The HIV-1 Nef protein suppresses multiple immune surveillance mechanisms to promote viral pathogenesis and is an attractive target for the development of novel therapeutics. A key function of Nef is to remove the CD4 receptor from the cell surface by hijacking clathrin- and AP2-dependent endocytosis. However, exactly how Nef does this has been elusive. Here, we describe the underlying mechanism as revealed by a 3.0Å crystal structure of a fusion protein comprised of Nef and the cytoplasmic domain of CD4 bound to the tetrameric AP2 complex. An intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. A pocket on Nef previously identified as crucial for recruiting class I MHC is also responsible for recruiting CD4, revealing a potential approach to inhibit two of Nef’s activities and sensitize the virus to immune clearance.

20The HIV-1 protein Nef suppresses multiple immune surveillance mechanisms to promote viral pathogenesis 1 . Individuals infected with HIV-1 encoding defective nef genes do not develop AIDS for decades 2,3 . A key target of Nef is the cellular receptor CD4. Although essential for viral entry into host cells, CD4 is problematic for the virus later in its replication cycle: CD4 disrupts processing of the viral glycoprotein, Env, inhibiting 25 infectivity 4 ; it interferes with the release of new virions 5,6 ; and it causes vulnerability to superinfection, causing premature cell death and limiting viral productivity 7 . Furthermore, binding of CD4 to Env exposes otherwise-concealed Env epitopes, rendering infected cells more susceptible to antibody-dependent cellular cytotoxicity and virus particles more susceptible to neutralizing antibodies 8-10 . HIV-1 has evolved strategies to mitigate these 30 problems. Newly synthesized CD4 is targeted in the endoplasmic reticulum by the viral Vpu protein for proteasomal degradation 11 . Surface-expressed CD4, in contrast, is targeted by Nef for endocytosis and lysosomal degradation 12-15 . Nef's effect on CD4 involves hijacking of clathrin adaptor complex 2 (AP2)-dependent endocytosis 16,17 . Although how Nef associates with a part of the tetrameric AP2 is understood 18 , a complete understanding 35 of the interaction, especially how CD4 is sequestered by Nef into a complex with AP2, has remained elusive. Here, we present a high-resolution crystal structure that describes the underlying mechanism. An intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. Strikingly, a pocket on Nef

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