Infection pressure in apes has driven selection for CD4 alleles that resist lentivirus (HIV/SIV) infection

Reviewer #1 (Public Review):

Summary:
The authors previously demonstrated that species-specific variation in primate CD4 impacts its ability to serve as a functional receptor for diverse SIVs. Here, Warren and Barbachano-Guerrero et al. perform population genetics analyses and functional characterization of great ape CD4 with a particular focus on gorillas, which are natural hosts of SIVgor. They first used ancestral reconstruction to derive the ancestral hominin and hominid CD4. Using pseudotyped viruses representing a panel of envelopes from SIVcpz and HIV strains, they find that these ancestral reconstructions of CD4 are more similar to human CD4 in terms of being a broadly susceptible entry receptor (in the context of mediating entry into Cf2Th cells stably expressing human CCR5). In contrast, extant gorilla and chimpanzee CD4 are functional entry receptors for a narrower range of HIV and SIVcpz isolates. Based on these differences, authors next surveyed gorilla sequences and identified several CD4 haplotypes, specifically in the region encoding the CD4 D1 domain, which directly contacts the viral glycoprotein and thus may impact the interaction. Consistent with this possibility, the authors demonstrated that gorilla CD4 haplotypes are, on average, less capable of supporting entry than human CD4, and that some are largely unable to function as SIV entry receptors. Interestingly, individual residues found at key positions in the gorilla CD4 D1 when tested in the context of human CD4 reduce entry of some virions pseudotyped with diverse SIVcpz envelopes, suggesting that individual amino acids can in part explain the observed differences across gorilla CD4 haplotypes. Finally, the authors perform statistical tests to infer that CD4 from great apes with endemic SIV (i.e., chimpanzees and gorillas) but not non-reservoirs (i.e., orangutans, bonobos) or recent spillover hosts (i.e., humans), have been subject to selection as a result of pressure from endemic SIV.

The conclusions of this paper are mostly well supported by data.

Strengths:
The functional assays are appropriate to test the stated hypothesis, and the authors use a broad diversity of envelopes from HIV and SIVcpz strains. The authors also partially characterize one potential mechanism of gorilla CD4 resistance - receptor glycosylation at the derived N15 found in 5/6 gorilla haplotypes.

Ancestral reconstruction provides a particularly interesting aspect of the study, allowing authors to infer the ancestral state of hominid CD4 relative to modern CD4 from gorillas and chimpanzees. This, coupled with evidence supporting SIV-driven selection of gorilla CD4 diversity and the characterization of functional diversity of extant haplotypes provides several interesting findings.

Weaknesses:
The major inference of the work is that SIV infection of gorillas drove the observed diversity in gorilla CD4. This is supported by the majority of SNPs being localized to the CD4 D1, which directly interacts with the envelope, and the demonstrated functional consequences of that diversity for viral entry. However, SIVgor (to the best of my knowledge) only infects Western lowland gorillas (Gorilla gorilla gorilla), and one Gorilla gorilla diehli and three Gorilla beringei graueri individuals were included in the haplotype and allele frequency analyses. The presence of these haplotypes or the presence of similar allele frequencies in Eastern lowland and mountain gorillas would impact this conclusion. It would be helpful for the authors to clarify this point.

The authors appear to use a somewhat atypical approach to assess intra-population selection to compensate for relatively small numbers of NHP sequences (Fig. 6). However, they do not cite precedence for the robustness of the approach or the practice of grouping sequences from multiple species for the endemic vs other comparison. They also state in the methods that some genes encoded in the locus were removed from the analysis "because they have previously been shown to directly interact with a viral protein." This seems to undercut the analysis and prevents alternative explanations for the observed diversity in CD4 (e.g., passenger mutations from selection at a neighboring locus).

Data in Figure 5 is graphed as % infected cells instead of virus titer (TDU/mL). It's unclear why this is the case, and prevents a comparison to data in Figure 2 and Figure 4.

The lack of pseudotyping with SIVgor envelope is a surprising omission from this study, that would help to contextualize the findings. Similarly, building gorilla CD4 haplotype SNPs onto the hominin ancestor (as opposed to extant human CD4) may provide additional insights that are meaningful toward understanding the evolutionary trajectory of gorilla CD4.

Reviewer #2 (Public Review):

Lentiviral infection of primate species has been linked to the rapid mutational evolution of numerous primate genes that interact with these viruses, including genes that inhibit lentiviruses as well as genes required for viral infection. In this manuscript, Warren et al. provide further support for the diversification of CD4, the lentiviral entry receptor, to resist lentiviral infection in great ape populations. This work builds on their prior publication (Warren et al. 2019, PMCID: PMC6561292 ) and that of other groups (e.g., Russell et al. 2021, PMCID: PMC8020793; Bibollet-Ruche et al. 2019, PMCID: PMC6386711) documenting both sequence and functional diversity in CD4, specifically within (1) the CD4 domain that binds to the lentiviral envelope and (2) great ape populations with endemic lentiviruses. Thus, the paper's finding that gorilla populations exhibit diverse CD4 alleles that differ in their susceptibility to lentiviral infection is well demonstrated both here and in a prior publication.

To bolster the argument that lentiviruses are indeed the causative driver of this diversification, which seems likely from a logical perspective but is difficult to prove, Warren et al. pursue two novel lines of evidence. First, the authors reconstruct ancestral CD4 genes that predate lentiviral infection of hominid populations. They then demonstrate that resistance to lentiviral infection is a derived trait in chimpanzees and gorillas, which have been co-evolving with endemic lentiviruses, but not in humans, which only recently acquired HIV. Nevertheless, the derived resistance could be stochastic or due to drift. This argument would be strengthened by demonstrating that bonobo and orangutan CD4, which also do not have endemic lentiviruses, resemble the ancestral and human susceptibility to great-ape-infecting lentiviruses.

Second, Warren et al. provide a population genetic argument that only endemically infected primates exhibit diversifying selection, again arguing for endemic lentiviruses being the evolutionary driver. The authors compare SNP occurrence in CD4 to neighboring genes, demonstrating that non-synonymous SNP frequency is only elevated in endemically infected species. Moreover, these amino-acid-coding changes are significantly concentrated in the CD4 domain that binds the lentiviral envelope. This is a creative analysis to overcome the problem of very small sample sizes, with very few great ape individuals sequenced. The additional small number of species compared (2-3 in each group) also limits the power of the analysis; the authors could consider expanding their analysis to Old World Monkey species that do or do not have endemic lentiviruses, as well as great apes.

Overall, this manuscript lends additional support to a well-documented example of a host-virus arms race: that of lentiviruses and the viral entry receptor.