Functional characterization of the human immunodeficiency virus type 1 Nef acidic domain.
The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef downregulates major histocompatibility complex class I (MHC-I) from the cell surface. It has been proposed that the direct interaction of the acidic cluster (AC) of Nef, (62)EEEE(65), with the furin binding region (fbr) of PACS-1 is crucial for this Nef function. Contrary to this proposal, evidence is presented here that the four glutamates in Nef do not functionally engage the PACS-1 fbr. (i) The binding of Nef to the PACS-1 fbr in vitro is much weaker than the binding of the canonical furin AC to the PACS-1 fbr. (ii) The mutation of two of the four glutamates in Nef's AC to alanines does not alter Nef's ability to downregulate MHC-I, and triply mutated Nefs exhibit 50% activity. (iii) The introduction of lysine into the AC has little effect on Nef function. (iv) The mutation of all four glutamates to alanine does debilitate Nef MHC-I downregulation, but this quadruple mutation also impairs the ability of Nef to regulate p21-activated protein kinase and enhance viral particle infectivity. (v) The replacement of the Nef AC with the bona fide AC from furin results in the loss of the expected regulatory properties of the furin AC. (vi) The insertion of the conformation-disrupting amino acid proline into the Nef AC does not disrupt MHC-I downregulation. Our results are consistent with an alternative model in which (62)EEEE(65) plays a stabilizing role in the formation of a ternary complex between Nef, the MHC-I cytoplasmic domain, and AP-1.
Baugh
Laura L
LL
Y9.206, Department of Internal Medicine, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
Garcia
J Victor
JV
Foster
John L
JL
D000595:Amino Acid Sequence; D015967:Gene Expression Regulation, Viral; D015693:Gene Products, nef / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D015395:Histocompatibility Antigens Class I / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018808:Transcription Factor AP-1 / Q000737:chemistry*
D015693:Gene Products, nef; D015395:Histocompatibility Antigens Class I; D018808:Transcription Factor AP-1