Conservation of Cytotoxic T Lymphocyte (CTL) Epitopes as a Host Strategy to Constrain Parasite Adaptation: Evidence from the nef

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1 Conservation of Cytotoxic T Lymphocyte (CTL) Epitopes as a Host Strategy to Constrain Parasite Adaptation: Evidence from the nef Gene of Human Immunodeficiency Virus 1 (HIV-1) Jack da Silva and Austin L. Hughes Department of Biology and Institute of Molecular Evolutionary Genetics, Pennsylvania State University Host cytotoxic T lymphocytes (CTLs) that recognize specific viral peptides (epitopes) are thought to provide the most effective control of viral replication and spread. However, viruses may escape this recognition through mutations in CTL epitopes. We tested the hypothesis that, as an adaptation on the part of the host to constrain parasite escape from immune control, class I major histocompatibility complex (MHC) molecules present peptides that are derived from conserved regions of foreign proteins to CTLs. We did this by estimating the relative conservation of CTL epitopes of the functionally important Nef protein of human immunodeficiency virus 1 (HIV-1) and relating this to the structure and function of the protein. In comparisons among sequences from several HIV-1 subtypes and both major groups, CTL epitopes had lower rates of nonsynonymous nucleotide substitution per site than did the remainder of the protein, indicating the relative conservation of these epitopes. In contrast, helper T-cell epitopes were as conserved as, and monoclonal antibody epitopes less conserved than, the remainder of the protein. The conservation of CTL epitopes is apparently due to their derivation from functionally important domains of Nef, since CTL epitopes coincide with these domains and these domains are conserved relative to the remainder of the protein, in contrast to secondary structural elements, which are not. Recent studies provide evidence of CTL selection on HIV-1 epitopes, but the variational range of viral escape mutants appears to be limited by functional constraints on the protein regions from which epitopes are derived. The presentation of conserved foreign peptides to CTLs by class I MHC molecules may be a general adaptation of vertebrate hosts to constrain the adaptation of their intracellular parasites. Introduction Two components of the vertebrate immune system function in specific molecular recognition. T-cell receptors (TCRs), present on both cytotoxic T lymphocytes (CTLs) and helper T cells (Th), recognize short peptides (epitopes) derived from intracellularly processed foreign proteins that are bound and presented on the cell surface by major histocompatibility complex (MHC) class I (for CTL) and class II (for Th) molecules. The second component consists of immunoglobulins (antibodies), which recognize unprocessed extracellular foreign antigens. CTLs, upon recognizing a foreign epitope, lyse the infected cell, and CTLs specific for viral epitopes are thought to provide the most effective control of viral replication and spread. However, parasites may escape CTL recognition when mutations in epitope regions interfere with either the presentation of the peptide by MHC molecules or the recognition of the bound epitope by TCRs (e.g., McMichael and Phillips 1997). In this context, it is interesting that human leukocyte antigen (HLA) (i.e., human MHC) class I molecules present self peptides that are largely derived from highly conserved proteins and that the peptides are generally more conserved than the remainder of their source protein (Hughes and Hughes 1995). Furthermore, the peptides tend to be hydrophobic, whereas the source proteins tend to be hydrophilic as a whole. This suggests that in the processing of peptides for class I MHC presentation, Key words: HIV-1, nef gene, conserved CTL epitopes, MHC, HLA. Address for correspondence and reprints: Austin L. Hughes, Department of Biology, Pennsylvania State University, 208 Mueller Laboratory, University Park, Pennsylvania austin@hugaus3.bio.psu.edu. Mol. Biol. Evol. 15(10): by the Society for Molecular Biology and Evolution. ISSN: peptides are selected from hydrophobic regions of otherwise hydrophilic proteins, and because hydrophobic regions tend to be functionally important, the peptides presented tend to be conserved relative to the rest of the protein. This is hypothesized to be an adaptation on the part of the host to constrain the evolution of escape mutants in parasites (Hughes and Hughes 1995). In partial support of the Hughes and Hughes (1995) hypothesis, Seibert et al. (1995) found that four out of five CTL epitopes from proteins encoded by the gag, env, and pol genes of human immunodeficiency virus 1 (HIV-1) were more hydrophobic than was the remainder of the protein, and that three out of the five epitopes were significantly more conserved than was the remainder of their protein. Bauer et al. (1997) point out that three epitopes presented by B7 molecules of class I HLA coincide with structurally and functionally important sites on the Nef protein of HIV-1, and that this may constrain amino acid replacement in these epitopes and thus curtail escape from CTL recognition. Also, authors describing CTL epitopes typically remark that they are from conserved regions of proteins. This is not because searches for epitopes are restricted to conserved regions; C. Zhang et al. (1997) have shown that class I HLA peptide-binding motifs, indicating putative CTL epitopes, are less densely distributed over variable regions than over conserved regions of HIV-1 env and gag proteins. They also note that these motifs tend to coincide with structurally and functionally important regions of the proteins, and that this probably constrains the peptides from varying. C. Zhang et al. (1997) propose an alternative explanation for the conservation of CTL epitopes. They suggest that CTL recognition selects amino acid replacements in HIV that eliminate class I MHC-binding motifs 1259

2 1260 da Silva and Hughes of epitopes, and that these motifs are more easily eliminated from protein regions that are not functionally constrained, resulting in epitopes being derived mainly from conserved regions. The implication is that the intracellular processing of peptides for presentation by class I MHC molecules is not selective with respect to peptide conservation and, therefore, that the conservation of CTL epitopes is an adaptation of the virus rather than the host. However, this hypothesis is clearly not tenable, since human self peptides presented by class I HLA molecules are also conserved relative to the remainder of their source protein (Hughes and Hughes 1995), implicating selective intracellular processing of peptides by the host. We describe natural selection on well-defined CTL epitopes of the Nef protein of HIV-1 and relate our findings to the structure and function of the protein. These observations are contrasted with natural selection on putative Th and monoclonal antibody (MAb) epitopes of Nef. The nef gene has a central role in the pathogenesis of HIV-1 and simian immunodeficiency virus (SIV). A nonfunctional nef gene appears to severely reduce the in vivo infectivity and replication of the virus, and deletions in nef may be responsible for disease nonprogression in humans and other primates (reviewed by Collette 1997). From in vitro studies, the mechanisms of Nef functions appear to involve interactions with various cellular signal transduction proteins, including inducing endocytosis of cell surface CD4 receptor molecules and interacting with the src-homology domain 3 (SH3) of the protein tyrosine kinases Lck, Hck, and Lyn (reviewed by Greenway and McPhee 1997); the amino acid residues of Nef that are putatively involved in these interactions have been identified through mutational studies (Saksela, Cheng, and Baltimore 1995) and analysis of the protein s three-dimensional structure (Grzesiek et al. 1996; Lee et al. 1996; Arold et al. 1997). Another important function of Nef may be the induction of class I MHC endocytosis (Schwartz et al. 1996), which reduces the efficacy of CTL lysis of infected cells in culture (Collins et al. 1998) and may explain the reduction of cell surface class I HLA in vivo by HIV-1 (Puppo et al. 1997). As a consequence of the importance of nef to HIV pathogenesis, a large amount of information has accumulated on the gene s sequence variation and the protein s structure, function, and immunology. We used this rich database to investigate the conservation of CTL epitopes in relation to protein functional domains. Our approach to describing selection on epitopes was to compare rates of synonymous and nonsynonymous nucleotide substitutions per site, a powerful method of discriminating between positive Darwinian selection and neutral polymorphism (Hughes anei 1988, 1989). Positive selection favoring diversity at the amino acid level, such as overdominant selection or negative frequency-dependent selection, results in the rate of nonsynonymous substitutions exceeding that of synonymous substitutions, whereas purifying selection results in the rate of synonymous substitutions being higher, and the absence of selection results in equal rates of synonymous and nonsynonymous substitutions. The relative evolutionary conservation of different regions of a protein can be determined by comparing nonsynonymous substitution rates between regions, with more conserved regions exhibiting lower nonsynonymous substitution rates (e.g., Seibert et al. 1995). The effect of mutation independent of selection on protein variation can be analyzed by comparing synonymous substitution rates, since a synonymous substitution does not change the amino acid and, therefore, cannot be affected directly by selection on the protein. Materials and Methods Sequences Analyzed CTL, Th, and MAb epitopes (fig. 1) were collected from the HIV Molecular Immunology Database 1996 (Korber et al. 1996). CTL epitopes were used only if CTLs recognized the naturally processed epitope and both the optimal epitope and the restricting HLA molecule were defined; this subset of CTL epitopes is given in Brander and Walker (1996). To investigate the relationship between epitopes and the structure and function of the protein, data on structural and functional sites were also collected. The three-dimensional structures of the C-terminal core region (Lee et al. 1996; Arold et al. 1997; Grzesiek et al. 1997) and the N-terminal 25 residues (Barnham et al. 1997) of HIV-1 Nef were recently resolved. Secondary structural elements and other structural and functional sites of the protein were mapped onto its amino acid sequence (fig. 1). The protein is cleaved into an N-terminal cellular-membrane-anchoring tail (tail) and a core region (core) by viral protease between sites W57 and L58. The tail contains a myristylation site that functions in targeting the protein to cellular membranes (Yu and Felsted 1992), and an alpha helix (H1) that contains putative RNA-binding sites. The core is highly structured. Proceeding from its N-terminus, the core contains a type-ii polyproline helix (PPII), two long alpha helices (H2 and H3) (separated by a short beta strand [ 1]) that form a hydrophobic crevice between them, a beta sheet composed of four antiparallel beta strands ( 2 5), and, finally, two short helices (H4 and H5). A long, unstructured, exposed loop links the last two strands of the beta sheet ( 4 and 5). Some tertiary structure is imposed by contact between the protease cleavage site and the hydrophobic crevice. There are two main types of functional sites within the core: sites that bind the cytoplasmic tail of CD4, which overlap the cleavage site and the hydrophobic crevice, and sites that interact with the SH3 domain of cellular signal transduction proteins, which mostly overlap the polyproline helix and the two long alpha helices (H2 and H3). A site on the second long alpha helix (H3) is also involved in the protein s association with cellular serine/threonine kinase. Aligned DNA sequences for HIV-1 nef were collected from the Human Retroviruses and AIDS 1996 database (Myers et al. 1996). Seventy-six sequences, representing distinct isolates, were available, but 13 were excluded from analysis because they were incomplete or had in-frame stop codons, 2 (MAL and Z321)

3 Evolutionarily Conserved CTL Epitopes 1261 were excluded because their positions relative to established HIV-1 subtypes were not well supported in our phylogeny (possibly because they are hybrids), and the closely-related chimpanzee sequence CPZGAB was excluded in order to restrict the study to human sequences. The remaining 60 sequences are identified by common name and GenBank accession number in the phylogenetic tree (fig. 2) (see below). FIG. 1. Amino acid sequence of protein encoded by nef from the LAI isolate of HIV-1 (Wain-Hobson et al. 1985). Secondary structural elements (Lee et al. 1996; Arold et al. 1997; Barnham et al. 1997; Grzesiek et al. 1997) are underscored; straight lines indicate helices, and wavy lines indicate beta strands; PPII is a type II polyproline helix; the region between 4 and 5 is an unstructured loop. Symbols indicate other structural sites and putative functional sites: myristylation signal (Shugars et al. 1993) ( ), RNA-binding (Echarri, Gonzalez, and Carrasco 1997) ( ), HIV-1 protease cleavage site (Freund et al. 1994; Welker et al. 1996) ( ), cleavage site contact with hydrophobic pocket (Grzesiek et al. 1997) (*), binding to CD4 cytoplasmic tail (Grzesiek et al. 1996) ( ), interaction with SH3 domains (Saksela, Cheng, and Statistical Methods Since the form of selection detected may depend on the evolutionary distance between sequences, presumably because of selective constraints on nonsynonymous sites (Hughes anei 1988), we reconstructed a phylogeny of nef sequences and made comparisons at and between different levels of the phylogeny. A phylogenetic tree was reconstructed by the neighbor-joining method (Saito anei 1987) on the basis of the proportion of amino acid differences between sequences; amino acid sequences were used because synonymous nucleotide sites were saturated in comparisons between Group O sequences and other sequences (see fig. 2 and table 1). Alignment gaps and missing information in any sequence were excluded from all sequences so that a comparable data set was used in each comparison of a pair of sequences. The tree was rooted arbitrarily at the midpoint between the two farthest points, forming clusters that corresponded to established HIV-1 groups and subtypes (Leitner 1996). Support for nodes within the tree was determined by running 1,000 bootstrap replicates (Felsenstein 1985). Bootstrap support was high (100%) for groups (M and O) and low (61%) to high (100%) for subtypes (A, B, and D). The relationships among subtypes are consistent with results from analyses with other HIV-1 genes (Leitner 1996). Mean numbers of nucleotide substitutions per synonymous site (d S ) and per nonsynonymous site ( ) (Nei and Gojobori 1986) were estimated from all possible pairwise comparisons between sequences, and standard errors of d S and were estimated by Nei and Jin s (1989) method. To test for natural selection acting on the nef-encoded protein in the context of its interaction with the immune system, we estimated d S and for CTL, Th, and MAb epitopes separate from the remainder of the gene in each case. To investigate the relationship between selection on epitopes and the structure and function of the protein, we also estimated d S and for secondary structural elements and for combined tertiary structural sites and functional sites separate from the remainder of the gene in each case. Baltimore 1995; Lee et al. 1996; Arold et al. 1997) ( ), association with cellular serine/threonine kinase (Sawai et al. 1995) ( ), and cooperative in binding Hck SH3 domain (Saksela, Cheng, and Baltimore 1995) ( ). Epitopes are delineated by brackets (including site below each bracket). A, Helper T-cell epitopes (Th) (antigenic domain ant dom was used in analysis) and CTL epitopes (identified by restricting HLA allele). B, Monoclonal antibody epitopes.

4 1262 da Silva and Hughes FIG. 2. Phylogenetic tree of HIV-1 nef sequences based on proportion of amino acid differences (p). Bootstrap values (%) are shown for major sequence families. Sequences are identified by common name and GenBank accession number (in brackets). Classification follows established subtypes (Sub) and groups (Grp). Results There is little overlap between CTL and Th epitopes, with the latter spanning the protease cleavage site and the hydrophobic crevice, while CTL epitopes coincide mostly with other structural and functional sites (fig. 1A). Neither type of T-cell epitope is found on the long, unstructured, exposed loop. MAb epitopes are located over all regions of the folded protein predicted to be exposed, and are conspicuously absent from the predicted interior regions, which include the hydrophobic crevice and the three N-terminal strands ( 2 4) of the beta sheet (fig. 1B). d S and were estimated for each subtype and group as well as within and between nested clusters of subtypes and groups (e.g., subtypes B and D vs. subtype A). This was done because d S and increase at different rates and, therefore, their relationships depend on the amount of divergence between the sequences being compared. For instance, synonymous sites were saturated in all comparisons between group O and group M sequences, and the statistical significance of differences in comparisons of d S and varied with the family of sequences being analyzed (see tables 1 5). d S and were estimated separately for CTL epitopes combined and the remainder of the protein (table 1). In all comparisons between d S and within CTL epitopes and within the remainder of the protein, d S exceeded, and the difference was statistically significant in every comparison for epitopes and in 6 of 10 comparisons for the remainder of the protein. Comparisons

5 Evolutionarily Conserved CTL Epitopes 1263 Table 1 Mean Numbers of Synonymous (d S ) anonsynonymous ( ) Nucleotide Substitutions per 100 Sites ( SE) in Comparisons Between HIV-1 nef Sequences for Cytotoxic T-Lymphocyte Epitopes and Remainder of Gene EPITOPES VS. EPITOPES FAMILY d S d S d S Group M Subtype B... Subtype D... Subtype B vs. subtype D... Subtypes B D... Subtype A... Subtypes B D vs. subtype A.. Group O... Group M vs. O... Groups M O * * NOTE. Families are as in figure 2. Z-tests of hypotheses that d S for epitopes or the remainder, and that d S(epitopes) d S(remainder) or (epitopes) (remainder) : * P 0.05; P 0.01; P a Undefined Jukes-Cantor-corrected distance (Jukes and Cantor 1969) due to saturation of sites. * * were also made between combined epitopes and the remainder of the protein for mean d S and separately; in none of the comparisons was d S significantly different between epitopes and the remainder of the protein, whereas was significantly lower for the epitopes in all comparisons but one (group O). This pattern indicates that CTL epitopes are conserved relative to the remainder of the protein and that this is not due to a lower mutation rate in epitopes, since the synonymous substitution rate did not differ between epitopes and the remainder of the protein. In contrast to CTL epitopes, Th epitopes are not conserved relative to the remainder of the protein. As with the CTL epitope analysis, d S consistently exceeded for both epitopes and the remainder of the protein, and the differences were generally significant (table 2). Also, d S did not generally differ between epitopes and the remainder of the protein; only two comparisons showed significant differences, and these were in opposite directions. However, was lower for epitopes than for the remainder of the protein in only 2 of 10 comparisons, and in only one of these was the difference substantial and significant. MAb epitopes are less conserved than the remainder of the protein, in direct contrast to CTL epitopes. As in the other analyses, d S consistently exceeded for both epitopes and the remainder of the protein, and there was generally no difference in d S between epitopes and the remainder of the protein (table 3). However, was consistently higher for epitopes than for the remainder of the protein, and the differences were significant in all but one comparison (subtype B vs. subtype D). This could be explained by the observation that MAb epitopes include all but the predicted interior of the folded protein (fig. 1B) and that the interior is highly conserved relative to the rest of the protein. To investigate the cause of the relative conservation of CTL epitopes, d S and were compared between secondary structural elements combined and the remainder of the protein, since CTL epitopes overlap all of the secondary structural elements to some extent (fig. 1A). Within elements and the remainder of the protein, d S Table 2 Mean Numbers of Synonymous (d S ) anonsynonymous ( ) Nucleotide Substitutions per 100 Sites ( SE) in Comparisons Between HIV-1 nef Sequences for Helper T-Cell Epitopes and Remainder of Gene EPITOPES FAMILY d S d S Group M Subtype B... Subtype D... Subtype B vs. subtype D... Subtypes B D... Subtype A... Subtypes B D vs. subtype A... Group O... Group M vs. O... Groups M O * * * * * * EPITOPES VS. d S * NOTE. Families are as in figure 2. Z-tests of hypotheses that d S for epitopes or remainder, and that d S(epitopes) d S(remainder) or (epitopes) (remainder) :* P 0.05; P 0.01; P a Undefined Jukes-Cantor-corrected distance (Jukes and Cantor 1969) due to saturation of sites.

6 1264 da Silva and Hughes Table 3 Mean Numbers of Synonymous (d S ) anonsynonymous ( ) Nucleotide Substitutions per 100 Sites ( SE) in Comparisons Between HIV-1 nef Sequences for Monoclonal Antibody Epitopes and Remainder of Gene EPITOPES VS. EPITOPES FAMILY d S d S d S Group M Subtype B... Subtype D... Subtype B vs. subtype D... Subtypes B D... Subtype A... Subtypes B D vs. subtype A... Group O... Group M vs. O... Groups M O * * NOTE. Families are as in figure 2. Z-tests of hypotheses that d S for epitopes or remainder, and that d S(epitopes) d S(remainder) or (epitopes) (remainder) :* P 0.05; P 0.01; P a Undefined Jukes-Cantor-corrected distance (Jukes and Cantor 1969) due to saturation of sites. consistently exceeded, and these differences are generally significant (table 4). Between elements and the remainder of the protein, d S did not differ significantly, but was significantly higher for elements in four of five comparisons with significant differences. Therefore, the overlap of CTL epitopes with secondary structural elements could not completely explain the conservation of these epitopes. To further investigate the cause of the relative conservation of CTL epitopes, d S and were compared between tertiary structural sites and functional sites combined and the remainder of the protein, since CTL epitopes overlap most of the functional sites (fig. 1A). Since only 2 of 31 sites are involved exclusively in maintaining tertiary structure, these 31 sites are henceforth referred to as functional domains. For functional domains, d S consistently exceeded, but differences were significant in only 4 of 10 comparisons (table 5). For the remainder of the protein, d S exceeded significantly in all comparisons that could be made. For comparisons between the domains and the remainder, d S was consistently lower in the domains, but the differences were significant in only four of eight comparisons. Similarly, was consistently lower in the domains than in the remainder of the protein, but the differences were significant in 7 of 10 comparisons. The similarity between d S and within domains and between domains and the remainder may reflect that not all important functional sites have been identified. Nevertheless, these functional domains are generally conserved relative to the remainder of the protein, and the binding of peptides from these regions by class I HLA molecules may explain the conservation of CTL epitopes. Most of the sequences analyzed are from virus that had been propagated in cell culture. Since HIV-1 may adapt to culture conditions (reviewed by Wain-Hobson 1992), these sequences may not be representative of isolates sequenced directly from patients. Therefore, we repeated the analyses with nine subtype B sequences that were clearly identified in the database as being from uncultured peripheral blood mononuclear cells (P102A13, P164A22, P166A10, P175A01, P226A12, P227A16, P248A01, P357A01, and YU10; fig. 2). The results are qualitatively identical to those from the anal- Table 4 Mean Numbers of Synonymous (d S ) anonsynonymous ( ) Nucleotide Substitutions per 100 Sites ( SE) in Comparisons Between HIV-1 nef Sequences for Secondary Structural Elements and Remainder of Gene ELEMENTS VS. ELEMENTS FAMILY d S d S d S Group M Subtype B... Subtype D... Subtype B vs. subtype D... Subtypes B D... Subtype A... Subtypes B D vs. subtype A... Group O... Group M vs. O... Groups M O * * * NOTE. Families are as in figure 2. Z-tests of hypotheses that d S for elements or remainder, and that d S(elements) d S(remainder) or (elements) (remainder) : * P 0.05; P 0.01; P a Undefined Jukes-Cantor-corrected distance (Jukes and Cantor 1969) due to saturation of sites.

7 Evolutionarily Conserved CTL Epitopes 1265 Table 5 Mean Numbers of Synonymous (d S ) anonsynonymous ( ) Nucleotide Substitutions per 100 Sites ( SE) in Comparisons Between HIV-1 nef Sequences for Tertiary Structural and Functional Site Domains and Remainder of Gene DOMAINS VS. DOMAINS FAMILY d S d S d S Group M * * Subtype B... Subtype D... Subtype B vs. subtype D... Subtypes B D... Subtype A... Subtypes B D vs. subtype A.. Group O... Group M vs. O... Groups M O * * NOTE. Families are as in figure 2. Z-tests of hypotheses that d S for domains or remainder, and that d S(domains) d S(remainder) or (domains) (remainder) :* P 0.05; P 0.01; P a Undefined Jukes-Cantor-corrected distance (Jukes and Cantor 1969) due to saturation of sites. * yses with all 52 subtype B sequences: in comparisons between domains and the remainder of the protein, d S did not differ significantly, whereas was significantly lower for CTL epitopes and functional domains, did not differ significantly for Th epitopes, and was higher for MAb epitopes and secondary structural elements (table 6). Discussion The hypothesis that class I MHC molecules present conserved peptides as an adaptation on the part of the host to constrain parasite escape from immune control (Hughes and Hughes 1995) predicts that CTL epitopes will be conserved relative to the remainder of the protein and that they will coincide with functionally important sites. This hypothesis requires that CTLs play an important role in immune function and that parasites are occasionally successful in escaping CTL recognition; otherwise, there would be no selection for the presentation of conserved peptides. We have shown evidence that CTL epitopes are conserved relative to the remainder of Nef and that these epitopes are derived from functionally constrained regions. Evidence for CTL escape mutants and their importance in HIV-1 disease progression has been accumulating and is discussed below in the context of the conservation of CTL epitopes. We found that CTL, Th, and MAb epitopes and the remainder of the protein are subject to purifying selection. However, CTL epitopes were more conserved than the remainder of the protein, in contrast to Th epitopes, which were about equally conserved, and MAb epitopes, which were less conserved than the remainder of the protein. In analyzing sequences from various isolates, Seibert et al. (1995) also observed purifying selection on putative T-cell epitopes from the proteins encoded by gag, pol, and env of HIV-1, HIV-2, and SIVs, but they found evidence of positive selection on some of the hypervariable domains of the envelope (env) glycoprotein gpl20, which are putatively recognized by host antibodies. In the case of Nef, we did not observe positive selection on MAb epitopes, possibly because the protein is not commonly found outside of the virion, where it would be exposed to antibodies. Our evidence suggests that the conservation of CTL epitopes is not due to a lower mutation rate for epitopes, since this would have manifested itself as a lower synonymous substitution rate for epitopes than for the remainder of the protein, whereas there was no sig- Table 6 Mean Numbers of Synonymous (d S ) anonsynonymous ( ) Nucleotide Substitutions per 100 Sites ( SE) in Comparisons Between Sequences from Nine Uncultured Subtype B Isolates of HIV-1 nef for Domains and Remainder of Gene DOMAIN CTL epitopes... Th epitopes... MAb epitopes... Secondary structural elements... Tertiary structural and functional sites... DOMAINS d S d S * * DOMAINS VS. d S NOTE. Z-tests of hypotheses that d S for domains or the remainder, and that d S(domains) d S(remainder) or (domains) (remainder) :*P 0.05; P 0.01; P

8 1266 da Silva and Hughes nificant difference between the two regions. Rather, CTL epitopes seem to be conserved because they coincide with functional sites, which were also more conserved than the remainder of the protein. Not all of the CTL epitopes that we used map onto functional sites, however, but this is probably because not all functional sites have been identified. For example, Hua et al. (1997), through a mutational study of their own combined with a previous study, delineate broad regions of Nef (amino acid residues and ) that are involved in the reduction of cell surface CD4, regions which overlap several CTL epitopes, but for parts of which there are no functional sites indicated (fig. 1A); we did not incorporate these data into our analysis, because specific functional residues could not be identified. Although sequences from cultured virus may reflect adaptation to culture conditions (reviewed by Wain- Hobson 1992) and hence may not be appropriate for the study of selection in vivo, our results were the same whether we analyzed all 52 subtype B sequences or only 9 of these sequences that were from uncultured virus. This may suggest that the regions of the gene that we studied are either under similar selection in vivo and in culture, or under weak selection in culture. Both explanations may apply: functionally important regions may be under stronger purifying selection than nonfunctional regions both in vivo and in culture, and selection on these and other regions may be generally weaker under the permissive conditions of culture than in vivo. Cell lysis by viral-specific CTLs may be important in controlling HIV-1 infection (Klenerman et al. 1996; Ogg et al. 1998), which would imply selection for CTL escape mutants. McMichael and Phillips (1997) review the evidence for such mutants of HIV-1. One of these studies (Price et al. 1997) shows increasing to exceed d S for an HLA-B8-restricteef epitope over about 6 months postinfection in a patient with CTLs specific for the epitope, resulting in some epitope variants that were not recognized by the patient s CTLs. Interestingly, the 14 epitope variants that were described exhibited amino acid replacement at all sites (amino acids 90 97) except the three C-terminal sites, which putatively function in CD4 binding (fig. 1A). This is consistent with variation in CTL epitopes being constrained by protein function. We did not observe d S for CTL epitopes in comparisons among isolates that are mostly from different patients, possibly because functional constraints allow d S to increase at a higher rate than over the long term, and because CTL selection on specific epitopes is expected to vary among patients even if they have identical class I HLA genotypes (Goulder et al. 1997b). Several earlier studies have also shown evidence of selection on CTL epitopes of HIV-1 Nef. Couillin et al. (1994) observed amino acid replacements at three of four HLA-A11-binding anchor sites of two epitopes in each of two patients and showed that these replacements interfere with the binding of the peptides to HLA-A11 molecules, which would prevent the presentation of the peptides to CTLs. Both peptides overlap several putative SH3-binding sites (fig. 1A), and for one of the peptides, both anchor residues are also SH3-binding sites. One of these sites showed a replacement and represents the only SH3-binding site to be replaced. The other site showed no replacement and is the only anchor residue to not be replaced in either patient. These results seem to show CTL selection for replacement of anchor residues and that the advantage to the virus of such replacement may compensate for some loss of protein function, but that the functional role of an anchor residue may also constrain its replacement. In another study, Koenig et al. (1995) transferred to an HIV-1-infected patient the patient s own CTLs, specific for an HLA-A3.1-restricteef epitope, after the CTLs had been expanded in vitro. The end result of two such infusions was the apparent selection of large deletions of the target epitope and adjacent residues (103 sequences with deletions out of 497 in the final sample). These deletions almost certainly interfered with the protein s function, since they deleted most or all of the polyproline helix (PPII) and the first (H2) and second (H3) long alpha helices of the core region, thereby removing most of the sites putatively involved in SH3 and CD4 binding (fig. 1A) and changing the protein s tertiary structure. This result is likely due to artificially strong selection on one epitope, which may have forced a substantial reduction in Nef function in order to achieve CTL escape. However, cases of large deletions in Nef in HIV-1-infected long-term survivors (Deacon et al. 1995) may indicate the natural occurrence of such strong CTL responses. Haas et al. (1996) describe what may be a more typical scenario for CTL selection in slow progressors. They observed CTLs specific for five Nef epitopes in four patients and, over a 2-year period, the replacement of the original variants of some of the epitopes, followed by the expansion of CTLs specific for the new variants. However, for two epitopes, one restricted by HLA-A2 and the other by HLA-B7, a single variant tended to remain the most common over the 2 years despite its recognition by CTLs. The persistence of the B7-restricted epitope (amino acid residues 68 77) within a single patient may be explained by the six putative SH3-binding sites contained within it (fig. 1A); a second HLA- B7-restricted epitope (residues ) does not contain described functional sites and was completely replaced in the same isolates. Therefore, it is tempting to speculate that the epitopes that were replaced are from regions of Nef that are less functionally constrained than those of the persistent epitopes. Our study is limited to a single protein, but evidence of CTL escape mutants and functional constraints on them exists for other HIV-1 proteins. For example, Borrow et al. (1997) observed the emergence of variants of an HLA-B44-restricted epitope of env gpl20 that escaped CTL detection, and they observed the expansion of CTL specific for the new variants, much like Haas et al. (1996) reported for Nef epitopes. Goulder et al. (1997a) observed the emergence of a CTL escape mutant of an HLA-B27-restricted epitope of gag p24 and subsequent progression to disease in two patients after 9 12 years of no stable epitope variation. The authors propose three explanations for the late emergence of the

9 Evolutionarily Conserved CTL Epitopes 1267 escape mutant: (1) escape mutants had occurred earlier but had been controlled by other aspects of the immune system, (2) CTL control became less effective late in infection, and (3) a mutation compensating for the loss of function caused by the escape mutation had to occur in order for the virus to remain viable. The last explanation is supported by observations of mutations required to compensate for protease function loss in variants of HIV-1 resistant to protease inhibitors (e.g., Y. M. Zhang et al. 1997). Nietfield et al. (1995) describe a point mutation in the epitope studied by Goulder et al. (1997a) that renders the peptide unrecognizable to CTLs but also abrogates viral infectivity. HIV-1 typically manages to escape immune control despite CTL recognition. This could be partly due to Nef s apparent reduction of cell-surface class I MHC, which would interfere with CTL recognition of viral peptides. In addition, HIV-1 s large within-host population size, high mutation rate, and short generation time should allow the virus to adapt quickly to host individuals (Coffin 1995), as appears to be the case for many pathogens, especially viruses (Bell 1993). Therefore, the immune system is expected to have evolved to constrain the adaptation of parasites. One way in which the vertebrate immune system appears to do this is through the generation of CTLs specific for peptide variants as they arise. We have provided evidence of another strategy: CTLs are presented with peptides that are derived from functionally constrained regions of foreign proteins, thereby forcing the pathogen to trade off escape from CTL recognition for viability. We are suggesting not that this is an adaptation to HIV-1 specifically, but that it is a general strategy of vertebrate hosts to limit the adaptation of their intracellular parasites. Acknowledgments We thank F. Verra, M. 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