Functional constraints of influenza A virus epitopes limit escape from cytotoxic T lymphocytes

Abstract

Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in CTL epitopes. Also for influenza A viruses a number of amino acid substitutions in the nucleoprotein (NP) have been associated with escape from CTL. However, other previously identified influenza A virus CTL epitopes are highly conserved, including the immunodominant HLA-A*0201-restricted epitope from the matrix protein, M1(58-66). We hypothesized that functional constraints were responsible for the conserved nature of influenza A virus CTL epitopes, limiting escape from CTL. To assess the impact of amino acid substitutions in conserved epitopes on viral fitness and recognition by specific CTL, we performed a mutational analysis of CTL epitopes. Both alanine replacements and more conservative substitutions were introduced at various positions of different influenza A virus CTL epitopes. Alanine replacements for each of the nine amino acids of the M1(58-66) epitope were tolerated to various extents, except for the anchor residue at the second position. Substitution of anchor residues in other influenza A virus CTL epitopes also affected viral fitness. Viable mutant viruses were used in CTL recognition experiments. The results are discussed in the light of the possibility of influenza viruses to escape from specific CTL. It was speculated that functional constraints limit variation in certain epitopes, especially at anchor residues, explaining the conserved nature of these epitopes.

FIG. 1.

Effect of amino acid substitutions in the M1_58-66 epitope on viral fitness. Upon transfection of 293T cells and subsequent rescue in MDCK cells, infectious virus titers were determined for the wild-type (WT) and mutant influenza viruses with alanine replacements for each of the nine amino acids of the M1_58-66 epitope (A) and with the more conservative substitutions at position 59 (C). Influenza virus could be rescued with alanine replacements at all positions within the M1_58-66 epitope, except for position 59 (A). Influenza A virus tolerated the more conservative substitutions M1 I59L and I59V to a certain extent (C). The data represent the average of three experiments. Subsequently, growth curves were generated (B and D) postinfection (p.i.) of MDCK cells at an MOI of 0.001. Virus replication kinetics for wild-type virus (•) and M1 G58A (▴), L60A (▪), G61A (▾), F62A (⧫), V63A (▵), F64A (□), T65A (▿), L66A (⋄) (B), and wild-type virus (•), I59L (▴), and I59V (▪) (D) are shown. The data represent the average of three experiments. The error bars indicate the standard deviation. An asterisk indicates statistical significance (P < 0.05, Student's t test).

FIG. 2.

Effect of amino acid substitutions in the NP_418-426 epitope on viral fitness. Upon transfection of 293T cells and subsequent rescue in MDCK cells, infectious virus titers were determined for the wild-type and mutant influenza viruses with alanine replacements at position 419 or 426 or with the more conservative substitutions NP P419G and M426I (A). The data represent the average of three experiments. Subsequently, growth curves of wild-type (WT) virus (•) and influenza virus A/NL/95-NP M426I (▴) were generated postinfection (p.i.) of MDCK cells at an MOI of 0.001 (B). The data represent the average of three experiments. The error bars indicate the standard deviation. An asterisk indicates statistical significance (P < 0.05, Student's t test).

FIG. 3.

Effect of amino acid substitutions on viral fitness. Upon transfection of 293T cells and subsequent rescue in MDCK cells, infectious virus titers were determined for the wild-type (WT) and mutant influenza viruses with the conservative amino acid substitution PB1 D593N, NP E46Q, or NP R175K in epitopes PB1_591-599, NP_44-52, and NP_174-184, respectively (A). The data represent the average of three experiments. Subsequently, growth curves of wild-type virus (•) and mutant viruses A/NL/95-NP E46Q (▴) and R175K (▪) were generated postinfection (p.i.) of MDCK cells at an MOI of 0.001 (B). The data represent the average of three experiments. The error bars indicate the standard deviation. An asterisk indicates statistical significance between the wild type and mutant influenza virus A/NL/95-NP E46Q at 6 and 12 h postinfection and between the wild type and influenza virus A/NL/95-NP R175K at 48 and 72 h postinfection (P < 0.05, Student's t test).

FIG. 4.

Effect of alanine replacements in the M1_58-66 epitope on recognition by specific CTL. Reactivity of CTL clone, directed against the M1_58-66 epitope, with stimulator cells infected with wild type (WT) (B) or influenza virus A/NL/95-M1 G58A (C), L60A (D), G61A (E), F62A (F), V63A (G), T65A (H), or L66A (I) was determined by intracellular IFN-γ staining and flow cytometry. PE, phycoerythrin conjugate; FITC, fluorescein isothiocyanate. GILGFVFTL-peptide pulsed cells were included as a positive control (J). Untreated cells were used as a negative control (A). Indicated is the percentage IFN-γ-positive cells within the CD8⁺-T-cell population. The data are also presented as the number of IFN-γ-positive spots, as measured in an IFN-γ-specific ELISPOT assay (K). In addition, the percentage of specific lysis, as measured in chromium release assays, is shown (M). Effector cells were added at an effector/target cell ratio of 10, and specific lysis was calculated. A CTL clone specific for the NP_418-426 epitope was used as a control (L, N). The recognition of A/NL/95-M1 I59A and F64A could not be tested, since these mutant viruses could not be propagated to sufficiently high titers (*). Data from representative experiments are shown.

FIG. 5.

Effect of selected amino acid substitutions in the M1_58-66 epitope on recognition by specific CTL. Reactivity of the CTL clone, directed against the M1_58-66 epitope, with stimulator cells infected with the wild type (WT) (B) or influenza virus A/NL/95-M1 I59L (C) or I59V (D) was determined by intracellular IFN-γ staining and flow cytometry. PE, phycoerythrin conjugate; FITC, fluorescein isothiocyanate. GILGFVFTL-peptide-pulsed cells were included as a positive control (E). Untreated cells were used as a negative control (A). Indicated is the percentage IFN-γ⁺ cells within the CD8⁺-T-cell population. The data are also presented as the number of IFN-γ-positive spots, as measured in an IFN-γ-specific ELISPOT assay (F). In addition, the percentage of specific lysis, as measured in chromium release assays, is shown (H). Effector cells were added at different effector/target cell ratios as indicated, and specific lysis was calculated. A CTL clone specific for the NP_418-426 epitope was used as a control (G, I). Data from representative experiments are shown.

FIG. 6.

Amino acid substitution NP M426I affects recognition by specific CTL. Reactivity of the CTL clone, directed against the NP_418-426 epitope, with stimulator cells infected with the wild type (B) or influenza virus A/NL/95-NP M426I (C) was determined by intracellular IFN-γ staining and flow cytometry. PE, phycoerythrin conjugate; FITC, fluorescein isothiocyanate. LPFEKSTVM-peptide-pulsed cells were included as a positive control (D). Untreated cells were used as a negative control (A). Indicated is the percentage of IFN-γ-positive cells within the CD8⁺-T-cell population. The data are also presented as the number of IFN-γ-positive spots, as measured in an IFN-γ-specific ELISPOT (F). In addition, the percentage of specific lysis, as measured in chromium release assays, is shown (H). Effector cells were added at different effector/target cell ratios as indicated, and specific lysis was calculated. A CTL clone specific for the M1_58-66 epitope was used as a control (E, G). The recognition of A/NL/95-NP P419A, M426A, and P419G could not be tested, since these mutations prevented rescue of viable virus. Data from representative experiments are shown.