Frequent and variable cytotoxic-T-lymphocyte escape-associated fitness costs in the human immunodeficiency virus type 1 subtype B Gag proteins

Abstract

Cytotoxic-T-lymphocyte (CTL) escape mutations undermine the durability of effective human immunodeficiency virus type 1 (HIV-1)-specific CD8(+) T cell responses. The rate of CTL escape from a given response is largely governed by the net of all escape-associated viral fitness costs and benefits. The observation that CTL escape mutations can carry an associated fitness cost in terms of reduced virus replication capacity (RC) suggests a fitness cost-benefit trade-off that could delay CTL escape and thereby prolong CD8 response effectiveness. However, our understanding of this potential fitness trade-off is limited by the small number of CTL escape mutations for which a fitness cost has been quantified. Here, we quantified the fitness cost of the 29 most common HIV-1B Gag CTL escape mutations using an in vitro RC assay. The majority (20/29) of mutations reduced RC by more than the benchmark M184V antiretroviral drug resistance mutation, with impacts ranging from 8% to 69%. Notably, the reduction in RC was significantly greater for CTL escape mutations associated with protective HLA class I alleles than for those associated with nonprotective alleles. To speed the future evaluation of CTL escape costs, we also developed an in silico approach for inferring the relative impact of a mutation on RC based on its computed impact on protein thermodynamic stability. These data illustrate that the magnitude of CTL escape-associated fitness costs, and thus the barrier to CTL escape, varies widely even in the conserved Gag proteins and suggest that differential escape costs may contribute to the relative efficacy of CD8 responses.

Fig 1

The impact on replication capacity (RC) of CTL escape mutations in HIV-1B Gag. The mean replication capacity (RC; dots) with 95% confidence interval (CI; whiskers) is shown for the benchmark RT M184V mutation (A), the 12 CTL escape mutations associated with a statistically significant reduction in RC (P ≤ 0.05) (B), the 8 escape mutations associated with reduction in RC that exceeds that caused by the benchmark M184V mutation (C), the 6 escape mutations that had minimal impact on RC and the 1 that increased it (D), the 3 CTL negatope escape mutations (E), and the A163G mutation in the absence and presence of the S165N compensatory mutation (F). For all mutations, the susceptible amino acid residue is listed first and the escape residue is listed last. The null hypothesis that RC_escape = RC_susceptible is rejected at P < 0.05 if the 95% CI of RC_escape does not overlap RC_susceptible (horizontal dashed line), which is equal to 1.0 by definition. The mutations that withstand Bonferroni correction for multiple comparisons (P ≤ 0.002) are denoted by an asterisk.

Fig 2

CTL escape in HIV-1 Gag epitopes restricted by protective HLA class I alleles is associated with a greater reduction in replication capacity. The mean RCs of the 29 CTL escape mutations are shown grouped according to whether the associated HLA class I allele is enriched among HIV controllers (protective) or not (nonprotective). The horizontal bars denote the mean RC of mutations in epitopes restricted by protective alleles (0.80) and nonprotective alleles (0.98). The data are normally distributed (P = 0.445, Shapiro-Wilk test), and the difference in mean RC between the two groups is statistically significant (P = 0.02, Student's t test).

Fig 3

The impact on replication capacity (RC) of CTL escape mutation in HIV-1 Gag is not correlated with position or genetic conservation (Shannon entropy). (A) The mean replication capacity (RC; dots) with 95% confidence interval (CI; whiskers) is shown for the benchmark RT M184V mutation and for the 29 individual HIV-1B Gag CTL escape mutations in order of their linear position in the Gag polyprotein. For all mutations, the susceptible residue is listed first and the escape residue is listed last. The horizontal dashed line denotes RC_susceptible, which is equal to 1.0 by definition. (B) The CTL escape mutations are plotted from greatest to least reduction in replication (lowest to highest RC value) (white bars) opposite the Shannon entropy of the mutant position (gray bars). (C and D) The lack of correlation between Shannon entropy and RC is shown for all mutations tested (r = 0.16, P = 0.39; Pearson product moment test) (C) and for only those mutations that caused a statistically significant impact on RC (r = 0.27, P = 0.37; Pearson product moment test) (D).

Fig 4

Structural locations of the CTL escape mutations in HIV-1 Gag p17 and p24. The CTL escape mutations mapped onto two rotations of the ribbon representation of the three-dimensional monomer structures of p17 (PDB: 1HIW) (A) and p24 (PDB: 3GV2) (B). The locations of CTL escape mutations are denoted by the consensus amino acid and the Gag amino acid position. Mutations that caused a statistically significant impact on virus replication are denoted by an asterisk and by red highlighting of the ribbon. All other mutations are denoted by blue highlighting.

Fig 5

In silico estimation of the impact on protein stability of CTL escape mutations in HIV-1B Gag p17 and p24. (A) Protein stability is defined by the difference in Gibbs free energy (ΔG) between the unfolded and folded states. The FOLDEF energy function (51) was used to estimate the ΔG for the “wild-type” (WT) protein sequence and for each CTL escape mutant sequence (MUT), and the stability impact of a mutation was quantified as the absolute value of the difference between WT ΔG and MUT ΔG (|ΔΔG|). (B) The impact on protein stability (|ΔΔG|) was estimated for the 21 classical CTL escape mutations located within the Gag p17 trimer and p24 pentamer and hexamer crystal structures. (C) The CTL escape-associated impact on protein stability (|ΔΔG|) and reduction in replication capacity (RC) were negatively correlated when the outlier p24 R264K mutation was excluded from the analysis (rho = −0.46, P = 0.02; Spearman rank correlation). CTL escape mutations in Gag p17 and p24 are denoted by circles and triangles, respectively.