Dynamic evolution of the human immunodeficiency virus type 1 pathogenic factor, Nef

Abstract

The human immunodeficiency virus type 1 (HIV-1) early gene product Nef is a multifunctional protein that alters numerous pathways of T-cell function, including endocytosis, signal transduction, vesicular trafficking, and immune modulation, and is a major determinant of pathogenesis. Individual Nef functions include PAK-2 activation, CD4 downregulation, major histocompatibility complex (MHC) class I downregulation, and enhancement of viral particle infectivity. How Nef accomplishes its multiple tasks presents a difficult problem of mechanistic analysis because of the complications associated with multiple, overlapping functional domains in the context of significant sequence variability. To address these issues we determined the conservation of each Nef residue based on 1,643 subtype B Nef sequences. Mutational analysis based on conservative substitutions and Nef sequence data allowed us to search for amino acids on the surface of Nef that are specifically required for PAK-2 activation. We found residues 85, 89, and 191 to be highly significant determinants for Nef's PAK-2 activation function but functionally unlinked to CD4 and MHC class I downregulation or enhancement of infectivity. These residues are not conserved across HIV-1 subtypes but are confined to separate sets of surface elements within a subtype. Thus, L85/H89/F191 and F85/F89/R191 are dominant in subtype B and subtype E or C, respectively. Our results provide support for developing subtype-specific interventions in HIV-1 disease.

FIG. 1.

Compilation of subtype B Nef sequences. The amino acid positions 1 through 206 of the Nef protein are shown. For each position, the number of different sequences containing the indicated amino acid residue is shown followed by its letter designation. Gaps are represented as a dash. Adding up all the different residues and gaps observed for each position totals 1,643, the number of sequences analyzed.

FIG. 2.

Functional and structural relevance of residues R106 and D123 of Nef. (A) The indicated amino acid substitutions were derived by site-directed mutagenesis, cloned into pcDNA, and transfected into 293T cells. Lysates were immunoprecipitated with anti-Nef antiserum. Immunocomplexes were subjected to an in vitro kinase assay. Left, in vitro kinase assays for SF2Nef and SF2Nef mutants R106Q, R106K, R106A, R106L, R105A, and R105Q. Western blot analysis of Nef proteins present in cell lysates is shown in the lower panel. Right, in vitro kinase assays for SF2Nef and SF2NefD123E. Western blot analysis of Nef proteins present in cell lysates is shown in the lower panel. (B) CEM cells were transduced with the LXSN (empty) retrovirus vector or LXSN expressing SF2Nef and derived mutants. CD4 and MHC class I cell surface downregulation of transduced cells was then determined. Only transduced cells expressing high levels of Nef showed downregulation of MHC I. (C) SF2Nef-derived mutants were cloned into the HIV-1 SF2 provirus, and the infectivity of the resultant viruses was compared to that of SF2 and SF2ΔNef using HeLa-Magi cells. SF2 infectivity was set to 1.00.

FIG. 3.

The phenylalanine residue at position 191 of subtype B SF2Nef is essential for PAK-2 activation. SF2Nef and mutants were cloned into pcDNA (control) and transfected into 293T cells. Lysates were subjected to immunoprecipitation with Nef antiserum followed by an in vitro kinase assay (top). Western blot analysis of Nef proteins is shown in the lower panel.

FIG. 4.

Analysis of PAK-2 activation by subtype E Nefs. (A) Subtype E Nef-mediated activation of PAK-2. Subtype E Nefs cloned into the pcDNA expression vector were cotransfected with pcDNAHA-PAK-2 (7) into 293T cells. Lysates were subjected to immunoprecipitation with HA antiserum followed by in vitro kinase assay. As controls, pcDNA and pcDNAHA-PAK-2 were transfected alone. Western blot analysis of Nef proteins present in cell lysates is in the middle panel. Western blot analysis of HAPAK-2 present in cell lysates is in the lower panel. (B) CD4 and MHC class I cell surface downregulation of CEM cells transduced with LXSN (vector control) or LXSN with SF2 nef and the different subtype E nefs as indicated. (C) nefs from the indicated subtype E isolates were cloned into the SF2 provirus, and the infectivity of the resultant viruses was compared to that of SF2 and SF2ΔNef using HeLa-Magi cells. SF2 infectivity was set to 1.00.

FIG. 5.

Restoration of PAK-2 activation of SF2NefF191R. (A) Left, PAK-2 activation by subtype B Nef isolate RP-1-9 (accession number AAA87489). Top: amino acids observed at positions 85, 89, 188, and 191 for SF2Nef and RP-1-9 Nef. Middle: lysates were subjected to immunoprecipitation with Nef antiserum followed by an in vitro kinase assay. Bottom: Western blot analysis of Nef proteins present in cell lysates. Right, amino acids substituted at positions 85, 89, 188, and 191 in SF2Nef. Middle and bottom, same as for panel A, left. (B) CD4 and MHC class I cell surface downregulation of transduced CEM cells expressing SF2Nef and derived mutants. Vector (control) and LXSN with SF2 nef are included as references. L85F, H89F, K188A, and F191R are SF2Nef single mutants; LFKR is an SF2Nef double mutant; FFAR is an SF2Nef quadruple mutant. (C) SF2Nef-derived mutants were cloned into the HIV-1 SF2 provirus, and the infectivity of the resultant viruses was compared to that of SF2 and SF2ΔNef using HeLa-Magi cells. SF2 infectivity was set to 1.00.

FIG. 6.

Restoration of PAK-2 activation in SF2NefF191Y and SF2NefF191H. Lysates of transiently transfected 293T cells expressing SF2Nef (wild type), SF2NefF191Y, SF3NefL85R/H89F/F191Y, SF2NefF191H, and SF3NefL85F/H89F/K188H/F191H were subjected to immunoprecipitation with Nef antiserum followed by an in vitro kinase assay. Western blot analysis of Nef proteins present in cell lysates is shown in the lower panel.