 subtype C versus subtype B protease [220]. This phenotypic data suggests that dominance of subtype C in the HIV-1 epidemic could be due to increased replicative capacity of subtype C isolates over other HIV-1 subtype isolates. In addition to these phenotypic differences, subtype C isolates rarely switch from a NSI/R5 phenotype to an SI/CXCR4 phenotype during late disease [29]. However, a recent study has compared the fitness of nine subtype B and six subtype C HIV-1 isolates in a pair-wise competition experiment in PBMC culture [4]. The relative fitness values were not statistically different in pair-wise competitions involving isolates of the same subtype (intrasubtype B or C competitions). In contrast, almost all of the subtype C isolates were out-competed by the subtype B isolates in the pair-wise competitions. In contrast to previous reports with recombinant HIV-1 clones containing fragments of the subtype C genome, this data suggests that subtype C isolates are likely less fit than subtype B isolates [4]. The poor relative fitness of subtype C isolates can also fit into model for subtype C dominance in the epidemic. Decreased fitness is linked to slower disease progression, which could result in increased in transmission time [188].
Is the fitness and pathogenicity of HIV-1 decreasing?
It is apparent that some simian immunodeficiency viruses (SIV) have become highly adapted to their host species over an extended period of time, resulting in asymptomatic, non-pathogenic infections [227]. Based on the subtype C fitness results, is it possible that HIV-1 is evolving to an attenuated state? This hypothesis was proposed by Temin more than ten years ago [213]. Rapid attenuation of virus was first observed in the myxoma virus infection of Australian rabbits [78]. Several introductions of this highly lethal virus resulted in a decrease of nearly half of the rabbit population followed by rapid evolution of a less virulent (fit) virus [81]. When a virus "jumps" and infects a new species, an increase in the initial virulence can only be supported in minor pandemics (e.g. Ebola pandemics). It is evident that to survive, viruses must continue to propagate in a living host and that a low/attenuated level of pathogenesis represents a tradeoff between virulence and transmissibility. Ewald [78] suggested that more virulent viruses would rapidly kill their hosts reducing the time for transmission and permitting the expansion of less virulent viruses that were still capable of establishing new infections. In the case of HIV-1, the continual spread of this lethal virus is a consequence of long asymptomatic but transmissible periods following initial infection. Individuals infected with a more pathogenic (high replication) strain will progress faster to HIV-1 disease, decreasing the probability of viral transmission. In contrast, attenuated HIV-1 strains (i.e. lower replicative capacity) would in theory delay disease progression and increase the likelihood of transmission. Based on this theory, changes in HIV-1 pathogenesis in the population may not be easily identified by common correlates of disease progression (e.g. viral load and CD4 cell counts). For example, the nonpathogenic infection of Sooty mangabeys monkeys with SIV [193] results in extremely high viral load while maintaining transmissibility in the absence of symptomatic disease. Thus, the higher viral loads observed in patients infected with subtypes B or A as compared to subtypes C or D (respectively) may be unrelated to the effect of subtype on disease progression.
Acknowledgments
We would like to thanks Drs. Esteban Domingo, Daniel R. Kuritzkes, and Richard D'Aquila for their critical reading and valuable comments to this review. M.E.Q-M was supported by research grants from the National Heart, Lung, and Blood Institute, NIH (5-KO1-HL67610-02) and the NIH Center for AIDS Research (AI36219) at Case Western Reserve University. E.J.A. was supported by research grants from the National Institute of Child Health and Development, NIH (NO1-HD-0-3310-502-02) and from the National Institute of Allergy and Infectious Diseases, NIH (AI49170)
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