CD4 receptor diversity in chimpanzees protects against SIV infection

Abstract

Human and simian immunodeficiency viruses (HIV/SIVs) use CD4 as the primary receptor to enter target cells. Here, we show that the chimpanzee CD4 is highly polymorphic, with nine coding variants present in wild populations, and that this diversity interferes with SIV envelope (Env)-CD4 interactions. Testing the replication fitness of SIVcpz strains in CD4⁺ T cells from captive chimpanzees, we found that certain viruses were unable to infect cells from certain hosts. These differences were recapitulated in CD4 transfection assays, which revealed a strong association between CD4 genotypes and SIVcpz infection phenotypes. The most striking differences were observed for three substitutions (Q25R, Q40R, and P68T), with P68T generating a second N-linked glycosylation site (N66) in addition to an invariant N32 encoded by all chimpanzee CD4 alleles. In silico modeling and site-directed mutagenesis identified charged residues at the CD4-Env interface and clashes between CD4- and Env-encoded glycans as mechanisms of inhibition. CD4 polymorphisms also reduced Env-mediated cell entry of monkey SIVs, which was dependent on at least one D1 domain glycan. CD4 allele frequencies varied among wild chimpanzees, with high diversity in all but the western subspecies, which appeared to have undergone a selective sweep. One allele was associated with lower SIVcpz prevalence rates in the wild. These results indicate that substitutions in the D1 domain of the chimpanzee CD4 can prevent SIV cell entry. Although some SIVcpz strains have adapted to utilize these variants, CD4 diversity is maintained, protecting chimpanzees against infection with SIVcpz and other SIVs to which they are exposed.

Keywords: CD4; SIV; chimpanzee; envelope glycoprotein; glycan restriction.

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.

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.

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.

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.

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).

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).

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.