Structural constraints on human immunodeficiency virus type 1 Nef function.
HIV-1 Nef is a multifunctional protein that exerts its activities through interactions with multiple cellular partners. Nef uses different domains and mechanisms to exert its functions including cell surface down-modulation of CD4 and MHC-I receptors and activation of the serine/threonine kinase PAK-2. We inserted tags at the C-terminus and proximal to the N-terminus of Nef and the effects on Nef's structure/function relationships were examined. We discovered significant defects in MHC-I down-modulation with the insertion of HA/FLAG tags at either region. We also found impaired PAK-2 activation with a C-terminal fusion with GFP. Interestingly, Nef-GFP and Nef-GH(7) induced MHC-I down-modulation, suggesting that the negative charge of the HA/FLAG tag could contribute to the observed defect. Together, these observations highlight elements of Nef's functional complexity and demonstrate previously unsuspected structural requirements for PAK-2 activation and MHC-1 down-modulation in Nef's flexible N- and C-terminal regions.
Raney
Alexa
A
University of Texas Southwestern Medical Center at Dallas, TX 75390, USA.
Shaw
Alice Y
AY
Foster
John L
JL
Garcia
J Victor
JV
D015704:CD4 Antigens / Q000378:metabolism*; D002460:Cell Line; D015536:Down-Regulation; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D011493:Protein Kinase C / Q000378:metabolism*; D017433:Protein Structure, Secondary; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D015704:CD4 Antigens; D015395:Histocompatibility Antigens Class I; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; C061570:protein kinase N; D011493:Protein Kinase C