Separable functions of Nef disrupt two aspects of T cell receptor machinery: CD4 expression and CD3 signaling.
The Nef protein alters T cell receptor (TCR) signaling in T cells and is critical for the pathogenesis of AIDS. We used a transient expression assay in a human CD4+ T cell line to analyze the interaction of Nef with the TCR machinery. We show that, in addition to down-regulating CD4 expression on the cell surface, Nef blocks a receptor-proximal event in CD3 signaling. Analysis of a large number of mutant Nef proteins demonstrated that the effects of Nef on CD4 expression and on CD3 signaling are separable. The ability of Nef to block CD3 signaling was selectively abolished by mutations in the central part of the Nef protein and in particular by those known to disrupt the SH3 binding surface in the structured core of Nef. In contrast, the ability of Nef to down-regulate CD4 expression was selectively abolished by two clusters of mutations, one in the N-terminal and one in the C-terminal region of Nef. These two regions correspond to the two flexible loops in Nef as predicted by solution NMR analysis. We show that this general functional organization is conserved between the Nef proteins of the human and simian immunodeficiency viruses (HIV-1 and SIV). Our data demonstrate that Nef has at least two independent mechanisms to alter TCR function and thus may interfere with a range of T cell responses.
Iafrate
A J
AJ
Department in Molecular Genetics and Microbiology, SUNY at Stony Brook, NY 11794, USA.
Bronson
S
S
Skowronski
J
J
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics; D017252:CD3 Complex / Q000235:genetics / Q000502:physiology*; D015704:CD4 Antigens / Q000032:analysis*; D019556:COS Cells; D004268:DNA-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D015693:Gene Products, nef / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015497:HIV-1; D006801:Humans; D019169:Jurkat Cells; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D015302:Simian Immunodeficiency Virus; D051758:Transcription Factor TFIIH; D019473:Transcription Factors, TFII; D054311:nef Gene Products, Human Immunodeficiency Virus; D000099192:CD69 Antigens
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D015704:CD4 Antigens; D004268:DNA-Binding Proteins; D015693:Gene Products, nef; D037181:Lectins, C-Type; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011948:Receptors, Antigen, T-Cell; D051758:Transcription Factor TFIIH; D019473:Transcription Factors, TFII; D054311:nef Gene Products, Human Immunodeficiency Virus; D000099192:CD69 Antigens; C072546:GTF2H1 protein, human