Interaction of HIV-1 Nef with the cellular dileucine-based sorting pathway is required for CD4 down-regulation and optimal viral infectivity

Abstract

The HIV-1 Nef protein is important for pathogenesis, enhances viral infectivity, and regulates the sorting of at least two cellular transmembrane proteins, CD4 and major histocompatibility complex (MHC) class I. Although several lines of evidence support the hypothesis that the Nef protein interacts directly with the cellular protein sorting machinery, the sorting signal in HIV-1 Nef has not been identified. By using a competition assay that functionally discriminates between dileucine-based and tyrosine-based sorting signals, we have categorized the motif through which Nef interacts with the sorting machinery as dileucine-based. Inspection of diverse Nef proteins from HIV-1, HIV-2, and simian immunodeficiency virus revealed a well-conserved sequence in the central region of the C-terminal, solvent-exposed loop of Nef (E/DXXXLphi) that conforms to the consensus sequence of the dileucine-based sorting motifs found in cellular transmembrane proteins. This sequence in NefNL4-3, ENTSLL, functioned as an endocytosis signal when appended to the cytoplasmic tail of a heterologous protein. The leucine residues in this motif were required for the interaction of full-length Nef with the dileucine-based sorting pathway and were required for Nef-mediated down-regulation of CD4. These leucine residues were also required for optimal viral infectivity. These data indicate that a dileucine-based sorting signal in Nef is utilized to address the cellular sorting machinery. The data also suggest that an influence on the distribution of cellular transmembrane proteins may mechanistically unite two previously distinct properties of Nef: down-regulation of CD4 and enhancement of viral infectivity.

Figure 1

Effect of Nef on the surface expression of indicator proteins containing dileucine- or tyrosine-based sorting motifs. 293 cells were transfected with plasmids encoding the transmembrane indicator proteins Tac, IL-2 receptor-α chain (0.25 μg) (A); TTMb, a chimera containing the extracellular and transmembrane domains of Tac fused to a cytoplasmic domain containing a tyrosine-based motif (2.5 μg) (B and D); or Tac-DKQTLL, a fusion protein in which the dileucine-based motif DKQTLL is appended to the C terminus of the Tac cytoplasmic domain (2 μg) (C). A plasmid encoding the GFP (0.5 μg) was included as a transfection marker. Cells were stained with anti-CD25 (which recognizes Tac) conjugated to PE and analyzed by flow cytometry. The mean PE fluorescence (surface level of Tac) for each GFP channel number (transfection efficiency) is plotted for 293 cells transfected with each of the Tac indicator proteins with and without competitors. +Nef cells were transfected with 15 μg of Nef expression vector. +Lamp cells were transfected with 15 μg of Lamp1 expression vector.

Figure 2

The Nef sequence ENTSLL functions as an endocytosis signal. (A) Endocytosis rates of Tac-based indicator proteins. Tac and Tac-DKQTLL are described in the legend of Fig. 1. Tac-ENTSLL contains the putative dileucine-motif in Nef appended to the C terminus of Tac. Tac-ENTSAA is identical to Tac-ENTSLL except that the leucines have been replaced with alanines. 293 cells were transfected with plasmids encoding Tac (5 μg), Tac-DKQTLL (10 μg), Tac-ENTSLL (6 μg), or Tac-ENTSAA (4 μg) and with a GFP-expression plasmid (0.5 μg), then stained with PE-conjugated anti-CD25 and incubated at 37°C for the indicated times before removal of label associated with the cell surface by acid wash and analysis by flow cytometry. The percentage of internalized label at each time point was calculated as described. The following slope values were calculated by using linear regression analysis: Tac, 0.23; Tac-DKQTLL, 0.64; Tac-ENTSLL, 0.36; Tac-ENTSAA, 0.13. (B) Effect of Nef on the surface expression of Tac-ENTSLL and Tac-ENTSAA. The mean PE fluorescence (Tac surface level) for each GFP channel number (transfection efficiency) is plotted for 293 cells transfected with Tac-ENTSLL and Tac-ENTSAA. +Nef cells were transfected with 15 μg of Nef expression vector. The amounts of indicator plasmids were as follows: Tac-ENTSLL, 0.5 μg; and Tac-ENTSAA, 0.1 μg.

Figure 3

The effect of mutations in the ENTSLL sequence of Nef on surface displacement of Tac-DKQTLL. 293 cells were transfected with a plasmid encoding Tac-DKQTLL (1 μg), a GFP-expression plasmid (0.5 μg), and the indicated amounts of the Nef expression plasmids. Transfected cells were analyzed by flow cytometry. The mean PE fluorescence (Tac surface levels) of GFP-positive cells is graphed versus the amount of Nef-expression plasmid. pCI-NL encodes wild-type Nef. pCI-LL164/165AA encodes a mutant in which the leucine residues within the sequence ENTSLL (residues 164 and 165 of HIV-1 Nef_NL4-3) are replaced with alanine residues. pCI-E160A encodes a mutant in which the glutamic acid residue within the sequence ENTSLL (residue 160) is replaced by an alanine residue.

Figure 4

The effect of mutations in the ENTSLL sequence of Nef on Nef-mediated down-regulation of CD4. (A) CD4 down-regulation. 293 cells were transfected with a CD4 expression plasmid (1 μg), a GFP-expression plasmid (0.5 μg), and the indicated amounts of the Nef-expression plasmids. Cells were stained with PE-conjugated anti-CD4 and analyzed by flow cytometry. The mean PE fluorescence (CD4 surface levels) of GFP-positive cells is graphed versus the amount of Nef expression plasmid. (B) Western blot analysis of Nef expression. Cells (5 × 10⁴) analyzed in A and transfected with 2 μg of each Nef expression vector were lysed, separated electrophoretically, and analyzed as described (32).

Figure 5

The effect of mutations in the ENTSLL sequence of Nef on Nef-mediated enhancement of viral infectivity. The relative infectivities of cell-free virions produced from the indicated genomes were determined by using an infectious center assay in which CD4-positive HeLa cells were used as targets. The infectivities of the mutant virions were determined as described and are expressed relative to the wild type. WT, wild-type HIV-1_NL4-3; nef−, nef-negative mutant containing two premature termination codons in the 5′ terminus of the nef-ORF (7); E160A, mutant encoding alanine substitution of E160; LL164/165AA, mutant encoding alanine substitutions of L164A and L165A.