A single gp120 residue can affect HIV-1 tropism in macaques

<div><p>Species-dependent variation in proteins that aid or limit virus replication determines the ability of lentiviruses to jump between host species. Identifying and overcoming these differences facilitates the development of animal models for HIV-1, including models based on chimeric SIVs that express HIV-1 envelope (Env) glycoproteins, (SHIVs) and simian-tropic HIV-1 (stHIV) strains. Here, we demonstrate that the inherently poor ability of most HIV-1 Env proteins to use macaque CD4 as a receptor is improved during adaptation by virus passage in macaques. We identify a single amino acid, A281, in HIV-1 Env that consistently changes during adaptation in macaques and affects the ability of HIV-1 Env to use macaque CD4. Importantly, mutations at A281 do not markedly affect HIV-1 Env neutralization properties. Our findings should facilitate the design of HIV-1 Env proteins for use in non-human primate models and thus expedite the development of clinically relevant reagents for testing interventions against HIV-1.</p></div

Amino acid changes in the CD4 binding site of gp120.

<p><b>(A)</b> Amino acid sequence alignments of the CD4 binding site of HIV-1 gp120 proteins prior to (top) and following passage in macaques of 1054. Clones obtained from animal P1A at weeks 2, 9 and 35 post-inoculation are represented by sequences P1(Aw2), P1(Aw9) and P1A(w35) respectively. Individual clones from animal P1A obtained from week 17 post-inoculation and subsequent weeks and passages are shown. Residues are numbered as in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006572#ppat.1006572.ref037" target="_blank">37</a>] and residues shown to interact directly with CD4 are in black. (B) Amino acid sequence alignments as in (A) of AD8, SF162, 89.6, SF33 and C109 HIV-1 Env aa sequences prior to (top) and following <i>in vivo</i> adaptation (bottom). <b>(C)</b> Structure of gp120 bound to soluble human CD4 (modified from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006572#ppat.1006572.ref037" target="_blank">37</a>] using PyMol). Surface representation of HIV-1 gp120 is shown in grey, with aa A281 with in red. Stick representation of huCD4 in light purple with residue N39 shown as green spheres. Calculated distance between gp120-A281 and huCD4-N39 is shown. The magenta asterisk marks Phe43 in CD4 (that is conserved between human and macaque proteins) involved in the interaction with gp120.</p

Role of aa 281 in HIV-1 gp120 use of macaque CD4.

<p><b>(A)</b> SHIV infectivity on cells expressing human or macaque CD4 variants. SHIVs expressing parental or A281T mutant 1054 Env proteins were titrated on indicator Helios cells expressing hu/rh/pgtCD4. Infectivity was quantified by measuring luciferase expression in infected cells as Relative Light Units (RLU) and plotted as the ratio of RLU obtained in rh/pgtCD4-Helios over RLU obtained in huCD4Helios. Average and standard deviation from two independent experiments is shown. (<b>B)</b> Replication of SHIVs expressing the parental or mutant 1054 Env proteins in huPBMC, rhPBMC and rhesus 221 immortalized T cells. SHIV stocks were normalized for RT and 100pg RT of each virus was used per 1x10⁵ activated hu/rhPBMC. For 221 cells 50pg RT of each virus was used per 1x10⁵ cells. Replication was measured by RT in samples collected at the indicated times post-inoculation. <b>(C)</b> Infectivity of SHIVs expressing parental or mutant AD8 Env proteins as in (A). The differences between AD8 and AD8-N283T reached statistical significance *P = 0.02 and **P = 0.004. <b>(D)</b> Replication of SHIVs expressing the parental or mutant AD8 Env proteins as in (B).</p

Env protein neutralization profile.

<p>Neutralization sensitivity of parental and mutant HIV-1 Env proteins used in this study. Neutralization tier phenotype was determined as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006572#ppat.1006572.ref043" target="_blank">43</a>]. Neutralization sensitivity/resistance to soluble huCD4 (sCD4) and antibodies targeting the epitopes indicated is shown as the concentration (μg/ml) at which relative luminescence units were reduced 50% compared to virus control wells. For PG9 and PG16, 5μg/ml was the highest antibody concentration tested.</p

<i>In vitro</i> replication of SHIVs expressing adapted 1054 Env proteins.

<p>Replication of SHIVs expressing the parental or adapted 1054 Env proteins in huPBMC, rhPBMC and rhesus 221 immortalized T cells. SIV_MAC was used as control. Individual SHIV stocks were normalized for RT and 100pg RT of each virus was used per 1x10⁵ activated hu/rhPBMC. For 221 cells 50pg RT of each virus was used per 1x10⁵ cells. Replication was measured by RT in samples collected at the indicated times post-inoculation.</p

Role of aa 281 in macCD4 use of divergent HIV-1 envelope proteins.

<p><b>(A)</b> SHIV infectivity on cells expressing human or macaque CD4 variants. SHIVs expressing the indicated parental or A281T mutant HIV-1 Env proteins were titrated on indicator Helios cells expressing hu/rh/pgtCD4. Infectivity was quantified by measuring luciferase expression in infected cells as Relative Light Units (RLU) and plotted as the ratio of RLU obtained in rh/pgtCD4-Helios over RLU obtained in huCD4Helios. Average and standard deviation from two independent experiments is shown. (<b>B)</b> Replication of SHIVs expressing the parental or mutants Env proteins indicated in huPBMC and rhesus 221 immortalized T cells. SHIV stocks were normalized for RT and 100pg RT of each virus was used per 1x10⁵ activated hu/rhPBMC. For 221 cells 50pg RT of each virus was used per 1x10⁵ cells. Replication was measured by RT in samples collected at the indicated times post-inoculation.</p

Adaptation of 1054 Env <i>in vivo</i>.

<p><b>(A)</b> Schematic representation of <i>in vivo</i> passaging of SHIV₁₀₅₄. Animals indicated by inverted cartoons were euthanized for clinical cause including AIDS defining symptoms and the time of euthanasia (weeks post-inoculation) is indicated under each animal. Colors indicate passage and animal designation A/B and the same color scheme is maintained throughout the figures. (<b>B)</b> Plasma viremia in SHIV₁₀₅₄ infected rhesus macaques. Grey arrow indicates the time point at which blood was taken to initiate the subsequent passage. (<b>C)</b> Phylogenetic analysis of Env sequences isolated from plasma samples of macaques infected with SHIV₁₀₅₄ and subsequent animal passages outlined in (A). Clones analyzed in this study are highlighted.</p