Cell-based Fluorescence Complementation Reveals a Role for HIV-1 Nef Protein Dimerization in AP-2 Adaptor Recruitment and CD4 Co-receptor Down-regulation

被引:16
作者
Shu, Sherry T. [1 ]
Emert-Sedlak, Lori A. [1 ]
Smithgall, Thomas E. [1 ]
机构
[1] Univ Pittsburgh, Dept Microbiol & Mol Genet, Sch Med, Bridgeside Point 2,Ste 523,450 Technol Dr, Pittsburgh, PA 15219 USA
基金
美国国家卫生研究院;
关键词
VIRUS TYPE-1 NEF; CRYSTAL-STRUCTURE; SH3; DOMAIN; T-LYMPHOCYTES; INFECTION; SURFACE; REPLICATION; DETERMINANT; BINDING; AIDS;
D O I
10.1074/jbc.M116.770016
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The HIV-1 Nef accessory factor enhances viral infectivity, immune evasion, and AIDS progression. Nef triggers rapid down-regulation of CD4 via the endocytic adaptor protein 2 ( AP-2) complex, a process linked to enhanced viral infectivity and immune escape. Here, we describe a bimolecular fluorescence complementation ( BiFC) assay to visualize the interaction of Nef with AP-2 and CD4 in living cells. Interacting protein pairs were fused to complementary non-fluorescent fragments of YFP and co-expressed in 293T cells. Nef interactions with both CD4 and AP-2 resulted in complementation of YFP and a bright fluorescent signal by confocal microcopy that localized to the cell periphery. Co-expression of the AP-2 alpha subunit enhanced the Nef.AP-2 sigma 2 subunit BiFC signal and vice versa, suggesting that the AP-2 alpha-sigma 2 hemicomplex interacts cooperatively with Nef. Mutagenesis of Nef amino acids Arg-134, Glu-174, and Asp-175, which stabilize Nef for AP-2 alpha-sigma 2 binding in a recent co-crystal structure, substantially reduced AP-2 interaction without affecting CD4 binding. A dimerization-defective mutant of Nef failed to interact with either CD4 or AP-2 in the BiFC assay, indicating that Nef quaternary structure is required for CD4 and AP-2 recruitment as well as CD4 down-regulation. A small molecule previously shown to bind the Nef dimerization interface also reduced Nef interactions with AP-2 and CD4 and restored CD4 expression to the surface of HIV-infected cells. Our findings provide a mechanistic explanation for previous observations that dimerization-defective Nef mutants fail to down-regulate CD4 and validate the Nef dimerization interface as a target site for antiretroviral drug development.
引用
收藏
页码:2670 / 2678
页数:9
相关论文
共 41 条
[1]   NEF INDUCES CD4 ENDOCYTOSIS - REQUIREMENT FOR A CRITICAL DILEUCINE MOTIF IN THE MEMBRANE-PROXIMAL CD4 CYTOPLASMIC DOMAIN [J].
AIKEN, C ;
KONNER, J ;
LANDAU, NR ;
LENBURG, ME ;
TRONO, D .
CELL, 1994, 76 (05) :853-864
[2]   Interaction with the Src Homology (SH3-SH2) Region of the Src-family Kinase Hck Structures the HIV-1 Nef Dimer for Kinase Activation and Effector Recruitment [J].
Alvarado, John Jeff ;
Tarafdar, Sreya ;
Yeh, Joanne I. ;
Smithgall, Thomas E. .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2014, 289 (41) :28539-28553
[3]   The crystal structure of HIV-1 Nef protein bound to the Fyn kinase SH3 domain suggests a role for this complex in altered T cell receptor signaling [J].
Arold, S ;
Franken, P ;
Strub, MP ;
Hoh, F ;
Benichou, S ;
Benarous, R ;
Dumas, C .
STRUCTURE, 1997, 5 (10) :1361-1372
[4]   DOWN-REGULATION OF CELL-SURFACE CD4 EXPRESSION BY SIMIAN IMMUNODEFICIENCY VIRUS NEF PREVENTS VIRAL SUPER INFECTION [J].
BENSON, RE ;
SANFRIDSON, A ;
OTTINGER, JS ;
DOYLE, C ;
CULLEN, BR .
JOURNAL OF EXPERIMENTAL MEDICINE, 1993, 177 (06) :1561-1566
[5]   A Basic Patch on α-Adaptin Is Required for Binding of Human Immunodeficiency Virus Type 1 Nef and Cooperative Assembly of a CD4-Nef-AP-2 Complex [J].
Chaudhuri, Rittik ;
Mattera, Rafael ;
Lindwasser, O. Wolf ;
Robinson, Margaret S. ;
Bonifacino, Juan S. .
JOURNAL OF VIROLOGY, 2009, 83 (06) :2518-2530
[6]   HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes [J].
Collins, KL ;
Chen, BK ;
Kalams, SA ;
Walker, BD ;
Baltimore, D .
NATURE, 1998, 391 (6665) :397-401
[7]   Interaction of HIV-1 Nef with the cellular dileucine-based sorting pathway is required for CD4 down-regulation and optimal viral infectivity [J].
Craig, HM ;
Pandori, MW ;
Guatelli, JC .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1998, 95 (19) :11229-11234
[8]   GENOMIC STRUCTURE OF AN ATTENUATED QUASI-SPECIES OF HIV-1 FROM A BLOOD-TRANSFUSION DONOR AND RECIPIENTS [J].
DEACON, NJ ;
TSYKIN, A ;
SOLOMON, A ;
SMITH, K ;
LUDFORDMENTING, M ;
HOOKER, DJ ;
MCPHEE, DA ;
GREENWAY, AL ;
ELLETT, A ;
CHATFIELD, C ;
LAWSON, VA ;
CROWE, S ;
MAERZ, A ;
SONZA, S ;
LEARMONT, J ;
SULLIVAN, JS ;
CUNNINGHAM, A ;
DWYER, D ;
DOWTON, D ;
MILLS, J .
SCIENCE, 1995, 270 (5238) :988-991
[9]   Role of the CD4 Down-Modulation Activity of Nef in HIV-1 Infectivity [J].
Doria, Margherita .
CURRENT HIV RESEARCH, 2011, 9 (07) :490-495
[10]   Effector Kinase Coupling Enables High-Throughput Screens for Direct HIV-1 Nef Antagonists with Antiretroviral Activity [J].
Emert-Sedlak, Lori A. ;
Narute, Purushottam ;
Shu, Sherry T. ;
Poe, Jerrod A. ;
Shi, Haibin ;
Yanamala, Naveena ;
Alvarado, John Jeff ;
Lazo, John S. ;
Yeh, Joanne I. ;
Johnston, Paul A. ;
Smithgall, Thomas E. .
CHEMISTRY & BIOLOGY, 2013, 20 (01) :82-91