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


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ViralZone:HIV-1
PDB: 2NEF (HIV-1 NEF)
SwissProt: P04601 (HIV-1 HXB2 NEF)
Chime Tutorial: Online Macromolecular Museum
Los Alamos HIV structure DB: NEF
EMBL: K03455 [EMBL/GenBank/DDBJ]
BioAfrica: Nef Protein Data Mining Tool


Isoforms:

  • p27-p25 (123 amino acids)


Cleavage site:


Localization:

  • Virion
  • Cell plasma membrane
  • Cell cytoplasm

Function:

  • GTP-binding activity
  • autophosphorylating activity
  • down-regulates CD4(T4) antigen (Ref. #7)
  • increases rate of CD4 endocytosis and lysosomal degradation (Ref. #8)
  • induce expression of the Fas ligand (Ref. #15)
  • prevent apoptosis by interacting with Apoptosis Signal-Regulating Kinase 1 (ASK1), which is involved in apoptotic signaling by TNF and Fas-ligand (Ref. #15)
  • increases efficiency of reverse transcription
  • enhances serine phosphorylation of HIV Matrix protein

Additional Information:

  • also known as F-protein
  • also known as 3'ORF
  • Nef is packaged into virions, where it is cleaved by viral protease during virion maturation (Ref. #13)
  • Nef has been shown to bind to a subset of the Src family of kinases (Ref. #5)
  • Nef is expressed from multiply-spliced mRNA (therefore is Rev-independent)
  • Nef is expressed early in HIV life cycle
  • Nef is the first viral protein to accumulate to detectable levels in cell following cellular infection (Ref. #6)
  • CD4 downregulation is beneficial for HIV infectivity, since excess CD4 on the cell surface inhibits Env incorporation and virion budding (Ref. #9 & #10)
  • Class I MHC downregulation is beneficial, since it decreases the efficiency of death by cytotoxic T cells (Ref. #11)
  • Nef has an negative effect on the induction of NF-kappa-beta (transcription factor) on IL-2 expression (Ref. #12)
  • CD4 downregulation and increased viral infectivity are genetically distinct, and relate to different domains (Ref. #14)
  • Apoptosis of uninfected cells may be induced through the expression of the Fas ligand on the surface of HIV-infected cells, stimulating the Fas-dependent apoptotic pathway in cells that come in contact with the ligand (Ref. #15)
  • Interactions between Nef and ASK1 prevent phosphorylation of downstream MAP kinases and JNK kinases involved in apoptotic signaling (Ref. #15)
  • 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

Genomic Location: [TOP]

Reference Sequences:

HIV-1 (HXB2):

          10         20         30         40         50         60         70
| | | | | | |
MGGKWSKSSV IGWPTVRERM RRAEPAADRV GAASRDLEKH GAITSSNTAA TNAACAWLEA QEEEEVGFPV
80 90 100 110 120
| | | | |
TPQVPLRPMT YKAAVDLSHF LKEKGGLEGL 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:

Protein-Protein Interactions: [TOP]

  • Protein kinase C phosphorylation at residues 15-17 (_TvR_), 80-82 (_TyK_), and 103-105 (_SqR_). [Prosite: PS00005]
  • Casein kinase II phosphorylation at residues 15-18 (_TvrE_). [Prosite: PS00006]
  • N-myristoylation at residues 2-7 (_GGkwSK_), 41-46 (_GAitSS_), 95-100 (_GGleGL_), and 99-104 (_GLihSQ_). [Prosite: PS00008]
  • Nef interacts with SH3 domain of Fyn cellular tyrosine kinase (subset of Src family of kinases) - (Ref. #5)
  • The SH3 domain forms H-bonds, ionic bonds and hydrophobic interactions with the polyproline alpha-helix of Nef (Ref. #16 & #17)
  • Some of the cellular signalling proteins that interact with Nef: Hck, Lck, CD4, MAPK, c-Raf1 kinase, p62/PAK/Serine kinase
  • A negatively charged Glutamic acid in Nef interacts with the cytoplasmic tail of CD4 and a dileucine repeat in its membrane proximal region - (Ref. #8)
  • Residues 60-71, 96-144, and 177-190 are critical for CD4 downregulation
  • The Nef/CD4 binding site overlaps the second alpha-helix, and links alpha-helix 1 and alpha-helix 2 of Nef. This region contains a hydrophobic patch, which is centered at the HIV protease cleavage site (Trp57, Leu58, Glu59)
  • Nef interacts with Apoptosis Signal-Regulating Kinase (ASK1) (Ref. #15)
  • Nef binds to clathrin adaptor complexes
  • Nef binds to NBP1, hACTE-III and NAF1, to mediate CD4 downregulation

Primary and Secondary Database Entries: [TOP]

Identifiers:

ViralZone: HIV-1
PDB/MMDB: Search for HIV-1 & NEF

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

PIR: UNKNOWN
HIV: K03455; NEF$HXB2
InterPro: IPR001558
Pfam: PF00469 - "F-protein"
Prints: none
ProDom: PD271875 (residues 1 - 38) / PD494424 (residues 1 - 23) / PD000034 (residues 1 - 34) /
PD592900 (residues 1 - 41) / PD614609 (residues 1 - 41) / PD000031 (residues 57 - 123) /
PD686891 (residues 62 - 103) /
SCOP: SSF55671 Regulatory factor Nef / SSF81624 N-terminal domain of MutM-like DNA repair protein
BLOCKS: P04601
Prosite: P04601
ProtoNet: P04601
ProtoMap: P04601
PRESAGE: P04601
Database of Interacting Proteins: P04601
ModBase: P04601
Swiss-2DPAGE: 2D gel

BioAfrica Tools:
- Nef Protein Data Mining Tool provides real-time analysis of HIV-1 Nef isolates
- HIV Structure BLAST searches for similar HIV sequences that have known structures
- HIV Proteomics Resource contains protein sequence and structure analysis tools

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: 00005286]
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]



Page last updated by Tulio de Oliveira.