Identification of Owl Monkey CD4 Receptors Broadly Compatible with Early-Stage HIV-1 Isolates

Abstract

Most HIV-1 variants isolated from early-stage human infections do not use nonhuman primate versions of the CD4 receptor for cellular entry, or they do so poorly. We and others have previously shown that CD4 has experienced strong natural selection over the course of primate speciation, but it is unclear whether this selection has influenced the functional characteristics of CD4 as an HIV-1 receptor. Surprisingly, we find that selection on CD4 has been most intense in the New World monkeys, animals that have never been found to harbor lentiviruses related to HIV-1. Based on this, we sampled CD4 genetic diversity within populations of individuals from seven different species, including five species of New World monkeys. We found that some, but not all, CD4 alleles found in Spix's owl monkeys (Aotus vociferans) encode functional receptors for early-stage human HIV-1 isolates representing all of the major group M clades (A, B, C, and D). However, only some isolates of HIV-1 subtype C can use the CD4 receptor encoded by permissive Spix's owl monkey alleles. We characterized the prevalence of functional CD4 alleles in a colony of captive Spix's owl monkeys and found that 88% of surveyed individuals are homozygous for permissive CD4 alleles, which encode an asparagine at position 39 of the receptor. We found that the CD4 receptors encoded by two other species of owl monkeys (Aotus azarae and Aotus nancymaae) also serve as functional entry receptors for early-stage isolates of HIV-1.

Importance: Nonhuman primates, particularly macaques, are used for preclinical evaluation of HIV-1 vaccine candidates. However, a significant limitation of the macaque model is the fact that most circulating HIV-1 variants cannot use the macaque CD4 receptor to enter cells and have to be adapted to these species. This is particularly true for viral variants from early stages of infection, which represent the most relevant vaccine targets. In this study, we found that some individuals from captive owl monkey populations harbor CD4 alleles that are compatible with a broad collection of HIV-1 isolates, including those isolated from early in infection in highly affected populations and representing diverse subtypes.

FIG 1

Positive natural selection has shaped the CD4 D1 domain, particularly in the New World monkeys. (A) A schematic of the interaction between CD4 and MHC-II, alongside its cocrystal (PDB code: 1JL4) (49), where the sites under positive selection in CD4 (3) are represented by red spheres. APC, antigen-presenting cell. (B) Schematic of the interaction between CD4 and HIV-1 gp120, alongside its cocrystal (PDB code: 1RZJ) (50), where the sites under positive selection in CD4 (3) are represented by red spheres. (C) Evolutionary analysis of the D1 domain of CD4 showing the number of nonsynonymous and synonymous mutations (in parentheses; N:S) predicted to have occurred along each branch of a 31-species primate phylogeny. Branches with red text have a dN/dS ratio of >1, and this ratio is shown before the parentheses. This ratio cannot be calculated in cases where dS is 0. Primate species shown in bold text are known to be naturally infected with a simian or human immunodeficiency virus (51). Only one CD4 sequence per species was utilized in this analysis. Accession numbers for primate CD4 sequences can be found in Materials and Methods.

FIG 2

There are many nonsynonymous SNPs in the portion of CD4 encoding the D1 domain. (A) An amino acid alignment of the D1 domain from species included in the population study. Nonsynonymous SNPs identified are indicated with slashes, where the two alternately encoded amino acids are given on either side. Dotted lines separate the major clades of simian primates (Hom, hominoids; OWMs, Old World monkeys; NWMs, New World monkeys). The number of individuals analyzed from each species is shown in parentheses adjacent to the species name. Amino acid positions highlighted in yellow were previously identified to be evolving under positive selection (3). Numbering along the top is relative to the mature CD4 protein, after cleavage of the 25-amino-acid N-terminal signal peptide. Species shown in bold indicate populations that were sequenced in this study. Arrows indicate three sites that have been shown previously to affect HIV-1 entry (14, 17). (B) A table showing amino acids in the CD4 D1 domain that contact gp120 (blue) or MHC-II (orange), are under positive selection (red) (3), or are polymorphic for nonsynonymous mutations in human or nonhuman primate (NHP) populations included in the present study (gray). Asterisks along the bottom denote residue positions that are completely conserved in the 31 primate species shown in Fig. 1C. The table format is modified from reference .

FIG 3

Some Spix's owl monkey CD4 alleles encode receptors permissive for entry by early, nonadapted HIV-1 isolates. (A) Amino acid alignment of the D1 domain from six CD4 receptors tested for virus entry. Sites that are polymorphic in Spix's owl monkeys are highlighted in yellow. (B) Expression levels of CD4s stably introduced into Cf2Th/syn CCR5 cells as measured by flow cytometry using an APC-conjugated anti-CD4 antibody. (C) Cell lines expressing different CD4 alleles (indicated along the x axis) were infected with the indicated Env-pseudotyped virions. Env variants are labeled such that the first letter represents the subtype, followed by the strain name. Infection is indicated as percent GFP-positive cells measured by flow cytometry 72 h postinfection. All pseudotyped virions were generated using the subtype A-derived Q23ΔEnvGFP proviral clone. Error bars represent the standard deviations of the means from three independent experiments conducted in duplicate.

FIG 4

Some Spix's owl monkey CD4 receptors support entry by representative viruses of the major clades of HIV-1 group M. Cf2Th/syn CCR5 cells expressing various CD4 alleles (indicated along the x axis) were infected with the indicated Env-pseudotyped virions. Env variants are labeled such that the first letter represents the subtype, followed by the strain name. Infection was measured by flow cytometry as percentage GFP-positive cells 72 h postinfection. All pseudotyped virions were generated in the Q23ΔEnvGFP background. Error bars represent the standard deviations of the means from three independent experiments conducted in duplicate.

FIG 5

Subtype C Envs differentially utilize owl monkey CD4 for cellular entry. (A) Cf2Th/syn CCR5 cells expressing various CD4 alleles (indicated along the x axis) were infected with the indicated Env-pseudotyped virions. Infection was measured by flow cytometry as percent GFP-positive cells 72 h postinfection. All pseudotyped virions were generated in the Q23ΔEnvGFP background. Error bars represent the standard deviations of the means from three independent experiments conducted in duplicate. (B) A partial amino acid alignment is shown for the nine subtype C Envs used in this study, along with the reference subtype B HXB2 strain. Numbering is relative to HXB2. Amino acids that make direct contact with CD4 (22) are highlighted in gray. Asterisks indicate positions that are completely conserved. Stars indicate amino acid residues that were identified to be evolving under positive selection in previous analyses (7–9).

FIG 6

CD4 genotypes are shown for the Spix's owl monkey colony at the KCCMR. (A) Two Spix's owl monkeys from the KCCMR colony (photo credit: Julio Ruiz). (B) A pedigree illustrates the structure of the Aotus vociferans colony housed at the KCCMR. The CD4 genotype was determined for most living individuals, with a plus indicating the presence of a permissive CD4 allele and a minus indicating the presence of a nonpermissive CD4 allele. Squares indicate males, and circles indicate females. Diagonal slashes indicate deceased individuals. Genotypes in gray are deduced from progeny or parents, which in some cases yielded two possible genotypes as indicated.

FIG 7

CD4 from Azara's and Nancy Ma's owl monkeys are also permissive for HIV-1 entry. (A) Flow cytometry scatter plots showing CD4 expression of various primate alleles (indicated along the x axis) and human CCR5 expression (indicated along the y axis) in 293T cells. (B) 293T cells from panel A were infected with the indicated Env-pseudotyped virions. Infection was measured by flow cytometry as percent GFP-positive cells 72 h postinfection. All pseudotyped virions were generated in the Q23ΔEnvGFP background. Error bars represent the standard deviations obtained from duplicate wells. The results are representative of three independent experiments.