Chimpanzee CD4⁺ T Cell Cultures Differ in Their Susceptibility to SIVcpz Infection.

Generating infectious molecular clones (IMCs) of SIVcpz, we previously noted that some viruses that replicated efficiently in human CD4⁺ T cells were unable to infect chimpanzee CD4⁺ T cells (44). To examine this surprising phenotype, we obtained leftover blood samples from 28 healthy chimpanzees housed at US primate centers and infected their CD4⁺ T cells with a panel of eight chimpanzee viruses representing both SIVcpzPtt (MT145, EK505, MB897, LB715, and GAB2) and SIVcpzPts (BF1167, TAN2, and TAN13) strains (21, 44⇓⇓–47). SIVcpz IMCs were transfected, normalized based on infectivity in a permissive cell line (TZM-bl), and used to infect CD4⁺ T cells at a multiplicity of infection of 0.1 (Fig. 1). As observed previously (21, 44, 47), all SIVcpz strains replicated efficiently in human CD4⁺ T cells (Fig. 1A). However, their ability to establish a productive infection in chimpanzee CD4⁺ T cells varied considerably. Of the 28 chimpanzees tested, only 9 supported the replication of all, or nearly all, SIVcpz strains (e.g., Melissa in Fig. 1 B and D) while 18 others were refractory to six of the eight viruses (e.g., Dona in Fig. 1 C and D). Although not all viruses could be tested in cells from all animals, 15 chimpanzees supported replication of only TAN2 and MT145 while 3 others supported replication of only EK505 and MT145 (Fig. 1D). Cells from one chimpanzee (Chip) supported the replication of MT145, EK505, GAB2, and LB715, but not MB897, BF1167, and TAN2 (Fig. 1D). These infectivity patterns were reproducible in all instances where repeat blood samples from the same individuals were available (SI Appendix, Table S1). Only a single SIVcpz strain, MT145, was able to replicate in CD4⁺ T cells from all 28 chimpanzees (Fig. 1D). These results showed that, unlike human CD4⁺ T cells, chimpanzee CD4⁺ T cells were refractory to a subset of viruses, with susceptibility influenced by both host- and virus-specific determinants.

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Fig. 1.

Chimpanzee CD4⁺ T cells differ in their susceptibility to SIVcpz infection. (A–C) The replication potential of eight SIVcpz strains is shown in activated CD4⁺ T cells from one human (A) and two chimpanzees (B and C). Viral replication was monitored in culture supernatants by determining reverse transcriptase (RT) activity (ng/mL). (D) CD4⁺ T cells from 28 captive chimpanzees (columns) were infected with different strains of SIVcpz (rows) and monitored for viral replication. The chimpanzee-adapted HIV-1 SG3 strain was used for positive control (SI Appendix, SI Materials and Methods). RT activity at day 13 was used to classify each culture as supporting robust (>5 ng/mL RT, dark circles), weak (1 to 5 ng/mL RT, grey circles), or no (<1 ng/mL RT, white circles) viral replication (SI Appendix, Table S1). Replication results are shown in relation to the respective chimpanzee CD4 genotype, with the position of polymorphic amino acid residues highlighted. (E) Protein sequences of five chimpanzee CD4 variants shown in comparison with human CD4 (residues are numbered according to their position in the mature CD4 protein). Extracellular (D1 to D4), transmembrane (TM), and intracytoplasmic domains of the protein are indicated relative to their coding exons (highlighted by alternating black and blue text), with the D1 domain underlined in red. Dots indicate amino acid identity to the human sequence, with the polymorphic sites in the D1 domain highlighted in red. Conserved and polymorphic potential N-linked glycosylation sites (PNGSs) are shaded in gray and red, respectively.

Refractory CD4⁺ T Cells Exhibit an Entry Block.

To determine at which step in the life cycle SIVcpz was blocked, we generated a replication-competent SIVcpz reporter virus by inserting an enhanced green fluorescent protein (eGFP) gene between the env and nef genes of the SIVcpz MB897 molecular clone (SI Appendix, Fig. S2A). A transfection-derived stock of this reporter virus was then used to infect activated human and chimpanzee CD4⁺ T cells. Consistent with results obtained for the WT MB897 strain (Fig. 1D), eGFP expression was detected in CD4⁺ T cells from humans and susceptible chimpanzees, but not in CD4⁺ T cells from refractory chimpanzees (SI Appendix, Fig. S2B). However, complementation of the MB897 reporter virus with the vesicular stomatitis virus G protein (VSV-G) resulted in productive infection of all cultures (SI Appendix, Fig. S2B). These data indicated that SIVcpz was inhibited at the level of cell entry.

CD4 Polymorphisms Govern the Susceptibility of CD4⁺ T Cells to SIVcpz Infection.

In addition to CD4, SIVcpz utilizes the chemokine receptor CCR5 to infect target cells (21, 44). Since the VSV-G complementation studies suggested a block at the receptor and/or coreceptor level, we sequenced the CD4 and CCR5 genes of all 28 chimpanzees. In contrast to the CCR5 gene, which comprises a single coding exon, the CD4 gene is expressed from nine exons, some of which have been reported to be polymorphic (42). Since previous studies did not ensure linkage of variable sites and failed to guard against PCR artifacts (42), we extracted RNA from chimpanzee CD4⁺ T cells and used limiting dilution RT-PCR to amplify single CD4 transcripts (48). These analyses showed that none of the 28 chimpanzees exhibited mutations in their CCR5 gene. However, analysis of their CD4 sequences revealed several single nucleotide polymorphisms (SNPs), four of which changed the amino acid sequence of the mature CD4 protein (SI Appendix, Fig. S3A). These nonsynonymous SNPs caused substitutions at positions 40 (Q/R), 52 (N/K), 55 (V/I), and 68 (P/T), the combination of which resulted in five CD4 variants that differed solely in their D1 domain (Fig. 1E). Importantly, the P68T substitution created a potential N-linked glycosylation site (PNGS) at position 66, in addition to the invariant PNGS present at position 32 present in all chimpanzee CD4 alleles (Fig. 1E). Based on the amino acids present at positions 40, 52, 55, and 68, we designated the chimpanzee CD4 alleles QNIP, QNVP, QNVT, RNVP, and RKVT, respectively (SI Appendix, Table S2); the latter two had not previously been described (42).

Comparing the CD4 allelic diversity between permissive and refractory cultures, we found a remarkable association between the CD4 genotype and SIVcpz infection phenotype (Fig. 1D). CD4⁺ T cells from four chimpanzees, which were heterozygous for the QNVT and QNVP alleles, supported replication of all eight SIVcpz strains while CD4⁺ T cells from four other chimpanzees, which were heterozygous for QNVT and QNIP alleles, supported the same set of viruses, except for GAB2. In contrast, chimpanzees homozygous for the QNVT allele supported infection of only two of the eight SIVcpz strains. This was also true for chimpanzees heterozygous for the QNVT and RKVT alleles except this genotype inhibited TAN2 instead of EK505. In general, CD4⁺ T cells from chimpanzees with the same CD4 genotype were susceptible to infection by the same set of viruses while even a single amino acid substitution in one of the two CD4 alleles changed the number or types of viruses that were able to replicate (Fig. 1D).

Chimpanzee Subspecies Differ in Their CD4 Diversity.

The finding of new CD4 alleles among captive chimpanzees suggested that additional variants might exist in wild populations. To determine the full extent of CD4 diversity, we thus made use of our extensive collection of blood and fecal samples obtained previously from wild and sanctuary chimpanzees for molecular epidemiological studies of SIVcpz and ape Plasmodium infections (22, 49, 50). Samples were selected based on their geographic and subspecies origin, SIVcpz infection status, and individual information (SI Appendix, Table S3). Since the chimpanzee CD4 gene spans a 19-Kb region on chromosome 12, with a large intron (13.7 Kb) separating exons 2 and 3, we were unable to amplify the entire CD4 coding region from a single DNA template. However, since all CD4 polymorphisms were located in the D1 domain, we amplified exon 2 (247 bp) and exon 3 (222 bp) separately, sequencing their respective amplicons without fragmentation to maintain linkage between polymorphic sites. Homozygous loci were amplified up to eight times to exclude allelic dropout. Using this approach, we CD4 genotyped 60 P. t. verus, 41 P. t. ellioti, 246 P. t. troglodytes, and 197 P. t. schweinfurthii apes that were sampled at 58 sites throughout their range (SI Appendix, Fig. S1). Although exons 2 and 3 were sequenced separately, we were able to infer 13 different D1 domain haplotypes from individuals who were homozygous for one of the two exons (SI Appendix, Fig. S3B). These haplotypes contained one synonymous SNP (g/a) at the second nucleotide of exon 2 (nucleotide position 51 of the CD4 coding sequence) and five nonsynonymous SNPs, which resulted in amino acid substitutions at positions 25, 40, 52, 55, and 68 of the mature CD4 protein. Comparison of these to CD4 sequences from other ape species identified the (g)QQNVP allele as the ancestral state, which appeared to have diversified both by point mutations and recombination, including within exons 2 and 3 (SI Appendix, Fig. S3B). Thus, in contrast to the human CD4, for which the most common D1 variant occurs at a frequency of only 6.6 × 10⁻⁵ (SI Appendix, SI Materials and Methods), the chimpanzee CD4 is highly polymorphic in this domain, with nine coding variants of CD4 identified in wild populations (Fig. 2A).

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Fig. 2.

Allelic diversity of CD4 in chimpanzees. (A) CD4 coding variants identified in wild chimpanzee populations. D1 domain alleles are compared with the human CD4, with partial exons 2 and 3 derived sequences color-coded. Only the polymorphic region is shown (see Fig. 1E for an alignment of full-length CD4 coding sequences). Dots indicate identical amino acids while arrows highlight polymorphic sites, with their position in the mature CD4 protein indicated. (B) Frequencies of exon 2 and 3 alleles among members of the four chimpanzee subspecies. Exon 2 includes polymorphic amino acids at positions 25 (Q/R) and 40 (Q/R) while exon 3 includes polymorphic amino acids at positions 52 (N/K)), 55 (I/V), and 68 (P/T). The number of chimpanzees sequenced for each subspecies is indicated.

Chimpanzees are believed to have originated in west central Africa where they initially split into two lineages when the ancestor of P. t. verus and P. t. ellioti diverged from the ancestor of P. t. troglodytes and P. t schweinfurthii (51). Given these relationships and the fact that only central and eastern chimpanzees are SIVcpz-infected, we expected P. t. verus and P. t. ellioti to exhibit similar levels of CD4 diversity and to differ from P. t. troglodytes and P. t. schweinfurthii both in the number and distribution of CD4 alleles. However, this was not the case. Instead, we found P. t. ellioti and P. t. troglodytes to be most similar, with exon 2 QQ and QR alleles and exon 3 NVP, NIP, and KVT alleles present at comparable frequencies in both subspecies. Although P. t. troglodytes exhibited two additional exon 3 alleles (NVT and KVP), both were extremely rare and may thus have been missed in the less extensively sampled P. t. ellioti subspecies. P. t. schweinfurthii also encoded multiple CD4 alleles, but their frequencies differed markedly from those in P. t. troglodytes and P. t. ellioti. For example, using the CD4 exon 3 haplotype frequencies, G_ST (a measure of population differentiation) (52) between P. t. troglodytes and P. t. schweinfurthii was 0.31, compared with only 0.11 between P. t. troglodytes and P. t. ellioti. Moreover, none of the eastern chimpanzees encoded the R40 allele, but instead some encoded an R25 allele, which was found only in chimpanzees living in Gombe National Park in Tanzania (SI Appendix, Table S3). The most surprising finding was that P. t. verus apes did not exhibit any CD4 polymorphisms. Although 60 individuals were sampled at five different locations throughout West Africa (SI Appendix, Fig. S1), every single one was homozygous for the (a)QQNVT allele (Fig. 2B). Given the scarcity of the exon 3 NVT allele in the other three subspecies, the predominance of this allele is unlikely the result of a founder effect. Instead, P. t. verus apes appear to have undergone a selective sweep, possibly in response to an SIV-like pathogen that has since gone extinct. Taken together, these findings suggest that the various CD4 polymorphisms evolved before the introduction of present-day SIVcpz and likely already existed in the chimpanzee ancestor.

CD4 Polymorphisms Inhibit SIVcpz Infection.

To examine the interaction of the nine CD4 alleles with individual SIVcpz Envs, we developed a single round infection assay by transfecting 293T cells with vectors expressing human and chimpanzee CD4 alleles, along with the chimpanzee CCR5 gene, and then infecting these cells with pseudotyped viruses carrying the Envs of different SIVcpz strains. As the pseudotyping backbone, we used an env-deficient version of the GFP-expressing MB897 reporter virus, which allowed the use of flow cytometry to identify infected (GFP-expressing) cells (SI Appendix, Fig. S4). The results of these studies largely recapitulated the replication data obtained in primary CD4⁺ T cells (Figs. 1 and 3). First, most SIVcpz Envs used the human CD4 more efficiently than any of the chimpanzee CD4 alleles (Fig. 3), consistent with the universal susceptibility of human CD4⁺ T cells to SIVcpz infection. Second, the Env of the promiscuous MT145 strain utilized all CD4 alleles while cell entry of the more restricted MB897 Env was markedly reduced in QQNVT-expressing cells and nearly completely blocked in QRNVP- and QRKVT-expressing cells (Fig. 3). Analysis of new CD4 alleles from wild chimpanzees extended these results and identified R25, R40, and T68 as the most inhibiting substitutions. All chimpanzee CD4 alleles contain a PNGS at residue 32, not found in human CD4, while those with a threonine at position 68 (T68) contain two PNGSs. Interestingly, chimpanzee CD4 alleles with a P at position 68 (P68) reduced the infectivity of all SIVcpz Envs, compared with the human CD4, while chimpanzee CD4 alleles that encoded a threonine at position 68 (T68) reduced their infectivity even further (SI Appendix, Fig. S5A). This inhibition was not dependent on the baseline infectivity of these SIVcpz Envs, nor on the allelic context of the P68T substitution (SI Appendix, Fig. S6A), suggesting that the presence of two glycans in the D1 domain is more protective than the presence of only one.

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Fig. 3.

Chimpanzee CD4 polymorphisms govern SIVcpz Env-mediated cell entry. The infectivity of pseudoviruses carrying different SIVcpz Envs is shown for transiently transfected cells expressing human and chimpanzee CD4 alleles. The infectivity on human CD4-expressing cells is set to 100%. Bars represent the average of three independent transfections, each performed in triplicate, with standard deviations shown.

In contrast to the changes in PNGS numbers, other amino acid substitutions in the D1 domain affected only a subset of SIVcpz Envs. For example, the substitution of a glutamine to an arginine at position 25 (Q25R) reduced cell entry of BF1167, TAN2, and TAN13 by up to 18-fold but had little effect on the remaining SIVcpz Envs, except for GAB2, whose infectivity was slightly enhanced (SI Appendix, Figs. S5B and S6B). Similarly, the substitution of a glutamine to an arginine at position 40 (Q40R) reduced MB897, BF1167, TAN2, and TAN13 cell entry more than 100-fold but did not appear to inhibit any of the other SIVcpz Envs (SI Appendix, Figs. S5C and S6C). The change from an asparagine to a lysine at position 52 (N52K) inhibited all SIVcpz Envs modestly in the context of the QQNVP allele but had a slight enhancing effect in the context of the QQNVT allele (SI Appendix, Figs. S5D and S6D). Finally, the change from a valine to an isoleucine at position 55 (V55I) had no detectable effect in the context of both the QQNVP and QRNVP alleles (SI Appendix, Figs. S5E and S6E). Thus, while CD4 polymorphisms were able to block SIVcpz cell entry, the inhibitory effects were variable, context-dependent, and strain-specific.

Chimpanzee CD4 Polymorphisms Interfere with Env Binding by Altering Contact Residues at the CD4–Env Interface.

Having identified protective CD4 alleles, we next sought to examine the breadth of this protection and the associated mechanism(s). Both R25- and R40-containing CD4 alleles inhibited primarily SIVcpzPts strains (Figs. 3 and 4), but CD4 genotyping of 128 chimpanzees from Gombe National Park identified two SIVcpzPts-infected individuals that were R25-heterozygous. Since the virus of one of these individuals (TAN1) has been molecularly cloned (44), we tested its Env in the CD4 transfection assay (Fig. 4A). This analysis showed that TAN1, in contrast to the other SIVcpzPts Envs, was able to utilize the R25 allele (Fig. 4A). Similarly, of five SIVcpzPtt strains, four were resistant to the R40 inhibition, and all four were derived from chimpanzees that encoded at least one R40-containing allele (Fig. 4C). Thus, for both R25 and R40 polymorphisms, in vivo adaptation had generated SIVcpz strains that were capable of utilizing these CD4 variants.

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Fig. 4.

SIVcpz cell entry is blocked by charged residues at the CD4–Env interface. (A and C) Percentage of cells expressing the indicated CD4 alleles that are infected by SIVcpz Env-containing pseudoviruses. Points connected by lines represent paired averages of three independent experiments, each performed in triplicate. The parentheses indicate the CD4 genotype (amino acid at positions 25 in A and position 40 in C) of the naturally infected chimpanzee from which the respective SIVcpz Env was derived. (B and D) Modeling of chimpanzee CD4 residues 25 (B) and 40 (D) onto the crystal structure of the HIV-1 gp120 (yellow) bound to human CD4 (green). Polymorphic CD4 residues are shown in dark blue (Q) and red (R), respectively. Env residues in proximity to these polymorphic sites are highlighted. Partial Env protein alignments for the tested SIVcpz strains are shown, with the residue predicted to interact with the polymorphic CD4 residue highlighted (blue for Envs that can use both Q and R residues, red for Envs that are inhibited by the R residue). (E) Infectivity of WT (MB897, TAN2) and mutant (MB897 T455D, TAN2 K474V) SIVcpz Envs on cells expressing the indicated chimpanzee CD4 alleles. Circles represent average infectivity values from three independent experiments, each performed in triplicate, with mean and standard deviation shown. Significant differences are indicated (**P < 0.01, unpaired t test). (F) Replication potential of wild-type (wt) and mutant (T455D) MB897 and wild-type MT145 in chimpanzee CD4⁺ T cells heterozygous for QRNVP and QQNVT alleles. Viral replication was monitored in culture supernatants by determining reverse transcriptase (RT) activity.

To identify the mechanisms of inhibition mediated by R25 and R40, we mapped these CD4 residues onto an existing crystal structure of the human CD4 bound to HIV-1 gp120 (38). We then searched for Env amino acids that were in close proximity to these CD4 residues but differed between restricted and permissive SIVcpz strains (Fig. 4 B and D). For the R25 polymorphism, we found that Env residue 474 (HXB2 reference sequence numbering) was negatively charged or neutral in all viruses that were able to utilize the RQNVP allele, but positively charged in SIVcpz strains that were unable to utilize this allele (Fig. 4B). This finding suggested that the CD4 R25 repulsed positively charged Env amino acids at position 474, thus destabilizing the Env–CD4 interaction. Since the permissive TAN1 strain encoded a valine at this position, we mutagenized a restricted Env (TAN2) by replacing its lysine with a valine (K474V). This single amino acid substitution was sufficient to render the TAN2 Env infectious for cells expressing the RQNVP allele without altering its ability to utilize the QQNVP allele (Fig. 4E). Similarly, residue 455 tended to be negatively charged in Envs that could use R40-containing CD4 alleles, but neutral in Envs that were blocked by this allele (Fig. 4D). In this instance, the different orientation of the R40 side chain was incompatible with an interaction with Env residue T283, the contact residue for CD4 Q40. However, this loss was predicted to be potentially offset by the formation of long-range salt bridges between CD4 R40 and a negatively charged residue at Env position 455. In line with this prediction, replacing a threonine at position 455 with an aspartic acid (T455D) in the restricted MB897 Env was sufficient to restore its infectivity for QRNVP-expressing cells without altering its ability to utilize the QQNVP allele (Fig. 4E).

To examine the effect of the T455D mutation in the context of a replication-competent virus, we introduced this change into the MB897 infectious molecular clone and tested its infectivity in CD4⁺ T cells from a chimpanzee (Chip) that was heterozygous for the QRNVP and QQNVT alleles (Fig. 1). Remarkably, this single amino acid substitution rendered MB897 infectious in cells that were completely refractory to the WT virus (Fig. 4F), and the same results were obtained in CD4⁺ T cells from a chimpanzee that was heterozygous for QRKVT and QQNVT alleles (SI Appendix, Fig. S7A). Although Env amino acid 455 is unlikely the only residue impacted by the R40 substitution (the LB715 Env encodes a T at position 455 but is able to use the QRNVP allele), it seems clear that changes in the charge of Env/CD4 contact residues represent one mechanism by which CD4 polymorphisms prevent Env-mediated cell entry. Of note, R25 and R40 blocked CD4/Env interaction by different means since Env mutations that restored infectivity for one of these CD4 alleles did not restore infectivity for the other (Fig. 4E).

Chimpanzee CD4 Polymorphisms Cause Glycan–Glycan Steric Hindrance.

In contrast to the R25 and R40 alleles, the T68 polymorphism inhibited all SIVcpz Envs regardless of their baseline infectivity (Fig. 3). Since T68 creates a PNGS at position N66 in addition to the N32 that is present in all chimpanzee CD4 alleles, we asked whether these sites were indeed glycosylated. Immunoprecipitation of CD4 proteins from cells transfected with human and chimpanzee (QQNVP and QQNVT) alleles revealed differences in electrophoretic mobility consistent with glycan occupancy, which was confirmed by endoglycosidase H treatment (SI Appendix, Fig. S8). To determine whether these D1 domain glycans interfered with glycans on the various SIVcpz Envs, we modeled their position in the crystal structure of the human CD4 bound to HIV-1 gp120 (38). This analysis identified three glycans in the MB897 Env that were predicted to clash with glycans in the chimpanzee CD4 protein (Fig. 5A). Env glycan N460 was predicted to clash with CD4 glycan N32 while Env glycans N295 and N446 were predicted to clash with CD4 glycan N66, albeit on adjacent Env protomers. Interestingly, no substantial glycan–glycan clashes were predicted for the Envs of MT145 and TAN2, both of which were able to replicate efficiently in CD4⁺ T cells from QQNVT-homozygous chimpanzees (Fig. 1D).

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Fig. 5.

Steric hindrance between CD4- and SIVcpz Env-encoded glycans. (A) A model of two SIVcpz Envs (Top, MB897; Bottom, MT145) in complex with the chimpanzee CD4 QQNVT variant (containing both N32 and N66 glycans) is shown. Env residues are shown in purple, Env glycans in green, and CD4 glycans in red. (B) Infectivity of WT and mutant MB897 Envs lacking the indicated glycans in cells expressing different CD4 alleles (parentheses indicate the number of D1 domain glycans). Asterisks denote significant enhancement of the mutant Envs relative to the MB897 WT (orange) for that particular allele. (C–E) Replication of wild-type and mutant MB897 viruses in CD4⁺ T cells from one heterozygous chimpanzee encoding QQNVP and QQNVT alleles (C) and two homozygous chimpanzees encoding the QQNVT allele (D and E).

To examine whether removal of the clashing glycans in the MB897 Env would increase its infectivity, we changed the asparagine residues at positions 295, 446, and 460 to glutamines and then tested these mutants, alone and in combination, in the transient CD4 transfection assay (Fig. 5B). Interestingly, the N460Q and the triple mutant increased the infectivity of the MB897 Env in both QQNVP- and QQNVT-expressing cells (Fig. 5B). In contrast, removal of N295 and/or N446 had little effect in the context of either allele (Fig. 5B). Introduction of these same mutations into the MB897 infectious molecular clone were consistent with these results. Compared with WT MB897, both the N460Q and the triple mutant grew faster and to higher titers in CD4⁺ T cells from a QQNVP/QQNVT-heterozygous chimpanzee (Fig. 5C). The N460Q mutant also replicated in CD4⁺ T cells from one, but not all, QQNVT-homozygous chimpanzees (Fig. 5 D and E) while N295Q and/or N446Q mutants again had no effect. Thus, removal of an Env glycan predicted to clash with the conserved N32 increased the infectivity of the MB897 strain, both in transient transfection and replication studies, albeit not to the level of other QQNVT-permissive SIVcpz strains, such as MT145. Removal of the other two glycans failed to restore infectivity, although this was not due to a fitness cost, since all mutants replicated to the same extent in human CD4⁺ T cells (SI Appendix, Fig. S7B). Thus, in silico modeling of the CD4 and Env glycan shields did not fully recapitulate the extent of their interactions.

Chimpanzee CD4 Polymorphisms Protect Against SIVs Infecting Old World Monkeys.

To test whether the inhibitory effect of the chimpanzee CD4 polymorphisms extended to more diverse SIVs, we cloned env genes from several SIV lineages into expression vectors, generated pseudoviruses, and tested their functional integrity in TZM-bl cells. Infectious Envs were then tested for CD4 dependence, and only those that required CD4 for cell entry were further analyzed. Using the transient CD4 transfection assay, we identified nine Envs from SIVs infecting mustached monkeys (Cercopithecus cephus), l’Hoest’s monkeys (Cercopithecus lhoesti), western red colobus (Procolobus badius), red-tailed monkeys (Cercopithecus ascanius), sooty mangabeys (Cercocebus atys), and African green monkeys (Chlorocebus tantalus) that mediated entry into cells expressing the human CD4 (Fig. 6A). However, when these same Envs were tested in cells expressing the chimpanzee QQNVP allele, the great majority exhibited markedly reduced infectivity, with some of them inhibited even more in cells expressing the chimpanzee QQNVT allele (SI Appendix, Fig. S6F). To examine possible mechanisms, we mutagenized the conserved N32 glycan in both the QQNVP and QQNVT alleles. Changing the asparagine at position 32 to a glutamine (N32Q) in the QQNVP allele enhanced the infectivity of most SIV Envs, suggesting that their inhibition was primarily glycan-mediated (SI Appendix, Fig. S6F). However, the same mutation in the QQNVT allele did not restore Env infectivity, suggesting that one glycan in the D1 domain is sufficient to exert inhibition (Fig. 6B and SI Appendix, Fig. S6F). For six SIV Envs that retained infectivity for the chimpanzee QQNVP allele, we also tested the remaining CD4 polymorphisms (Fig. 6C). Both the N52K and V55I polymorphisms inhibited Envs of the SIVsmm lineage but had little effect on the other SIV Envs, except for SIVagm, which was enhanced by the K52 substitution (SI Appendix, Fig. S6G). Since the African green monkey CD4 encodes an R at position 52, this substitution likely generated a chimpanzee CD4 allele that resembled the cognate SIVagm receptor more closely. The R25 substitution inhibited one SIVagm and one SIVsmm Env, but none of the other SIV Envs (SI Appendix, Fig. S6G). The most pronounced protective effect was again observed for the R40 substitution, which reduced cell entry of all SIV Envs by ∼10-fold, except for the Env of SIVasc (SI Appendix, Fig. S6G). Thus, like for SIVcpz strains, CD4 polymorphism-mediated inhibition of SIV cell entry was strain-specific and context-dependent.

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Fig. 6.

Chimpanzee CD4 polymorphisms protect against monkey SIVs. (A) Percent infectivity of Env-carrying pseudoviruses from diverse SIV lineages (color-coded) of cells expressing the indicated CD4 alleles (parentheses indicate the number of D1 domain PNGSs). Bars represent the average of three independent experiments, each performed in triplicate. Infectivity values are shown relative to the human CD4, which was set to 100%. Asterisks indicate statistically significant differences between the chimpanzee QQNVP and the human CD4, as well as between the chimpanzee QQNVP and QQNVT alleles (SI Appendix, Fig. S6). (B) Effect of the N32 glycan on SIV Env infectivity. Average infectivity values are shown for each Env tested against CD4 alleles with intact or mutated N32. Solid lines indicate statistical significance (SI Appendix, Fig. S6). (C) Effect of CD4 polymorphisms on SIV Env infectivity. Percent infectivity values are compared between chimpanzee CD4 allele pairs that differ at one polymorphic site (highlighted in red). Solid lines indicate statistical significance (SI Appendix, Fig. S6).

CD4 Receptor Diversity Protects Wild Chimpanzees from SIVcpz Infection.

Finally, we asked whether the various chimpanzee CD4 alleles influenced SIVcpz acquisition in vivo. To address this, we selected chimpanzee communities at two field sites where CD4 diversity could be analyzed in the context of high SIVcpz prevalence rates (SI Appendix, Table S3). One included Gombe National Park, where members of the Mitumba, Kasekela, and Kalande communities have been monitored for SIVcpz for two decades and where the negative impact of this infection on chimpanzee health was first demonstrated (27, 28). The other included the Lobéké (LB) and Mambélé (MB) area in southeastern Cameroon where chimpanzees harbor the closest relatives of HIV-1 group M at high prevalence rates (22). Using logistic regression to examine the effect of CD4 substitutions on SIVcpz infection relative to the ancestral CD4 allele, we found a suggestion of a protective effect for the R25 polymorphism in the Gombe population although this did not reach statistical significance (Fig. 7A). The 70 chimpanzees from the Lobéké (LB) and Mambélé (MB) area lacked the R25 polymorphism but encoded CD4 alleles with other substitutions in exon 2 and exon 3. When these were analyzed, none of them reached statistical significance although there was a trend for R40 and K52 to be associated with lower, and for I55 to be associated with higher, SIVcpz infection rates (Fig. 7B). Surprisingly, the T68 substitution did not appear to have a detectable effect. However, since only 4 of 46 individuals encoded T68 in the absence of K52, the impact of these two substitutions could not be differentiated. Interestingly, when the LB/MB data were analyzed at the whole exon level, the KVT allele was associated with 3.4-fold lower odds of being infected (P = 0.03, 95% CI: 1.07- to 10.8-fold) (Fig. 7). These data thus suggest that K52 (alone or in combination with T68) and, possibly, also R25 protect wild chimpanzees against infection by locally circulating SIVcpz strains although definitive conclusions must await more extensive sampling of wild populations.

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Fig. 7.

Effect of CD4 polymorphisms on SIVcpz infection rates in wild chimpanzees. A logistic regression was used to estimate the effects of CD4 polymorphisms on SIVcpz infection in wild-living chimpanzees from (A) Gombe and (B) Lobéké/Mambélé. Dots indicate the estimated effect of the presence of the given residue (Left) or allele (Right) relative to the ancestral state, with horizontal lines indicating the 95% confidence intervals (substitutions/alleles that could not be estimated by the model due to insufficient variation in the population are not shown). Dashed vertical lines mark a fold change of 1, indicating no predicted change in SIVcpz infection rate relative to the ancestral state.