Down-modulation of CD8αβ is a fundamental activity of primate lentiviral Nef proteins

Abstract

It is well established that the Nef proteins of human and simian immunodeficiency viruses (HIV and SIV) modulate major histocompatibility complex class I (MHC-I) cell surface expression to protect infected cells against lysis by cytotoxic T lymphocytes (CTLs). Recent data supported the observation that Nef also manipulates CTLs directly by down-modulating CD8αβ (J. A. Leonard, T. Filzen, C. C. Carter, M. Schaefer, and K. L. Collins, J. Virol. 85:6867-6881, 2011), but it remained unknown whether this Nef activity is conserved between different lineages of HIV and SIV. In this study, we examined a total of 42 nef alleles from 16 different primate lentiviruses representing most major lineages of primate lentiviruses, as well as nonpandemic HIV-1 strains and the direct precursors of HIV-1 (SIVcpz and SIVgor). We found that the vast majority of these nef alleles strongly down-modulate CD8β in human T cells. Primate lentiviral Nefs generally interacted specifically with the cytoplasmic tail of CD8β, and down-modulation of this receptor was dependent on the conserved dileucine-based motif and two adjacent acidic residues (DD/E) in the C-terminal flexible loop of SIV Nef proteins. Both of these motifs are known to be important for the interaction of HIV-1 Nef with AP-2, and they were also shown to be critical for down-modulation of CD4 and CD28, but not MHC-I, by SIV Nefs. Our results show that down-modulation of CD4, CD8β, and CD28 involves largely overlapping (but not identical) domains and is most likely dependent on conserved interactions of primate lentiviral Nefs with cellular adaptor proteins. Furthermore, our data demonstrate that Nef-mediated down-modulation of CD8αβ is a fundamental property of primate lentiviruses and suggest that direct manipulation of CD8+ T cells plays a relevant role in viral immune evasion.

Fig 1

Nef-mediated down-modulation of human CD8αβ is conserved between primate lentiviruses. (A) Flow cytometric analysis of CEM cells expressing A2-CD8β fusions, SupT1 cells, and primary CD8⁺ T cells transduced with HIV-1 recombinants expressing EGFP alone or together with the indicated nef alleles. The ranges of EGFP expression used to calculate the MFI of A2-CD8β fusion or CD8αβ expression are indicated. (B) Correlations between the efficiencies of Nef-mediated down-modulation of CD8αβ in primary CD8⁺ T cells and of A2-CD8β fusions in CEM cells or CD8αβ in SupT1 cells. (C) Correlations between the efficiencies of Nef-mediated down-modulation of CD8αβ in primary CD8⁺ T cells and of CD28, CD4, and MHC-I in primary CD4⁺ T cells. Each symbol represents average n-fold down-modulation (n = 3) of the indicated receptor molecule by one individual nef allele analyzed. An overview of the HIV and SIV nef alleles used is provided in Table S1 in the supplemental material.

Fig 2

Modulation of A2-CD8β fusions and other receptors by nef alleles from HIV-1 and its closest SIV counterparts. (A) Efficiencies of A2-CD8β fusion down-modulation by nef alleles from the indicated HIV-1 and SIV strains. Data shown are average values ± standard deviations (SD) derived from triplicate experiments. (B) Comparison of A2-CD8β fusion, MHC-I, CD4, and CD28 down-modulation by nef alleles from the indicated groups of primate lentiviruses. A2-CD8β fusion modulation was measured in CEM cells, and down-modulation of the remaining receptors was measured in primary CD4⁺ T cells. Each symbol represents the average for three independent measurements, and similar results were obtained in SupT1 cells.

Fig 3

Modulation of A2-CD8β fusions and other receptors by nef alleles from HIV-2, their SIVsmm and SIVmac counterparts, and other highly divergent primate lentiviruses. (A) Efficiencies of A2-CD8β fusion down-modulation by nef alleles from the indicated HIV-2 and SIV strains. (B) Comparison of A2-CD8β fusion, MHC-I, CD4, and CD28 down-modulation by nef alleles from the indicated groups of primate lentiviruses. oSIVs, SIVs that were not involved in the evolution of HIV-1 and HIV-2. A2-CD8β fusion modulation was measured in CEM cells, and down-modulation of the remaining receptors was measured in primary CD4⁺ T cells. Refer to the legend to Fig. 2 for further details.

Fig 4

Comparison of Nef-mediated receptor modulation by human and nonhuman primate lentiviruses. Quantitative assessment was performed to measure down-modulation of the A2-CD8β fusion (A), MHC-I (B), CD4 (C), and CD28 (D) by nef alleles, based on the species of origin (humans versus nonhuman primates) and biological properties (unable or able to down-modulate TCR-CD3) of the nef alleles examined. A2-CD8β fusion modulation was measured in CEM cells, and down-modulation of the remaining receptors was measured in primary CD4⁺ T cells. Abbreviations: Pr, precursor; Hu, human; NHP, nonhuman primate.

Fig 5

Importance of the EXXXLL motif in the C-terminal flexible loop for the modulation of cellular receptors by primate lentiviral Nef proteins. (A to D) CEM cells expressing the A2-CD8β fusion (A) or SupT1 cells (B to D) were transduced with VSV-G-pseudotyped vpu- and env-defective NL4-3-based constructs expressing the indicated nef alleles and assayed for surface expression of the A2-CD8β fusion (A), CD4 (B), CD28 (C), and MHC-I (D). Quantification was performed as described in Materials and Methods. Data shown are average values ± SD derived from two independent experiments. The inset in panel A shows an amino acid sequence comparison of the various EXXXLL-corresponding regions analyzed. The NL4-3 motif is shown as a reference at the top. Dots indicate amino acid identity. (E) Correlations between efficiencies of down-modulation of the A2-CD8β fusion on CEM cells and modulation of CD8αβ, CD4, CD28, and MHC-I on SupT1 cells.

Fig 6

Functional analysis of SIVagmSab Nef mutants. (A) The SIVagmSab Nef amino acid sequence is given in single-letter code. Mutated sequence motifs are highlighted by blue boxes, and positions of alanines are indicated. (B) CEM cells expressing A2-CD8β fusions or SupT1 cells were transduced with HIV-1 recombinants expressing EGFP alone or together with the indicated nef alleles and were assayed for surface expression of the A2-CD8β fusion, CD8αβ, CD4, CD28, and MHC-I. Quantification was performed as described in Materials and Methods. Data shown are average values ± SD derived from two independent experiments.

Fig 7

Functional analysis of SIVblu Nef mutants. (A) The SIVblu Nef amino acid sequence is given in single-letter code. The positions of single alanine substitutions are highlighted by blue boxes, and the domains affected by these mutations are indicated. (B and C) Jurkat T cells (B) or CEM cells (C) were transfected with pCGCG vectors expressing EGFP alone or together with the indicated nef alleles and were assayed for surface expression of CD4, CD28, MHC-I, and the A2-CD8β fusion. Quantification was performed as described in Materials and Methods. Data shown are average values ± SD derived from two independent experiments. (D) Correlations between efficiencies of down-modulation of the A2-CD8β fusion on CEM cells and of CD8αβ (left) or CD4 (right) on SupT1 cells. (E) Correlations between Nef-mediated down-modulation of CD8αβ on SupT1 cells and of CD4 (left) or CD28 (right) on Jurkat T cells.