In the present paper, the effect of amino acid substitutions in CTL epitopes on viral fitness and T-cell recognition was evaluated. It was concluded that functional constraints imposed on CTL epitopes limit escape from virus-specific CTL without loss of viral fitness.
The synonymous/nonsynonymous analysis revealed that in the 90 amino acids that constitute the 14 known epitopes located in the NP, relatively more nonsynonymous mutations occurred between 1969 and 2003 than in the rest of the protein. The hypervariable epitope NP418-426
had a major impact on the lower ds/dn ratio, and 5 out of the 14 partially overlapping epitopes were fully conserved. Some points in this analysis should be taken into consideration. First, since commonly old prototypic strains like A/Puerto Rico/8/34 have been used for the identification of influenza virus CTL epitopes, there is a bias towards the identification of conserved epitopes (12
). Recent work in our laboratory indicates that a significant number of epitopes are not conserved (Berkhoff et al., unpublished data). Second, the conserved epitopes and the variable epitopes, including the NP418-426
epitope, have in common that they all retained their anchor residues for binding to their corresponding HLA molecules. The only exception to this is an amino acid substitution at position 384 of the NP. The R384G substitution, which is at the anchor residues of the HLA-B*0801- and -B*2705-restricted epitopes NP380-388
, resulted in the loss of their epitopes and abrogated recognition of virus-infected cells by specific CTL (51
). However, introduction of a glycine at position 384 of the NP of influenza virus A/Hong Kong/2/68 was detrimental to viral fitness, and several comutations associated with the R384G substitution in epidemic influenza virus strains were required to functionally compensate for the detrimental effect of the R384G substitution (50
). Similar findings have been observed for CTL escape mutants of HIV and simian immunodeficiency virus (SIV), which also accumulated extraepitopic comutations in the gag protein for restoration of viral fitness in the presence of mutations in CTL epitopes (17
). Apparently, RNA viruses display sufficient flexibility to escape from CTL and retain viral fitness. For HIV and SIV, the selective pressure is mediated by CTL during the chronic infection of individual hosts, while for influenza viruses this takes place by CTL immunity at the population level (19
). It is of special interest that also for HIV, CTL escape mutants can be identified at the population level (45
), although transmission rates of this virus are much lower than those for influenza viruses. Thus, influenza virus CTL epitopes are either conserved, display variation at non-anchor residues, or lose their anchor residues at the cost of viral fitness, which is functionally compensated for by the accumulation of comutations. To assess the impact of amino acid substitutions in conserved epitopes on viral fitness and recognition by specific CTL, we conducted a mutational analysis of the epitope M158-66
(GILGFVFTL). This epitope is immunodominant and recognized by a large portion of individuals in the population, but is highly conserved. Replacement of the anchor residue at position 2 of the epitope (M1 I59A) was detrimental to viral fitness, whereas alanine replacements at the other eight positions did not prevent rescue of recombinant influenza virus and were tolerated to various extents. The M158-66
epitope is located in the fourth N-terminal α-helix of the M1 protein. Mutations in this region may disturb the functional and structural integrity of the protein, as has been described for mutations in the M1 “helix six” domain (8
). The reduced virus titers obtained with a number of these mutant M1 viruses correlated with the number of productively infected cells, as measured by immunofluorescence assay using an NP-specific monoclonal antibody 6 h postinfection of MDCK cells, suggesting that the virus replication cycle was affected at an early pretranscriptional stage (data not shown). Conservative amino acid substitutions at position 2 of the M158-66
epitope (M1 I59L and I59V) were less critical, although the kinetics of viral replication was somewhat affected. More importantly, the A/NL/95-M1 I59L and I59V mutant viruses were fully recognized by M158-66
specific CTL, which makes it unlikely that these variants would ever emerge in the human population. Although some of the other alanine replacements resulted in the partial loss of recognition by M158-66
-specific CTL, their impaired replication kinetics is not in favor of the emergence of these mutants. We speculate that there must be a trade-off between viral fitness and immune recognition of which we have little insight at present. The T-cell recognition patterns that were observed here with mutant virus-infected cells were in agreement with those observed with mutant M158-66
-peptides in previous studies (1
). Although the use of T-cell clones may not reflect the situation in vivo, the analysis of anchor residues boils down to recognition of the epitope or not, which is not different between clonal and polyclonal T-cell populations. In the analysis of T-cell receptor contact residues, as done for the M158-66
epitope, the situation is more complicated. However, the M158-66
-specific CTL response is oligoclonal in nature and dominated by T cells carrying the T-cell receptor with Vβ 17 chains (29
). Fitness costs also limit variation in the highly immunodominant Gag p11C, C-M CTL epitope of SIV and escape from specific CTL (43
). Therefore, this phenomenon may be more universal and apply to more RNA viruses, which are under selective pressure mediated by CTL. It even may contribute to shaping of the T-cell repertoire and have an influence on the hierarchy of epitope dominance.
Next, we wished to evaluate the conservative anchor residues of the otherwise hypervariable epitope NP418-426
(LPFEKSTVM). The relatively conservative NP P419A and P419G substitutions at position 2 of the epitope were both detrimental to viral fitness, indicating that the proline at this position is essential. Amino acid substitutions at position 9 of the epitope, the second anchor residue, yielded interesting results. First, the NP M426A substitution was detrimental to viral fitness. Second, with the conservative NP M426I substitution, the HLA-B*3501 binding motif was retained (16
) and viral fitness was not affected to a great extent. Of special interest, HLA-B*3501-positive cells infected with influenza virus A/NL/95-NP M426I were poorly recognized by NP418-426
-specific T-cell clones. Since the NP M426I mutant epitope retained its capacity to bind to HLA-B*3501, it may have undergone conformational changes in T-cell receptor contact residues, preventing recognition by CTL, as has been described previously for another HLA-B*3501-restricted epitope (15
). Conservative amino acid substitutions at the anchor residues of the epitopes PB1591-599
(CTELKLSDY), and NP174-184
(RRSGAAGAAVK) also affected viral fitness. The PB1 D593N substitution in particular was detrimental to viral fitness. Although the conservative NP E46Q substitution resulted in the loss of the anchor residue and would allow the virus to escape from specific CTL, the loss of viral fitness may limit the emergence of this variant in the human population.
Based on the data presented here, we speculate that influenza A viruses display a limited degree of variation in CTL epitopes despite selective pressure on these epitopes mediated by CTL. Functional constraints imposed on influenza virus CTL epitopes may limit efficient escape from CTL and could constitute the Achilles heel of these viruses, limiting the impact of epidemic and pandemic outbreaks of influenza on severe morbidity and mortality.