CD4 and major histocompatibility complex class I downregulation by the human immunodeficiency virus type 1 nef protein in pediatric AIDS progression

Abstract

The human immunodeficiency virus type 1 (HIV-1) nef gene is a crucial determinant in AIDS disease progression. Although several in vitro activities have been attributed to the Nef protein, identifying the one critical for in vivo pathogenicity remains elusive. In this study, we examined a large number of nef alleles derived at various time points from 13 perinatally infected children showing different progression rates: six nonprogressors (NPs), three slow progressors (SPs), and four rapid progressors (RPs). The patient-derived nef alleles were analyzed for their steady-state expression of a Nef protein, for their relative ability to downregulate cell surface expression of CD4 and major histocompatibility complex class I (MHC-I) and for their capacity to bind the clathrin adaptor AP-1 complex. We found that NP-derived nef alleles, compared to nef alleles isolated from SPs and RPs, had reduced CD4 and MHC-I downregulation activities. In contrast, SP- and RP-derived nef alleles did not differ and efficiently downregulated both CD4 and MHC-I. AP-1 binding was a conserved function of primary nef alleles not correlated with clinical progression. Defective Nef proteins from NPs, rather than sharing common specific changes in their sequences, accumulated various amino acid substitutions, mainly located outside the conserved domains previously associated with Nef biological properties. Our data indicate that Nef-mediated downregulation of cell surface CD4 and MHC-I significantly contributes to the expression of the pathogenic potential of HIV-1.

FIG. 1.

Expression levels of Nef proteins derived from NP, SP, and RP patients. Phoenix cells were transfected with an empty Pinco vector (lanes P), with a recombinant Pinco-nef vector expressing the nef gene of the NL4-3 viral strain (lanes N), or with Pinco vectors expressing nef alleles isolated at the indicated time points from NP, SP, and RP patients (numbers on lanes identify the clones). Lysates prepared from transfected cells were immunoblotted with anti-Nef serum (upper panels) as described in Materials and Methods. Following Nef protein detection, the blots were stripped and probed with anti-GFP antibodies to analyze transfection efficiency (lower panels). This figure is representative of three independent experiments.

FIG. 2.

Analysis of the expression of the RP4-5 and NP2-11 Nef variants. (A) Immunoblotting analysis of HA-tagged variants. Aliquots of total cell lysates from transfected Phoenix cells expressing NL4-3-derived, RP4-5, or NP2-11 Nef proteins, either without (−) or with an HA tag (+), were immunoblotted with anti-Nef serum (top), anti-HA (middle), and anti-GFP antibody (bottom). (B) Immunoblotting analysis of the RP4-5R124W and NP2-11L136P mutants. The R₁₂₄→W and L₁₃₆→P mutations were made in RP4-5 and NP2-11, respectively, giving RP4-5R124W and NP2-11L136P. The mutants were transfected, together with the original variants, in Phoenix cells, and lysates of transfected cells were analyzed by immunoblotting with anti-Nef serum (top) and anti-GFP antibody (bottom).

FIG. 3.

Downregulation of cell surface CD4 and MHC-I in cells expressing NL4-3 nef and six defective patient-derived nef alleles. HeLa-CD4⁺ cells (top panels) and RMAS-A2 cells (bottom panels) were infected with empty retrovirus (Pinco), with Pinco-nef (NL4-3), or with retroviruses expressing patient-derived nef alleles (two for each progression group) with aberrant activities (NP4-4, NP5-7, SP1-1, SP3-7, RP2-4, and RP4-11). After 40 h, expression on the cell surface of CD4 (top panels) or HLA-A2 (bottom panels) was analyzed together with GFP expression by two-color flow cytometry. The mean fluorescence intensity (MFI) values specific for CD4 or HLA-A2 of cells expressing medium levels of GFP fluorescence (gated in the R1 region) are indicated.

FIG. 4.

CD4 and MHC-I downregulation activity of Nef protein variants derived from NP, SP, and RP pediatric patients. Cells were infected with recombinant retroviruses expressing nef alleles isolated from nonprogressor (A), slow progressor (B), and rapid progressor patients (C), then stained, and analyzed as described in Materials and Methods and in the Fig. 3 legend. After gating for cells expressing medium GFP levels (R1), the geometric mean fluorescence for CD4 and MHC-I was determined for each sample as well as for Pinco- and Pinco-nef-infected cells. Values are expressed as a percentage of NL4-3 Nef activity of CD4 downregulation in HeLa-CD4⁺ cells (black bars) and MHC-I downregulation in RMAS-A2 cells (gray bars). The activity of each nef allele was tested in triplicate in three independent experiments. Reported values are the means and standard deviations (error bars) from three independent determinations in a representative experiment. For brevity, when two or three nef alleles from a patient predicted identical Nef proteins and consequently showed the same activity, data for only one allele are shown.

FIG. 5.

Two nef alleles from rapid progressor RP4 are highly active in MHC-I downregulation. RMAS-A2 cells were infected with small amounts of Pinco retroviruses expressing NL4-3, RP4-9, or RP4-11 Nef protein and analyzed by two-color flow cytometry (see Materials and Methods). The mean fluorescence intensity specific for HLA-A2 in cells expressing low and medium GFP levels (gated in R2 region) is reported. Data representative of one of three independent experiments are shown.

FIG. 6.

Binding of AP-1 to GST-Nef fusion proteins. GST alone, GST-Nef wild-type (NL4-3), or GST fused to the indicated Nef proteins derived from patients was immobilized on Sepharose beads and incubated with Jurkat cell lysates. Bound proteins were eluted, separated by SDS-PAGE, immunoblotted with anti-AP-1 γ subunit (top panels) or anti-GST antibody (lower panels), and quantified by densitometry. As a control, 20 μg of Jurkat cell lysate was loaded on the same gel. Relative AP-1 binding activity was calculated as the amount of AP-1 γ subunit normalized for the amount of GST fusion protein and expressed as a percentage of the value measured for GST-wild-type Nef. Data representative of one of three independent experiments are shown. CD4 and MHC-I downregulation activities relative to wild-type Nef are indicated.