Evolution toward beta common chain receptor usage links the matrix proteins of HIV-1 and its ancestors to human erythropoietin

Abstract

The HIV-1 matrix protein p17 (p17) is a pleiotropic molecule impacting on different cell types. Its interaction with many cellular proteins underlines the importance of the viral protein as a major determinant of human specific adaptation. We previously showed the proangiogenic capability of p17. Here, by integrating functional analysis and receptor binding, we identify a functional epitope that displays molecular mimicry with human erythropoietin (EPO) and promotes angiogenesis through common beta chain receptor (βCR) activation. The functional EPO-like epitope was found to be present in the matrix protein of HIV-1 ancestors SIV originated in chimpanzees (SIVcpz) and gorillas (SIVgor) but not in that of HIV-2 and its ancestor SIVsmm from sooty mangabeys. According to biological data, evolution of the EPO-like epitope showed a clear differentiation between HIV-1/SIVcpz-gor and HIV-2/SIVsmm branches, thus highlighting this epitope on p17 as a divergent signature discriminating HIV-1 and HIV-2 ancestors. P17 is known to enhance HIV-1 replication. Similarly to other βCR ligands, p17 is capable of attracting and activating HIV-1 target cells and promoting a proinflammatory microenvironment. Thus, it is tempting to speculate that acquisition of an epitope on the matrix proteins of HIV-1 ancestors capable of triggering βCR may have represented a critical step to enhance viral aggressiveness and early human-to-human SIVcpz/gor dissemination. The hypothesis that the p17/βCR interaction and βCR abnormal stimulation may also play a role in sustaining chronic activation and inflammation, thus marking the difference between HIV-1 and HIV-2 in term of pathogenicity, needs further investigation.

Keywords: HIV-1 and HIV-2 ancestors; HIV-1 evolutionary trajectory; HIV-1 matrix protein p17; common beta chain receptor; human erythropoietin.

Fig. 1.

Ability of different p17-derived peptides to induce angiogenesis and migration on HUVECs. (A) aa. sequence of eight p17-derived peptides. (B) HUVECs were cultured under stressed condition (EBM containing 0.5% fetal bovine serum [FBS]) for 16 h at 37 °C and then stimulated for 8 h at 37 °C with 10 ng/mL GST, p24, p17, or each p17-derived peptide (F1, F2, F3, F4, F5, F6, F7, F8). NT, not treated. (C) HUVECs were cultured under stressed condition for 16 h at 37 °C, and then the confluent cell monolayers were scratched and stimulated for 12 h at 37 °C with medium alone (NT) or with medium containing 10 ng/mL GST, p24, p17, or each p17-derived peptide (F1, F2, F3, F4, F5, F6, F7, F8). Images are representative of three independent experiments with similar results. (Original magnification, 10×.) Data are the mean ± SD of one representative experiment, of three with similar results, performed in triplicate. Statistical analysis was performed by one-way ANOVA, and the Bonferroni post hoc test was used to compare data (***P < 0.001).

Fig. 2.

EPO peptide and peptide F3-induced angiogenesis is mediated by βCR. (A) HUVECs were cultured under normal (EGM containing 10% FBS) or stressed conditions (EBM containing 0.5% FBS) for 16 h at 37 °C and then stimulated for 8 h at 37 °C with 5, 20, or 40 ng/mL EPO in complete medium. NT, not treated. (B, Upper) Peptide F3 has been modeled on the region of EPO showing the maximum rate of mimicry. (B, Lower) HUVECs were cultured and stimulated as above. NT, not treated. Values reported for tube formation are the mean ± SD of one representative experiment, of three with similar results, performed in triplicate. Statistical analysis was performed by one-way ANOVA, and the Bonferroni post hoc test was used to compare data (***P < 0.001). (C) Western blotting analysis (Left) performed 72 h after nucleofection of HUVECs with βCR siRNA (siβCR) and control siRNA (siScramble). The densitometric data (Right) are corrected by β-actin levels and expressed as percentage of siβCR (means ± SD, n = 4). Statistical analysis was performed by t test, ***P < 0.001 (siβCR vs. siScramble). (D) Seventy-two hours after nucleofection with siβCR or siScramble, HUVECs were stimulated for 8 h at 37 °C with 10 ng/mL CXCL8, EPO peptide, peptide F3S, or oligomeric p17. Values reported for tube formation are the mean ± SD of one representative experiment, of three with similar results, performed in triplicate. Statistical analysis was performed by one-way ANOVA, and the Bonferroni post hoc test was used to compare data (**P < 0.01, ***P < 0.001).

Fig. 3.

Phylogenetic tree of 37–52 p17 fragment. The tree of fragment 37–52 p17 was obtained using the representative fragment for each cluster. (A) The HIV-1/SIVcpz and SIVgor, OWM SIV, and HIV-2/SIVsmm branches are highlighted in pink, yellow, and azure, respectively. (B) The cluster of SIVcpzPts (orange) with two SIVgsn (purple) is shown in area 1. SIVcpzPts here pointed out is the SIV closely related to HIV-1, between HIV1/SIVcpz-gor and OWM SIV/HIV2-SIVsmm branches. Color code: black, HIV1; red, SIVcpzPtt and SIVgor; orange, SIVcpzPts; fuchsia, SIVcol/wrc (colobus genus); purple, SIVgsn/mon/mus/asc/deb/lst/den/syk/blu/sol (cercopithecus genus); green, SIVsab/tan/ver/grv/mal (chlorocebus genus); brown, SIVmnd/drl (mandrillus genus); silver, SIVrcm/agi (cercocebus genus); olive, SIVtal; blue, SIVsmm; azure, HIV-2.

Fig. 4.

Identification of the p17 functional epitope for angiogenesis. (A) The phylogenetic tree of S1 subfragment shows a nonhomogeneous assembling of viruses. Areas 1–6: highlighted some branches and clades of the tree; different OWM SIV and SIVsmm groups with HIV-1. Area 1: HIV-1 (black), SIV cercopithecus (purple), SIV cercocebus (gray). Area 2: HIV-1 (black), SIVcpz/gor (red). Area 3: HIV-1 (black), SIV cercopithecus (purple), SIVsmm (blue). Area 4: HIV-1 (black), SIV cercopithecus (purple), SIV chlorocebus (green). Area 5: HIV-1 (black), SIVsmm (blue). Area 6: HIV-1 (black), SIVsmm (blue), SIVcpzPtt (orange), HIV-2 (azure). (B) The phylogenetic tree of S2 subfragment shows the typical evolutionary path of HIV-1/SIVs. Areas 1–5 highlight SIVcpz and SIVgor grouping together with HIV-1. Area 6 highlights the clade formed by SIVcpzPts and two SIVgsn grouping with HIV-1. Areas 1–2: HIV-1 (black), SIVcpzPts (orange). Areas 3–5: HIV-1 (black), SIVcpz/gor (red). Area 4: HIV-1 (black), SIVcpz/gor (red), SIVcpzPts (orange). Area 6: SIVcpzPts (orange), SIV cercopithecus (purple). Color code: black, HIV-1 and HIV-1/SIVcpz-gor; red, SIVcpzPtt and SIVgor; orange, SIVcpzPts; blue, SIVsmm; azure, HIV-2; purple, SIVgsn/mon/mus/asc/deb/lst/den/syk/blu/sol (cercopithecus genus); green, SIVsab/tan/ver/grv/mal (chlorocebus genus); brown, SIVmnd/drl (mandrillus genus); silver, SIVrcm/agi (cercocebus genus); olive, SIVtal; fuchsia, SIVcol and SIVwrc (piliocolobus/colobus genus). (C) HUVECs were cultured under normal condition and then stimulated for 8 h at 37 °C with 10 ng/mL peptide ASRELERF, peptide ASRELERFLLE, peptide AANELDRF, or peptide AANELDRFLLE. NT, not treated. Values reported for tube formation are the mean ± SD of one representative experiment, of three with similar results, performed in triplicate. Statistical analysis was performed by one-way ANOVA and the Bonferroni post hoc test was used to compare data (****P < 0.0001).

Fig. 5.

Hypothesis for the role of βCR in SIV suboptimal adaptation to the human host.