HIV-1 Protein Nef Inhibits Activity of ATP-binding Cassette Transporter A1 by Targeting Endoplasmic Reticulum Chaperone Calnexin*

Background: HIV-1 Nef inhibits activity of ABCA1 and suppresses cholesterol efflux. Results: Nef binds to calnexin and disrupts its interaction with ABCA1 but enhances calnexin interaction with viral gp160. Conclusion: Nef regulates activity of calnexin to favor maturation of HIV gp160 at the expense of ABCA1. Significance: Learning how Nef regulates activity of calnexin is crucial for designing therapeutic strategies aimed at treating HIV infection and HIV-associated atherosclerosis. HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered high density lipoprotein profile exacerbated by down-modulation and impairment of ATP-binding cassette transporter A1 (ABCA1) activity by the HIV-1 protein Nef. However, the mechanisms of this Nef effect remain unknown. Here, we show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which regulates folding and maturation of glycosylated proteins. Nef disrupted interaction between calnexin and ABCA1 but increased affinity and enhanced interaction of calnexin with HIV-1 gp160. The Nef mutant that did not bind to calnexin did not affect the calnexin-ABCA1 interaction. Interaction with calnexin was essential for functionality of ABCA1, as knockdown of calnexin blocked the ABCA1 exit from the endoplasmic reticulum, reduced ABCA1 abundance, and inhibited cholesterol efflux; the same effects were observed after Nef overexpression. However, the effects of calnexin knockdown and Nef on cholesterol efflux were not additive; in fact, the combined effect of these two factors together did not differ significantly from the effect of calnexin knockdown alone. Interestingly, gp160 and ABCA1 interacted with calnexin differently; although gp160 binding to calnexin was dependent on glycosylation, glycosylation was of little importance for the interaction between ABCA1 and calnexin. Thus, Nef regulates the activity of calnexin to stimulate its interaction with gp160 at the expense of ABCA1. This study identifies a mechanism for Nef-dependent inactivation of ABCA1 and dysregulation of cholesterol metabolism.

[1]  D. Y. Thomas,et al.  Saccharomyces cerevisiae CNE1 Encodes an Endoplasmic Reticulum (ER) Membrane Protein with Sequence Similarity to Calnexin and Calreticulin and Functions as a Constituent of the ER Quality Control Apparatus (*) , 1995, The Journal of Biological Chemistry.

[2]  F. Kashanchi,et al.  Stimulation of the Liver X Receptor Pathway Inhibits HIV-1 Replication via Induction of ATP-Binding Cassette Transporter A1 , 2010, Molecular Pharmacology.

[3]  S. Yokoyama,et al.  Effects of Mutations of ABCA1 in the First Extracellular Domain on Subcellular Trafficking and ATP Binding/Hydrolysis* 210 , 2003, The Journal of Biological Chemistry.

[4]  J. Caramelo,et al.  Getting In and Out from Calnexin/Calreticulin Cycles* , 2008, Journal of Biological Chemistry.

[5]  J. Badimón,et al.  Cardiovascular implications of HIV-induced dyslipidemia. , 2011, Atherosclerosis.

[6]  H. Low,et al.  Cholesterol efflux assay. , 2012, Journal of visualized experiments : JoVE.

[7]  B. Peterlin,et al.  Structure–function relationships in HIV‐1 Nef , 2001, EMBO reports.

[8]  I. Braakman,et al.  Folding of the human immunodeficiency virus type 1 envelope glycoprotein in the endoplasmic reticulum. , 2001, Biochimie.

[9]  D. Keppler,et al.  Reconstitution of Transport-Active Multidrug Resistance Protein 2 (MRP2; ABCC2) in Proteoliposomes , 2002, Biological chemistry.

[10]  Angela Grant,et al.  Mutation of the ATP cassette binding transporter A1 (ABCA1) C-terminus disrupts HIV-1 Nef binding but does not block the Nef enhancement of ABCA1 protein degradation. , 2010, Biochemistry.

[11]  B. Moss,et al.  Calreticulin Interacts with Newly Synthesized Human Immunodeficiency Virus Type 1 Envelope Glycoprotein, Suggesting a Chaperone Function Similar to That of Calnexin (*) , 1996, The Journal of Biological Chemistry.

[12]  R. Swanstrom,et al.  Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo , 1993, Journal of virology.

[13]  D. Clarke,et al.  Prolonged association of temperature-sensitive mutants of human P-glycoprotein with calnexin during biogenesis. , 1994, The Journal of biological chemistry.

[14]  F. Kirchhoff,et al.  Human Immunodeficiency Virus Type 1 Inhibits DNA Damage-Triggered Apoptosis by a Nef-Independent Mechanism , 2005, Journal of Virology.

[15]  J. Riordan,et al.  Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator. , 1994, The Journal of biological chemistry.

[16]  M. Simm,et al.  Aberrant Gag protein composition of a human immunodeficiency virus type 1 vif mutant produced in primary lymphocytes , 1995, Journal of virology.

[17]  K. Collins,et al.  Human Immunodeficiency Virus Type 1 Nef: Adapting to Intracellular Trafficking Pathways , 2006, Microbiology and Molecular Biology Reviews.

[18]  M. Suico,et al.  Delta F508 CFTR pool in the endoplasmic reticulum is increased by calnexin overexpression. , 2003, Molecular biology of the cell.

[19]  F. Momburg,et al.  Multiple Residues in the Transmembrane Helix and Connecting Peptide of Mouse Tapasin Stabilize the Transporter Associated with the Antigen-processing TAP2 Subunit* , 2007, Journal of Biological Chemistry.

[20]  T. Nagase,et al.  Human ABCA1 contains a large amino-terminal extracellular domain homologous to an epitope of Sjögren's Syndrome. , 2001, Biochemical and biophysical research communications.

[21]  K. J. Henley,et al.  ABCA12 regulates ABCA1-dependent cholesterol efflux from macrophages and the development of atherosclerosis. , 2013, Cell metabolism.

[22]  F. Kashanchi,et al.  Liver X receptor agonist inhibits HIV-1 replication and prevents HIV-induced reduction of plasma HDL in humanized mouse model of HIV infection. , 2012, Biochemical and biophysical research communications.

[23]  Tracey M. Filzen,et al.  HIV-1 Nef Disrupts Intracellular Trafficking of Major Histocompatibility Complex Class I, CD4, CD8, and CD28 by Distinct Pathways That Share Common Elements , 2011, Journal of Virology.

[24]  R. Lempicki,et al.  Retinoic acid and liver X receptor agonist synergistically inhibit HIV infection in CD4+ T cells by up-regulating ABCA1-mediated cholesterol efflux , 2012, Lipids in Health and Disease.

[25]  M. Bukrinsky,et al.  The ABCA1 domain responsible for interaction with HIV-1 Nef is conformational and not linear. , 2014, Biochemical and biophysical research communications.

[26]  Y. Liu,et al.  Dissecting glycoprotein quality control in the secretory pathway. , 2001, Trends in biochemical sciences.

[27]  I. Tabas,et al.  ABCA1-mediated Cholesterol Efflux Is Defective in Free Cholesterol-loaded Macrophages , 2002, The Journal of Biological Chemistry.

[28]  M. Jackson,et al.  The molecular chaperone calnexin facilitates folding and assembly of class I histocompatibility molecules. , 1996, The EMBO journal.

[29]  Xiao-Fang Yu,et al.  Characterization of the Biosynthesis of Human Immunodeficiency Virus Type 1 Env from Infected T-cells and the Effects of Glucose Trimming of Env on Virion Infectivity* , 2001, The Journal of Biological Chemistry.

[30]  S. Matsushita,et al.  Characterization of a human immunodeficiency virus neutralizing monoclonal antibody and mapping of the neutralizing epitope , 1988, Journal of virology.

[31]  S. Reddy,et al.  HDL and cardiovascular disease: atherogenic and atheroprotective mechanisms , 2011, Nature Reviews Cardiology.

[32]  M. R. Leach,et al.  Localization of the Lectin, ERp57 Binding, and Polypeptide Binding Sites of Calnexin and Calreticulin* , 2002, The Journal of Biological Chemistry.

[33]  M. Robert-Guroff,et al.  Humoral immune response to the entire human immunodeficiency virus envelope glycoprotein made in insect cells. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[34]  P. A. Peterson,et al.  MHC class I molecules form ternary complexes with calnexin and TAP and undergo peptide-regulated interaction with TAP via their extracellular domains , 1996, The Journal of experimental medicine.

[35]  U. Danilczyk,et al.  The Lectin Chaperone Calnexin Utilizes Polypeptide-based Interactions to Associate with Many of Its Substrates in Vivo * , 2001, The Journal of Biological Chemistry.

[36]  R. Xavier,et al.  SPTLC1 binds ABCA1 to negatively regulate trafficking and cholesterol efflux activity of the transporter. , 2008, Biochemistry.

[37]  H. Kusuhara,et al.  Calreticulin facilitates the cell surface expression of ABCG5/G8. , 2006, Biochemical and biophysical research communications.

[38]  K. Ueda,et al.  The ABCA1 Q597R mutant undergoes trafficking from the ER upon ER stress. , 2008, Biochemical and biophysical research communications.

[39]  D. Y. Thomas,et al.  The Structure of calnexin, an ER chaperone involved in quality control of protein folding. , 2001, Molecular cell.

[40]  M. Bukrinsky,et al.  Circulating Nef induces dyslipidemia in simian immunodeficiency virus-infected macaques by suppressing cholesterol efflux. , 2010, The Journal of infectious diseases.

[41]  M. Desvarieux,et al.  Infection duration and inflammatory imbalance are associated with atherosclerotic risk in HIV-infected never-smokers independent of antiretroviral therapy , 2013, AIDS.

[42]  R. Guieu,et al.  Mapping of Domains on HIV Envelope Protein Mediating Association with Calnexin and Protein-disulfide Isomerase* , 2010, The Journal of Biological Chemistry.