Phylogenetic Reconstruction and Functional Characterization of the Ancestral Nef Protein of Primate Lentiviruses

Abstract

Nef is an accessory protein unique to the primate HIV-1, HIV-2, and SIV lentiviruses. During infection, Nef functions by interacting with multiple host proteins within infected cells to evade the immune response and enhance virion infectivity. Notably, Nef can counter immune regulators such as CD4 and MHC-I, as well as the SERINC5 restriction factor in infected cells. In this study, we generated a posterior sample of time-scaled phylogenies relating SIV and HIV Nef sequences, followed by reconstruction of ancestral sequences at the root and internal nodes of the sampled trees up to the HIV-1 Group M ancestor. Upon expression of the ancestral primate lentivirus Nef protein within CD4+ HeLa cells, flow cytometry analysis revealed that the primate lentivirus Nef ancestor robustly downregulated cell-surface SERINC5, yet only partially downregulated CD4 from the cell surface. Further analysis revealed that the Nef-mediated CD4 downregulation ability evolved gradually, while Nef-mediated SERINC5 downregulation was recovered abruptly in the HIV-1/M ancestor. Overall, this study provides a framework to reconstruct ancestral viral proteins and enable the functional characterization of these proteins to delineate how functions could have changed throughout evolutionary history.

Keywords: ancestral reconstruction; human immunodeficiency virus; negative effector protein; primate lentivirus.

Fig. 1.

The ancestral primate lentivirus (PLV) Nef protein potently downregulates cell-surface SERINC5, but only modestly downregulates cell-surface CD4. CD4${}^{+}$ HeLa cells coexpressing C-terminally eGFP-tagged ancestral PLV Nef, SIVmac239 Nef or the 2410 and 2391 primary HIV-1 Nef proteins with SERINC5.intHA were stained for cell-surface human SERINC5 and human CD4 and levels were quantified by flow cytometry. Single transfected CD4${}^{+}$ HeLa cells were determined by gating on eGFP${}^{+}$ cells using a fluorescence minus one (FMO) control. (A) Schematic of the gating strategy used to determine the transfected (eGFP${}^{+}$) cell population. (B) Representative pseudocolor plots illustrating cell-surface SERINC5 (AlexaFluor647; upper panel) and CD4 (PE; lower panel) levels of single cell populations. (C) Representative Western blots illustrating the expression of Nef-eGFP fusion proteins in the absence (upper panel) and presence (lower panel) of SERINC5 in CD4${}^{+}$ HeLa cells. (D) Representative histograms illustrating cell-surface SERINC5 and CD4 levels on CD4${}^{+}$ HeLa cells after gating on single and eGFP${}^{+}$ cells. Geometric MFIs of the cell-surface proteins are indicated on the x axis. (E) Summary of the fold Nef-eGFP expression (±SE) and the fold downregulation ability (±SE) for cell-surface SERINC5, and cell-surface CD4 from three independent experiments ($n=3$). Note—eGFP, enhanced green fluorescent protein; AF647, AlexaFluor647; PE, phycoerythrin; SE, standard error; MFI, mean fluorescence intensity; Mw, molecular weight; NT, nontransfected; kDa, kilodaltons; GAPDH, glyceraldehyde 3-phopshate dehydrogenase. Asterisks under dotted line: Significance fold difference with respect to eGFP only. Asterisks on top of boxplots: Significance fold difference with respect to PLV ancestor. Significance codes: $P<0.001$ “***”, $P<0.01$ “**”, $P<0.05$ “*” (adjusted P values reported from Tukey HSD).

Fig. 2.

Effect of ancestral Nef proteins on cell-surface SERINC5 and CD4 levels. CD4${}^{+}$ HeLa cells coexpressing the PLV Nef-eGFP protein, SIVmac239 Nef-eGFP, 2410 Nef-eGFP or intermediary nodes 51 (HIV-1/SIVsun), 52 (HIV-1/SIVcpz), 55 (HIV-1/SIVcpzptt), 56 (HIV-1/M, N/SIVcpzptt), and 59 (HIV-1/M) Nef-eGFP with SERINC5.intHA were stained for cell-surface human SERINC5 and human CD4. Subsequent cell-surface levels of SERINC5 and CD4 were then determined using flow cytometry. Single transfected CD4${}^{+}$ HeLa cells were determined by gating on eGFP${}^{+}$ cells using an FMO control. (A) and (B) Representative pseudocolor plots illustrating cell-surface SERINC5 (AlexaFluor647) and CD4 (PE) levels of single cell populations. (C) Representative Western blot illustrating the expression of Nef-eGFP fusion proteins in the presence of SERINC5 in CD4${}^{+}$ HeLa cells. (D) Fold Nef-eGFP expression (±SE) in CD4${}^{+}$ HeLa cells ($n=3$). (E) Representative histograms illustrating cell-surface SERINC5 and CD4 levels on CD4${}^{+}$ HeLa cells after gating on single, eGFP${}^{+}$ cells. Geometric MFIs of the cell-surface proteins are indicated on the x axis. Note—eGFP, enhanced green fluorescent protein; AF647, AlexaFluor647; PE, phycoerythrin; SE, standard error; FMO, fluroescence minus one; MFI, mean fluorescence intensity; Mw, molecular weight; kDa, kilodalton; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Asterisks under dotted line: Significance fold difference with respect to eGFP only. Asterisks on top of boxplots: Significance fold difference among adjacent groups. Significance codes: $P<0.001$ “***”, $P<0.01$ “**”, $P<0.05$ “*” (adjusted P values reported from Tukey HSD).

Fig. 3.

Predicting the evolution of Nef-mediated SERINC5 and CD4 cell-surface downregulation. (A) Maximum clade credibility tree (MCC) of 1,000 BEAST trees with consistent nodes used to obtain ancestral Nef sequences. (B) Fold downregulation ability (±SE) for cell-surface SERINC5 (orange), and cell-surface CD4 (blue) of the Nef ancestral proteins from the root (PLV Nef) to the tip (HIV-1/M Nef). Note—eGFP, enhanced green fluorescent protein. Asterisks under dotted line: Significance fold difference with respect to eGFP only. Asterisks on top of boxplots: Significance fold difference among adjacent groups. Significance codes: $P<0.001$ “***”, $P<0.01$ “**”, $P<0.05$ “*” (adjusted P values reported from Tukey HSD).