Human immunodeficiency virus type 1 Nef associates with lipid rafts to downmodulate cell surface CD4 and class I major histocompatibility complex expression and to increase viral infectivity.
Lipid rafts are membrane microdomains that are functionally distinct from other membrane regions. We have shown that 10% of human immunodeficiency virus type 1 (HIV-1) Nef expressed in SupT1 cells is present in lipid rafts and that this represents virtually all of the membrane-associated Nef. To determine whether raft targeting, rather than simply membrane localization, has functional significance, we created a Nef fusion protein (LAT-Nef) containing the N-terminal 35 amino acids from LAT, a protein that is exclusively localized to rafts. Greater than 90% of the LAT-Nef protein was found in the raft fraction. In contrast, a mutated form, lacking two cysteine palmitoylation sites, showed less than 5% raft localization. Both proteins were equally expressed and targeted nearly exclusively to membranes. The LAT-Nef protein was more efficient than its nonraft mutant counterpart at downmodulating both cell surface CD4 and class I major histocompatibility complex (MHC) expression, as well as in enhancing first-round infectivity and being incorporated into virus particles. This demonstrates that targeting of Nef to lipid rafts is mechanistically important for all of these functions. Compared to wild-type Nef, LAT-Nef downmodulated class I MHC nearly as effectively as the wild-type Nef protein, but was only about 60% as effective for CD4 downmodulation and 30% as effective for infectivity enhancement. Since the LAT-Nef protein was found entirely in rafts while the wild-type Nef protein was distributed 10% in rafts and 90% in the soluble fraction, our results suggest that class I MHC downmodulation by Nef may be performed exclusively by raft-bound Nef. In contrast, CD4 downmodulation and infectivity enhancement may require a non-membrane-bound Nef component as well as the membrane-bound form.
Alexander
Melissa
M
Myles H. Thaler Center for AIDS and Human Retrovirus Research, Carter Immunology Center, and the Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908, USA.
Bor
Yeou-cherng
YC
Ravichandran
Kodimangalam S
KS
Hammarskjöld
Marie-Louise
ML
Rekosh
David
D
D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism*; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D015536:Down-Regulation; D015693:Gene Products, nef / Q000378:metabolism*; D015497:HIV-1 / Q000472:pathogenicity*; D006367:HeLa Cells; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D054311:nef Gene Products, Human Immunodeficiency Virus
D015704:CD4 Antigens; D015693:Gene Products, nef; D015395:Histocompatibility Antigens Class I; D054311:nef Gene Products, Human Immunodeficiency Virus