Fitness cost of escape mutations in p24 Gag in association with control of human immunodeficiency virus type 1

Abstract

Mutational escape by human immunodeficiency virus (HIV) from cytotoxic T-lymphocyte (CTL) recognition is a major challenge for vaccine design. However, recent studies suggest that CTL escape may carry a sufficient cost to viral replicative capacity to facilitate subsequent immune control of a now attenuated virus. In order to examine how limitations can be imposed on viral escape, the epitope TSTLQEQIGW (TW10 [Gag residues 240 to 249]), presented by two HLA alleles associated with effective control of HIV, HLA-B*57 and -B*5801, was investigated. The in vitro experiments described here demonstrate that the dominant TW10 escape mutation, T242N, reduces viral replicative capacity. Structural analysis reveals that T242 plays a critical role in defining the start point and in stabilizing helix 6 within p24 Gag, ensuring that escape occurs at a significant cost. A very similar role is played by Thr-180, which is also an escape residue, but within a second p24 Gag epitope associated with immune control. Analysis of HIV type 1 gag in 206 B*57/5801-positive subjects reveals three principle alternative TW10-associated variants, and each is strongly linked to concomitant additional variants within p24 Gag, suggesting that functional constraints operate against their occurrence alone. The extreme conservation of p24 Gag and the predictable nature of escape variation resulting from these tight functional constraints indicate that p24 Gag may be a critical immunogen in vaccine design and suggest novel vaccination strategies to limit viral escape options from such epitopes.

FIG. 1.

Fitness cost resulting from T242N mutation. (A) Competition experiments in MT4 cells between HIV-1_NL4-3 and its site-directed T242N mutant. (B) Competition experiments in MT4 cells between p24 recombinant viruses from an HLA-B*57-negative child (SMH-05-Child) who was vertically infected by his HLA-B*57-positive mother. The only difference between both viral recombinants is the presence of Thr or Asn at Gag residue 242. (C) Infectivities of individual viral isolates, as assessed with Ghost reporter cells. The rate of infectivity is expressed as the percentage of GFP-positive cells at 24 h postinfection. The results shown represent the means ± standard deviations from two experiments.

FIG. 2.

Structural basis for fitness cost of T242N mutation. (a) Superimposition of capsid N-terminal domain structures. Gray rope, immature capsid structure (6, 31); purple rope, mature capsid structure (6, 31); green ribbon, CypA-bound structure (6, 14); cyan ribbon, CypA. Gray arrows indicate the observed conformational movements in the N-terminal hairpin, the CypA binding loop, and helix 6. Residue T242 is shown in magenta and drawn in stick representation. (b) Close-up of the TW10 epitope (magenta). The T(242)XXE(245) N-terminal cap for helix 6 is shown, with hydrogen bonds drawn as yellow dashes. Side chains (6) are drawn in stick representation. Hydrogen bonds between Trp-249, Glu-245, and the base of the CypA loop are shown together with Gly-248. (c) Ribbon representation as in panels a and b, with the mature capsid structure (6) shown as an orange rope with side chains depicted as sticks. The residues conferring escape are shown (T242, G248, N252, and M250). Note the close proximity of T242 to T180 (corresponding to T182 in SIV). T180 also forms an N-capping box; in this case, TXXD(183) stabilizes helix 3 and D183 also stabilizes the N-terminal hairpin. Residues I256 and M228, at which compensatory mutations occur, are also shown as sticks.

FIG. 3.

Polymorphisms within the Gag p24 N-terminal domain associated with TW10-linked mutations at Gag residues 242, 248, 250, and 252. * For H219X, X = Q in 71 cases, P in 2, and R in 1; for A248X, X = 20T, 5G, 3Q, 3N, 4D, and 1I. † Data for 2 by 2 tables is represented as [47/106/1/52], where 47 subjects had T242N and H219X, 106 subjects had T242N and H219, 1 subject had T242 and H219X, and 52 subjects had T242 and H219.