NEF - Negative Factor

HIV NEF interacts with host cell signal transduction proteins, downregulates expression of cell-surface proteins (CD4 and MHC Class I), and can induce apoptosis in non-infected cells.



PDB-2NEF: HIV-1 (isolate BH10) Nef protein fragment (Deletion of 2-39 and 159-173) with C206A mutation - produced using SwissModel/SPDBV/POVray

Key web links:
ViralZone: HIV-1, HIV replication cycle, HIV resource
PDB: 2NEF (HIV-1 NEF)
UniProt: P04601 (HIV-1 HXB2 NEF)
Chime Tutorial: Online Macromolecular Museum
HIV-1/Human Protein Interaction DB: HIV-1 Nef
Los Alamos HIV structure DB: NEF
EMBL: K03455 [EMBL/GenBank/DDBJ]


Isoforms:

  • p27-p25 (123 amino acids)


Cleavage sites:


Function:

  • Nef binds to the clathrin adaptor protein-1 (AP-1) and MHC-I to form a stable complex that allows AP-1 to direct MHC-I endocytosis and degradation.
  • Class I MHC downregulation is beneficial, since it decreases the efficiency of death by cytotoxic T cells .
  • Nef reduces MHC-I at the cell surface but synthesis and presense of MHC-I at the ER and Golgi apparatus occur normally .
  • Down-regulates CD4 (T4) antigen .
  • Increases rate of CD4 endocytosis and lysosomal degradation .
  • Nef binds to and modulates T-cell antigen receptor (TCR) signalling .
  • Nef prevents apoptosis by interacting with Apoptosis Signal-Regulating Kinase 1 (ASK1), which is involved in apoptotic signaling by TNF and Fas-ligand .
  • Induces the expression of the Fas ligand .

Localization:

  • Cell plasma membrane
  • Cell cytoplasm

Additional Information:

  • Early timing of expression.
  • Also known as F-protein or 3'ORF.
  • Nef has been shown to bind to a subset of the Src family of kinases .
  • Nef is expressed from multiply-spliced mRNA (therefore is Rev-independent).
  • Nef is the first viral protein to accumulate to detectable levels in the cell following cellular infection .
  • CD4 downregulation is beneficial for HIV infectivity, since excess CD4 on the cell surface inhibits Env incorporation and virion budding .
  • CD4 downregulation and increased viral infectivity are genetically distinct, and relate to different domains .
  • Nef mimics the action of a CD40 ligand, stimulating B cells to express cell surface receptors (CD22, CD34, and CD58) that induce resting CD4 cells to become active.
  • Nef is involved in the control of synctia formation.
  • GTP-binding and autophosphorylating activity.

Nef Function & Host-Virus Protein Interactions:[TOP]




Interactions highlighted in the image:


Potential interactions (not in the image):


Genomic Location & Protein Sequence: [TOP]

HIV-1 (HXB2):

          10         20         30         40         50         60         70         80         90        100
| | | | | | | | | |
MGGKWSKSSV IGWPTVRERM RRAEPAADRV GAASRDLEKH GAITSSNTAA TNAACAWLEA QEEEEVGFPV TPQVPLRPMT YKAAVDLSHF LKEKGGLEGL
110 120
| |
IHSQRRQDIL DLWIYHTQGY FPD
[download in fasta format]

Length: 123 amino acids
Molecular Weight: 13692 Da
Theoretical pI: 6.42
Gene Description: This isolate contains a mutation in position 124, which produces a stop codon. The HXB2 NEF isolate is much shorter than other isolates (~210 aa). Nef is encoded by a single exon that extends into the 3'LTR.


Protein Domains/Folds/Motifs: [TOP]

InterPro signature for Nef - IPR001558

Nef core domain

  • resembles the helix-turn-helix family of DNA-binding proteins
  • consists of 3 alpha-helices and a 5-stranded anti-parallel beta-sheet
  • one of the alpha-helices contains 4 prolines
  • hydrophobic packing is observed between alpha-helix 1 and alpha-helix 2
  • hydrophobic interactions are observed between alpha-helix 3 and beta-strand 1 and beta-strand 2

Secondary Structure prediction:

Low Complexity Regions - seg:


Antigenic Sites - EMBOSS:

Predicted Motifs: Printer-friendly version

N-glycosylation:
N-myristoylation:
Amidation:
Protein kinase C:
Casein kinase II:
Tyrosine kinase:
cAMP / cGMP kinase:
Cell attachment motif:
Asp Protease motif:
Asp Prot Retro motif:
Cysteine-rich Region:
Tryptophan-rich Region:
Zinc-finger CCHC motif:
Leucine Zipper motif:

Primary and Secondary Database Entries: [TOP]

Identifiers:



ViralZone: HIV-1, HIV replication cycle, HIV resource
PDB/MMDB: Search for HIV-1 & NEF

UniProt: P04601 (HIV-1 HXB2 NEF)
EMBL: K03455; AAB50263.1; [EMBL/GenBank/DDBJ]

InterPro: IPR001558
Pfam: PF00469 - "F-protein"
Prints: none
SCOP: SSF55671 Regulatory factor Nef / SSF81624 N-terminal domain of MutM-like DNA repair protein
Prosite: P04601
ProtoNet: P04601
Database of Interacting Proteins: P04601
ModBase: P04601
HIV-1/Human Protein Interaction DB: HIV-1 Nef
HIV-1 Sequence Database Los Alamos HIV Sequence Database


PDB:




Reviews and References: [TOP]

Cite the resource by citing the following paper:
Doherty R et al. BioAfrica's HIV-1 Proteomics Resource: Combining protein data with bioinformatics tools. Retrovirology (2005), 9;2(1):18.

1 - HIV Sequence Compendium 2000
Kuiken CL, Foley B, Hahn B, Korber B, Marx PA, McCutchan F, Mellors JW, Mullins JI, Sodroski J, Wolinksy S.
Theoretical Biol. & Biophys. Group, Los Alamos Nat Lab, LA-UR 01-3860 [Read it online: Compendium]
2 - Retroviruses
Coffin JM, Hughes SH, Varmus HE.
CD-ROM ed. (2002) Cold Spring Harbor Laboratory Press [Read it online: NCBI Bookshelf]
3 - Molecular Characteristics of HIV-1 Subtype C Viruses from KwaZulu-Natal, South Africa:
Implications for Vaccine and Antiretroviral Control Strategies.
Gordon M, De Oliveira T, Bishop K, Coovadia HM, Madurai L, Engelbrecht S, Janse van Rensburg E, Mosam A, Smith A, Cassol S.
Journal of Virology 77(4): 2587-2599 (2003) [pubmed: 12551997]
4 - HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product.
Guy B, Kieny M.P, Riviere Y, Le Peuch C, Dott K, Girard M, Montagnier L, Lecocq J.P.
Nature 330: 266-269 (1987) [pubmed: 3118220]
5 - 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 signalling.
Arnold S, Franken P, Strub M.P, Hoh F, Benichou S, Benarous R, Dumas C.
Structure 5: 1361-1372 (1997) [pubmed: 9351809]
6 - Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection:
Evidence for differential gene expression.
Kim SY, Byrn R, Groopman J.
J Virol 63: 3708-3713 (1989) [pubmed: 2760980]
7 - Downregulation of cell surface CD4 by nef.
Garcia JV, Miller AD.
Res Virol 143: 52-55 (1992) [pubmed: 1565858]
8 - Nef induces CD4 endocytosis: Requirement for a critical dileucine motif in the membrane-proximal
CD4 cytoplasmic domain.
Aiken C, Konner J, Landau NR.
Cell 76: 853-864 (1994) [pubmed: 8124721]
9 - Cell-surface expression of CD4 reduces HIV-1 infectivity by blocking Env incorporation
in a Nef- and Vpu-inhibitable manner.
Lama J, Mangasarian A, Trono D.
Curr Biol. 9: 622-31 (1999) [pubmed: 10375528]
10 - Inhibition of HIV-1 progeny virion release by cell-surface CD4 is relieved by expression
of the viral Nef protein.
Ross TM, Oran AE, Cullen BR.
Curr Biol. 9: 613-21 (1999) [pubmed: 10375525]
11 - Endocytosis of major histocompatibility complex class I molecules is induced by
the HIV-1 Nef protein.
Schwartz O, Marechal V, Le Gall S.
Nature Medicine 2: 338-342 (1996) [pubmed: 8612235]
12 - Expression of the type 1 human immunodeficiency virus Nef protein in T cells prevents
antigen receptor-mediated induction of interleukin 2 mRNA.
Luria S, Chambers I, Berg P.
Proc Natl Acad Sci USA 88: 5326-5330 (1991) [pubmed: 2052609]
13 - Producer-cell modification of human immunodeficiency virus type 1: Nef is a virion protein.
Pandori MW, Fitch NJ, Craig HM, et al.
J Virol 70: 4283-4290 (1996) [pubmed: 8676450]
14 - Dissociation of the CD4 downregulation and viral infectivity enhancement functions of
human immunodeficiency virus type 1 Nef.
Goldsmith MA, Warmerdam MT, Atchison RE.
J Virol 69: 4112-4121 (1995) [pubmed: 7769669]
15 - HIV-1 Nef: Negative effector of Fas?
Xu X, Screaton G.
Nature Immunology 2(5): 384-385 (2001) [pubmed: 11323689]
16 - Crystal Structure of the Conserved Core of HIV-1 Nef Complexed with a SRC Family SH3 Domain.
Lee C, Saksela K, Mirza UR, Chait BT, Kuriyan J.
Cell 16: 931-42 (1996) [pubmed: 8681387]
17 - The solution structure of HIV-1 Nef reveals an unexpected fold and permits delineation
of the binding surface for the SH3 domain of Hck tyrosine protein kinase.
Grzesiek S, Bax A, Clore GM, Gronenborn AM, Hu J, Kaufman J, Palmer I, Stahl SJ, Wingfield PT.
Nature Structural Biology 3(4): 340-45 (1996) [pubmed: 8599760]
18 - [HIV-1 Nef Review] Dynamic Nef and Nef dynamics: how structure could explain the complex
activities of this small HIV protein.
Arold ST, Baur AS.
Trends Biochem Sci 26(6): 356-363 (2001) [pubmed: 11406408]
19 - Domain assembly, surface accessibility and sequence conservation in full length HIV-1 Nef.
Geyer M, Peterlin BM.
FEBS Lett 496(2-3): 91-95 (2001) [pubmed: 11356189]
20 - Direct binding of human immunodeficiency virus type 1 Nef to the major histocompatibility
complex class I (MHC-I) cytoplasmic tail disrupts MHC-I trafficking.
Williams M, Roeth JF, Kasper MR, Fleis RI, Przybycin CG, Collins KL.
J Virol 76(23): 12173-12184 (2002) [pubmed: 12414957]
21 - Structure of the anchor-domain of myristoylated and non-myristoylated HIV-1 Nef protein.
Geyer M, Munte CE, Schorr J, Kellner R, Kalbitzer HR.
J Mol Biol 289(1):123-138 (1999) [pubmed: 10339411]
22 - HIV-1 accessory proteins adapt cellular adaptors to facilitate immune evasion.
Collins DR, Collins KL.
PLoS Pathog. 10(1):e1003851 (2014) [pubmed: 24465204]
23 - Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein.
Schwartz O, Maréchal V, Le Gall S, Lemonnier F, Heard JM.
Nat Med. 2(3):338-42 (1996) [pubmed: 8612235]
24 - Nef-arious goings-on at the Golgi.
Hurley JH, Bonifacino JS.
Nat Struct Mol Biol. 19(7):661-2 (2012) [pubmed: 22767237]
25 - Identification of a novel binding site between HIV type 1 Nef C-terminal flexible loop and AP2 required for Nef-mediated CD4 downregulation.
Jin YJ, Cai CY, Mezei M, Ohlmeyer M, Sanchez R, Burakoff SJ.
AIDS Res Hum Retroviruses 29(4):725-31 (2013) [pubmed: 23151229]
26 - HIV-1 Nef: a multifaceted modulator of T cell receptor signaling.
Abraham L, Fackler OT.
Cell Commun Signal. 10(1):39 (2012) [pubmed: 23227982]
27 - Direct in vitro binding of full-length human immunodeficiency virus type 1 Nef protein to CD4 cytoplasmic domain.
Preusser A, Briese L, Baur AS, Willbold D.
J Virol. 75(8):3960-4 (2001) [pubmed: 11264384]
28 - Cooperative binding of the class I major histocompatibility complex cytoplasmic domain and human immunodeficiency virus type 1 Nef to the endosomal AP-1 complex via its mu subunit.
Noviello CM, Benichou S, Guatelli JC.
J Virol. 82(3):1249-58 (2008) [pubmed: 18057255]
29 - Identification of a novel binding site between HIV type 1 Nef C-terminal flexible loop and AP2 required for Nef-mediated CD4 downregulation.
Jin YJ, Cai CY, Mezei M, Ohlmeyer M, Sanchez R, Burakoff SJ.
AIDS Res Hum Retroviruses 29(4):725-31 (2013) [pubmed: 23151229]
30 - Direct binding of human immunodeficiency virus type 1 Nef to the major histocompatibility complex class I (MHC-I) cytoplasmic tail disrupts MHC-I trafficking.
Williams M, Roeth JF, Kasper MR, Fleis RI, Przybycin CG, Collins KL.
J Virol. 76(23):12173-84 (2002) [pubmed: 12414957]
31 - Interaction of HIV-1 Nef protein with the host protein Alix promotes lysosomal targeting of CD4 receptor.
Amorim NA, da Silva EM, de Castro RO, da Silva-Januário ME, Mendonça LM, Bonifacino JS, da Costa LJ, daSilva LL.
J Biol Chem. 289(40):27744-56 (2014) [pubmed: 25118280]
32 - A novel acyl-CoA thioesterase enhances its enzymatic activity by direct binding with HIV Nef.
Watanabe H, Shiratori T, Shoji H, Miyatake S, Okazaki Y, Ikuta K, Sato T, Saito T.
Biochem Biophys Res Commun. 238(1):234-9 (1997) [pubmed: 9299485]
33 - An interdomain binding site on HIV-1 Nef interacts with PACS-1 and PACS-2 on endosomes to down-regulate MHC-I.
Dikeakos JD, Thomas L, Kwon G, Elferich J, Shinde U, Thomas G.
Mol Biol Cell 23(11):2184-97 (2012) [pubmed: 22496420]
34 - HIV-1 Nef binds PACS-2 to assemble a multikinase cascade that triggers major histocompatibility complex class I (MHC-I) down-regulation: analysis using short interfering RNA and knock-out mice.
Atkins KM, Thomas L, Youker RT, Harriff MJ, Pissani F, You H, Thomas G.
J Biol Chem. 283(17):11772-84 (2008) [pubmed: 18296443]
35 - A hydrophobic binding surface on the human immunodeficiency virus type 1 Nef core is critical for association with p21-activated kinase 2.
Agopian K, Wei BL, Garcia JV, Gabuzda D.
J Virol. 80(6):3050-61 (2006) [pubmed: 16501114]
36 - The human immunodeficiency virus type 1 Nef protein binds the Src-related tyrosine kinase Lck SH2 domain through a novel phosphotyrosine independent mechanism.
Dutartre H, Harris M, Olive D, Collette Y.
Virology 247(2):200-11 (1998) [pubmed: 9705913]
37 - SH3-mediated Hck tyrosine kinase activation and fibroblast transformation by the Nef protein of HIV-1.
Briggs SD, Sharkey M, Stevenson M, Smithgall TE.
J Biol Chem. 272(29):17899-902 (1997) [pubmed: 9218412]
38 - Human immunodeficiency virus impairs reverse cholesterol transport from macrophages.
Mujawar Z, Rose H, Morrow MP, Pushkarsky T, Dubrovsky L, Mukhamedova N, Fu Y, Dart A, Orenstein JM, Bobryshev YV, Bukrinsky M, Sviridov D.
PLoS Biol. 4(11):e365 (2006) [pubmed: 17076584]
39 - HIV-1 protein Nef inhibits activity of ATP-binding cassette transporter A1 by targeting endoplasmic reticulum chaperone calnexin.
Jennelle L, Hunegnaw R, Dubrovsky L, Pushkarsky T, Fitzgerald ML, Sviridov D, Popratiloff A, Brichacek B, Bukrinsky M.
J Biol Chem. 289(42):28870-84 (2014) [pubmed: 25170080]
40 - HIV's Nef interacts with Beta-catenin of the Wnt signaling pathway in HEK293 cells.
Weiser K, Barton M, Gershoony D, Dasgupta R, Cardozo T.
PLoS One 8(10):e77865 (2013) [pubmed: 24130899]
41 - Effect of myristoylation on p27 nef subcellular distribution and suppression of HIV-LTR transcription.
Yu G, Felsted RL.
Virology 187(1):46-55 (1992) [pubmed: 1736544]



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