Escape of HIV-1 from a small molecule CCR5 inhibitor is not associated with a fitness loss

Abstract

Fitness is a parameter used to quantify how well an organism adapts to its environment; in the present study, fitness is a measure of how well strains of human immunodeficiency virus type 1 (HIV-1) replicate in tissue culture. When HIV-1 develops resistance in vitro or in vivo to antiretroviral drugs such as reverse transcriptase or protease inhibitors, its fitness is often impaired. Here, we have investigated whether the development of resistance in vitro to a small molecule CCR5 inhibitor, AD101, has an associated fitness cost. To do this, we developed a growth-competition assay involving dual infections with molecularly cloned viruses that are essentially isogenic outside the env genes under study. Real-time TaqMan quantitative PCR (QPCR) was used to quantify each competing virus individually via probes specific to different, phenotypically silent target sequences engineered within their vif genes. Head-to-head competition assays of env clones derived from the AD101 escape mutant isolate, the inhibitor-sensitive parental virus, and a passage control virus showed that AD101 resistance was not associated with a fitness loss. This observation is consistent with the retention of the resistant phenotype when the escape mutant was cultured for a total of 20 passages in the absence of the selecting compound. Amino acid substitutions in the V3 region of gp120 that confer complete AD101 resistance cause a fitness loss when introduced into an AD101-sensitive, parental clone; however, in the resistant isolate, changes elsewhere in env that occurred prior to the substitutions within V3 appear to compensate for the adverse effect of the V3 changes on replicative capacity. These in vitro studies may have implications for the development and management of resistance to other CCR5 inhibitors that are being evaluated clinically for the treatment of HIV-1 infection.

Figure 1. Replication Kinetics of Clonal, Env-Chimeric Viruses with an AD101-Sensitive or an AD101-Resistant env Gene

The amount of p24 produced from PBMC culture infected with equal titers of CC1/85 cl.7 (squares) or CC101.19 cl.7 (circles), in the absence (filled symbols) or presence (open symbols) of 1 μM AD101, was determined at the indicated time points. Duplicate wells were used to derive each experimental value in each experiment, and the data shown are the average ± standard error of the mean (SEM) of values from five independent experiments.

Figure 2. The Use of the TaqMan QPCR Assay to Determine Replicative Fitness

(A) The sequences (3′ to 5′) of the vifX and vifY TaqMan probes are shown on top, the sequences of the vifX and vifY vif alleles are shown below. Each probe differs from its target sequence by one nucleotide because the vifX probe was designed to match the consensus for HIV-1 subtype B vif in this region. Each probe binds to the sense strand of proviral DNA. The probes are aligned to the target sequences with bars indicating positions of identity. The two probes and the two vif alleles differ at 11 nucleotides each. The vifY vif allele that was engineered to tag the reference viruses contains only synonymous changes. (B) The plot demonstrates the working range and reproducibility of the QPCR assay. The average threshold cycle (C_T) values obtained in six representative multiplexed assays are shown ± standard deviation (SD). In each assay, seven serial dilutions of the standard plasmid ranging from 5 × 10⁷ to 5 × 10¹ DNA templates were measured using TaqMan probes specific either for the vifX (Cy5 fluorescence, top panel), or the vifY sequence (FAM fluorescence, bottom panel). The correlation coefficients (R ²) of the two standard curves were >0.995 and the PCR efficiencies were >90%. (C) The specificity of the QPCR assay is depicted. Four independent PBMC cultures were singly infected with one of the CC1/85 cl.7 or the CC101.19 cl.7 viruses containing either the vifX or the vifY vif sequence. Genomic DNA from each culture was PCR amplified and then used in the multiplexed TaqMan QPCR assay, as described in Materials and Methods. The average number of copies of each vif allele detected per QPCR reaction for each of the four mono-infections from six representative experiments is shown ± SEM. The lower limit for copy number is set to 100 for plotting purposes, although 50 copies can be quantified reproducibly, and the allele that was not present in the infection was never detected in any quantifiable amount in singly infected cultures. (D) The vif tag has no effect on replicative fitness. Competitive replication assays were performed in which viruses bearing the NL4–3 (circles), CC1/85 cl.7 (squares), or CC101.19 cl.7 (triangles) env genes and the vifX vif allele were competed against viruses bearing the same env genes but the vifY sequence. The fitness differences (W_D) at each indicated MOI were calculated as described in Materials and Methods. The calculated fitness differences of each vifX virus relative to the vifY virus in each experiment are depicted, with the bar showing the mean value of three or four independent experiments. A fitness difference of 1 arises when the two competing viruses are of equal replicative fitness; a value < 1 indicates that the virus with vifY had the greater replicative fitness, > 1 that the virus with vifX was fitter.

Figure 3. Replication of Clonal, Env-Chimeric Viruses in Singly Infected Cultures

The extent of replication is shown as the mean copy number per QPCR reaction ± SEM from three to 47 independent, singly infected cultures. AD101 was absent (shaded bars) or was present at 1 μM (open bars). The initial MOI was 0.0005.

Figure 4. Replicative Fitness of the Clonal Env-Chimeric Viruses Relative to the CC1/85 cl.7 and CC101.19 cl.7 Reference Viruses

(A and B) The replicative fitness of each vifX clonal virus relative to the indicated vifY reference virus was calculated from the mean virus proportions from three to seven independent competition experiments, and is shown for MOIs of (A) 0.0001 and (B) 0.0005. Fitness differences < 1 indicate the vifX virus is less fit than the vifY reference virus, > 1 that the vifX virus is the more fit. The vifX viruses used were: CC1/85 cl.6 (open squares), CC1/85 cl.7 (open triangles), CC1/85 cl.8 (open diamonds), CC101.19 cl.3 (filled squares), CC101.19 cl.7 (filled triangles), CC101.19 cl.15 (filled diamonds), CCcon.20 cl.11 (open circles), CC1/85 cl.7 (HP) (X symbol), and CC1/85 cl.7 (V3) (filled circles). (C) The plotted fitness differences were averaged between reference viruses and MOIs from (A) and (B). Thus, the fitness differences shown are all relative to an arbitrary reference point (= 1.0). The three clones from the AD101-resistant CC101.19 isolate are shown in bold (C). (D) Independent fitness difference determinations generate reproducible results. The W_D values for CC1/85 cl.6 vifX relative to CC1/85 cl.7 vifY are plotted for three individual experiments at the MOIs indicated. This pairing was chosen because of the large difference in fitness between them. The W_D values were determined from the proportion of each virus within a single experiment. The bars represent the means of these W_D values (31 and 63 at MOIs of 0.0001 and 0.0005, respectively). The same data were analyzed in (A) and (B) by taking the average of the proportions between the three experiments before calculating the W_D, thereby minimizing the influence of outliers. The W_D values derived from (A) and (B) were 10 and 63 at MOIs of 0.0001 and 0.0005, respectively.

Figure 5. Replication and Replicative Fitness of the CC1/85 and CC101.19 Isolates

(A) PBMC cultures were singly infected with the indicated isolates at an MOI of 0.0005 in the presence (open bars) or absence (shaded bars) of 1 μM AD101. The vifX vif probe was used in the TaqMan QPCR assay to derive the copy number per QPCR reaction; the values shown are the means ± SEM from three independent experiments. (B) The fitness differences of the isolates CC1/85 (open symbols) or CC101.19 (filled symbols) as detected by the vifX probe are shown relative to the indicated vifY reference viruses at MOIs of 0.0001 (squares) or 0.0005 (triangles). The values are derived from the mean virus proportions from three independent experiments.

Figure 6. AD101 Sensitivity of AD101-Resistant Isolates after Extensive Passage in the Absence of AD101

PBMC cultures were infected with the parental isolate CC1/85 (open squares), the resistant isolate CC101.19 (filled squares), or isolates CC101.22R9 (filled diamonds), CC101.22R10 (filled triangles), CC101.22R15 (filled circles), and CC101.22R20 (X symbol) from the reversion cultures, in the presence of different concentrations of AD101. The amount of p24 produced in each AD101-containing culture after 7 d is shown as a percentage of that produced by the same isolate in the absence of inhibitor. The values shown are the means of four independent experiments ± SEM.

Figure 7. The Effect of AD101 on the Fitness of Clonal, Env-Chimeric AD101-Resistant Viruses

(A) Replicative fitness differences in PBMCs after 14 d of culture were determined at MOIs of 0.0001 (open squares) or 0.0005 (open triangles) for the vifX CC101.19 cl.7 virus relative to the vifY NL4–3 reference virus, in the presence of 20 nM AMD3100 and in the presence or absence of 20 μM AD101, as indicated. (B) Replicative fitness differences in PBMCs after 14 d of culture were determined at MOIs of 0.0001 (squares, circles) or 0.0005 (triangles, diamonds) for the vifX CC101.19 cl.3 virus (squares, triangles) or the vifX CC101.19 cl.15 virus (circles, diamonds), relative to the vifY CC101.19 cl.7 reference virus, and in the presence or absence of 20 μM AD101, as indicated. In both panels, the plotted values are derived from the mean virus proportions from three independent experiments.