Subunit H of the V-ATPase binds to the medium chain of adaptor protein complex 2 and connects Nef to the endocytic machinery.
Nef is an accessory protein of human and simian immunodeficiency viruses (HIV and SIV) that is required for efficient viral infectivity and pathogenicity. It decreases the expression of CD4 on the surface of infected cells. V1H is the regulatory subunit H of the vacuolar membrane ATPase (V-ATPase). Previously, the interaction between Nef and V1H has been found to facilitate the internalization of CD4, suggesting that V1H could connect Nef to the endocytic machinery. In this study, we demonstrate that V1H binds to the C-terminal flexible loop in Nef from HIV-1 and to the medium chain (mu2) of the adaptor protein complex 2 (AP-2) in vitro and in vivo. The interaction sites of V1H and mu2 were mapped to a central region in V1H from positions 133 to 363, which contains 4 armadillo repeats, and to the N-terminal adaptin-binding domain in mu2 from positions 1 to 145. Fusing Nef to V1H reproduced the appropriate trafficking of Nef. This chimera internalized CD4 even in the absence of the C-terminal flexible loop in Nef. Finally, blocking the expression of V1H decreased the enhancement of virion infectivity by Nef. Thus, V1H can function as an adaptor for interactions between Nef and AP-2.
Geyer
Matthias
M
Department of Medicine, University of California, San Francisco, California 94143-0703, USA.
Yu
Haifeng
H
Mandic
Robert
R
Linnemann
Thomas
T
Zheng
Yong-Hui
YH
Fackler
Oliver T
OT
Peterlin
B Matija
BM
D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033963:Adaptor Protein Complex 3; D033981:Adaptor Protein Complex delta Subunits; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001665:Binding Sites; D015704:CD4 Antigens / Q000737:chemistry; D016827:CD8 Antigens / Q000096:biosynthesis; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D002469:Cell Separation; D004705:Endocytosis; D005434:Flow Cytometry; D015693:Gene Products, nef / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*
C499064:AP1M2 protein, human; C510070:AP3M2 protein, human; D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033963:Adaptor Protein Complex 3; D033981:Adaptor Protein Complex delta Subunits; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D015704:CD4 Antigens; D016827:CD8 Antigens; D002352:Carrier Proteins; D015693:Gene Products, nef; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C499065:adaptor protein complex 1, mu 2 subunit; C438886:adaptor protein complex 2, mu 2 subunit; D025262:Vacuolar Proton-Translocating ATPases