Extraepitopic compensatory substitutions partially restore fitness to simian immunodeficiency virus variants that escape from an immunodominant cytotoxic-T-lymphocyte response

Abstract

Selection for escape mutant immunodeficiency viruses by cytotoxic T lymphocytes (CTL) has been well characterized and may be associated with disease progression. CTL epitopes accrue escape mutations at different rates in vivo. Interestingly, certain high-frequency CTL do not select for escape until the chronic phase of infection. Here we show that mutations conferring escape from immunodominant CTL directed against an epitope in the viral Gag protein are strongly associated with extraepitopic mutations in gag in vivo. The extraepitopic mutations partially restore in vitro replicative fitness of viruses bearing the escape mutations. Constraints on epitope sequences may therefore play a role in determining the rate of escape from CTL responses in vivo.

FIG. 1.

Extraepitopic amino acid substitutions associated with escape from Gag CM9-specific CTL. vRNA was isolated from Mamu-A*01-positive macaques in the chronic phase of infection with SIVmac239. vRNA was reverse transcribed, and cDNA was amplified and directly sequenced. Predicted amino acid sequences are shown for a region of the open reading frame encoding Gag. Amino acid residue numbers are indicated above the reference sequence. The Gag CM9 epitope sequence is boxed in yellow. Lowercase letters indicate the amino acid encoded by the variant codon at sites of mixed-base nucleotide heterogeneity.

FIG. 2.

Ontogeny of Gag CM9 escape and associated substitutions. Predicted amino acid sequences are shown for the region surrounding the Gag CM9 epitope in five Mamu-A*01-positive animals infected with SIVmac239. Plasma virus RNA was isolated, reverse transcribed, and directly sequenced as described for Fig. 1. Lowercase letters indicate the amino acid encoded by the variant codon at sites of mixed-base nucleotide heterogeneity.

FIG. 3.

Direct competition between wild-type SIV and variants reveals a fitness cost associated with escape from Gag CM9-specific CTL. Primary macaque lymphocytes were infected with mixtures of wild-type and epitope mutant SIV (5 ng of p27 for each virus). After the mixtures were cultured for 7 days, we quantified total vRNA and mutant vRNA in the culture supernatants by using QRT-PCR. The proportion of total viral sequences that were mutant could therefore be calculated for each sample (orange bars). The values shown are the averages of the results of duplicate independent experiments. Each error bar indicates the standard deviation from the average for each data point.