Reviewed, UniProtKB/Swiss-Prot P03406 (NEF_HV1BR)

Names and origin

Protein names

Recommended name:
    Protein Nef
Alternative name(s):
    Negative factor
      Short name=F-protein
    3'ORF
Cleaved into the following chain:
    1- Recommended name:
            C-terminal core protein

Gene names

Name:

nef

Organism

Human immunodeficiency virus type 1 (isolate BRU/LAI group M subtype B) (HIV-1)

Taxonomic identifier

11686 [NCBI]

Taxonomic lineage

Viruses › Retro-transcribing viruses › Retroviridae › Orthoretrovirinae › Lentivirus › Primate lentivirus group

Virus host

Homo sapiens (Human) [TaxID: 9606]

Protein attributes

Sequence length	206 AA.
Sequence status	Complete.
Sequence processing	The displayed sequence is further processed into a mature form.
Protein existence	Evidence at protein level.

General annotation (Comments)

Function	Factor of infectivity and pathogenicity, required for optimal virus replication. Alters numerous pathways of T-lymphocytes function and downregulates immunity surface molecules in order to evade host defense and increase viral infectivity. Alters the functionality of other immunity cells, like dendritic cells, monocytes/macrophages and NK cells. One of the earliest and most abundantly expressed viral proteins By similarity. In infected CD4+ T-lymphocytes, downregulates the surface MHC-I, mature MHC-II, CD4, CD28, CCR5 and CXCR4 molecules. Mediates internalization and degradation of host CD4 through the interaction of with the cytoplasmic tail of CD4, the recruitment of AP-2 (clathrin adapter protein complex 2), internalization through clathrin coated pits, and subsequent transport to endosomes and lysosomes for degradation. Diverts host MHC-I molecules to the trans-Golgi network-associated endosomal compartments by an endocytic pathway to finally target them for degradation. MHC-I down-regulation may involve AP-1 (clathrin adapter protein complex 1) or possibly Src family kinase-ZAP-70/Syk-PI3K cascade recruited by PACS2. In consequence infected cells are masked for immune recognition by cytotoxic T-lymphocytes. Decreasing the number of immune receptors also prevents reinfection by more HIV particles (superinfection). Bypasses host T-cell signaling by inducing a transcriptional program nearly identical to that of anti-CD3 cell activation. Interaction with TCR-zeta chain up-regulates the Fas ligand (FasL). Increasing surface FasL molecules and decreasing surface MHC-I molecules on infected CD4+ cells send attacking cytotoxic CD8+ T-lymphocytes into apoptosis By similarity. Ref.3 Ref.11 Ref.13 Ref.15 Ref.16 Plays a role in optimizing the host cell environment for viral replication without causing cell death by apoptosis. Protects the infected cells from apoptosis in order to keep them alive until the next virus generation is ready to strike. Inhibits the Fas and TNFR-mediated death signals by blocking MAP3K5. Interacts and decreases the half-life of p53, protecting the infected cell against p53-mediated apoptosis. Inhibits the apoptotic signals regulated by the Bcl-2 family proteins through the formation of a Nef/PI3-kinase/PAK2 complex that leads to activation of PAK2 and induces phosphorylation of Bad By similarity. Extracellular Nef protein targets CD4+ T-lymphocytes for apoptosis by interacting with CXCR4 surface receptors By similarity.
Subunit structure	Homodimer By similarity. Interacts with Nef associated p21-activated kinase (PAK2); this interaction activates PAK2. Associates with the Nef-MHC-I-AP1 complex; this complex is required for MHC-I internalization. Interacts (via C-terminus) with host PI3-kinase (via C-terminus). Interacts with host PACS1; this interaction seems to be weak. Interacts with host PACS2. Interacts with host LCK and MAPK3; these interactions inhibit the kinase activity of the latters. Interacts with host ATP6V1H; this interaction may play a role in CD4 endocytosis. Associates with the CD4-Nef-AP2 complex; this complex is required for CD4 internalization. Interacts with TCR-zeta chain; this interaction up-regulates the Fas ligand (FasL) surface expression. Interacts with various cellular proteins including MAP3K5, beta-COP, HCK, and PTE1 By similarity.
Subcellular location	Host cell membrane; Lipid-anchor; Cytoplasmic side By similarity. Host cytoplasm › host perinuclear region By similarity. Virion By similarity. Secreted By similarity. Note: Predominantly found in the paranuclear area, probably in the TGN. Correct localization requires PACS1. Also associates with the inner plasma membrane through its N-terminal domain. Nef stimulates its own export via the release of exosomes. Also incorporated in virions at a rate of about 10 molecules per virion, where it is cleaved By similarity. Ref.8 Ref.9
Domain	The N-terminal domain is composed of the N-myristoyl glycine and of a cluster of positively charged amino acids. It is required for inner plasma membrane targeting of Nef and virion incorporation, and thereby for infectivity. This domain is also involved in binding to p53 By similarity. The SH3-binding domain constituted of PxxP motifs mediates binding to several Src family proteins thereby regulating their tyrosine kinase activity. The same motifs also mediates the association with MAPK3, PI3-kinase and TCR-zeta By similarity. Ref.9 Ref.23 The di-leucine internalization motif and a diacidic motif seem to be required for binding to AP-2 By similarity. The acidic region may play a stabilizing role in the formation of a ternary complex between Nef, the MHC-I cytoplasmic domain, and AP1M1 By similarity.
Post-translational modification	The virion-associated Nef proteins are cleaved by the viral protease to release the soluble C-terminal core protein. Nef is probably cleaved concomitantly with viral structural proteins on maturation of virus particles By similarity. Ref.8 Ref.6 Phosphorylated on serine residues, probably by host PKC By similarity.
Miscellaneous	The infectious clone pNL4-3 is a chimeric provirus that consists of DNA from HIV isolates NY5 (5' half) and BRU (3' half). HIV-1 lineages are divided in three main groups, M (for Major), O (for Outlier), and N (for New, or Non-M, Non-O). The vast majority of strains found worldwide belong to the group M. Group O seems to be endemic to and largely confined to Cameroon and neighboring countries in West Central Africa, where these viruses represent a small minority of HIV-1 strains. The group N is represented by a limited number of isolates from Cameroonian persons. The group M is further subdivided in 9 clades or subtypes (A to D, F to H, J and K).
Sequence similarities	Belongs to the lentivirus primate group Nef protein family.

Ontologies

Keywords
Biological process	Apoptosis Host-virus interaction Viral immunoevasion Virulence
Cellular component	Host cell membrane Host cytoplasm Host membrane Membrane Secreted Virion
Developmental stage	Early protein
Disease	AIDS
Domain	SH3-binding
PTM	Lipoprotein Myristate Phosphoprotein
Technical term	3D-structure
Gene Ontology (GO)
Biological process	apoptosis Inferred from electronic annotation. Source: UniProtKB-KW evasion by virus of host immune response Inferred from electronic annotation. Source: UniProtKB-KW negative regulation by symbiont of host T-cell mediated immune response Inferred from direct assay. Source: UniProtKB negative regulation of CD4 biosynthetic process Inferred from direct assay. Source: UniProtKB pathogenesis Inferred from direct assay. Source: UniProtKB regulation of T cell activation Non-traceable author statement. Source: UniProtKB regulation of calcium-mediated signaling Inferred from direct assay. Source: UniProtKB suppression by virus of host MHC class I cell surface presentation Inferred from direct assay. Source: UniProtKB viral infectious cycle Inferred from direct assay. Source: UniProtKB
Cellular component	anchored to membrane Inferred from electronic annotation. Source: UniProtKB-SubCell cytoskeleton Inferred from direct assay. Source: UniProtKB cytosol Ref.12 Inferred from Experiment. Source: Reactome host cell perinuclear region of cytoplasm Inferred from electronic annotation. Source: UniProtKB-SubCell plasma membrane Inferred from direct assay. Source: UniProtKB virion Inferred from electronic annotation. Source: UniProtKB-SubCell
Molecular function	ATPase binding Inferred from physical interaction. Source: UniProtKB CD4 receptor binding Inferred from physical interaction. Source: UniProtKB GTP binding Inferred from electronic annotation. Source: InterPro MHC class I protein binding Inferred from physical interaction. Source: UniProtKB SH3 domain binding Ref.5 Ref.23 Inferred from physical interaction. Source: UniProtKB calmodulin binding Inferred from physical interaction. Source: UniProtKB protein kinase binding Ref.7 Ref.13 Inferred from physical interaction. Source: UniProtKB thioesterase binding Inferred from physical interaction. Source: UniProtKB
Complete GO annotation...

Sequence annotation (Features)

Feature key

Position(s)

Length

Description

Graphical view

Feature identifier

Molecule processing

Initiator methionine

1

1

Removed; by host

Chain

2 – 206

205

Protein Nef

PRO_0000038335

Chain

58 – 206

149

C-terminal core protein

PRO_0000038336

Regions

Region

2 – 57

56

N-terminal; associates with the host plasma membrane By similarity

Region

7 – 26

20

Necessary for MHC-I internalization By similarity

Region

62 – 65

4

Acidic; stabilizes the interaction of NEF/MHC-I with host AP1M1; necessary for MHC-I internalization and interaction with host PACS1 and PACS2 By similarity

Region

69 – 78

10

SH3-binding; interaction with Src family tyrosine kinases By similarity

Region

108 – 124

17

Mediates dimerization, Nef-PTE1 interaction, Nef-induced CD4 and MHC-I down-regulation and enhancement of infectivity By similarity

Region

148 – 180

33

Binding to ATP6V1H By similarity

Motif

72 – 75

4

PxxP; stabilizes the interaction of NEF/MHC-I with host AP1M1; necessary for MHC-I internalization By similarity

Motif

164 – 165

2

Di-leucine internalization motif; necessary for CD4 internalization By similarity

Motif

174 – 175

2

Diacidic; necessary for CD4 internalization By similarity

Compositional bias

62 – 65

4

Poly-Glu

Sites

Site

20

1

Might play a role in AP-1 recruitment to the Nef-MHC-I complex

Site

57 – 58

2

Cleavage; by viral protease

Amino acid modifications

Lipidation

2

1

N-myristoyl glycine; by host Probable

Natural variations

Natural variant

11

1

V → I in strain: Clone pNL4-3.

Natural variant

15

1

T → A in strain: Clone pNL4-3.

Natural variant

33

1

A → V in strain: Clone pNL4-3.

Natural variant

51

1

T → N in strain: Clone pNL4-3.

Experimental info

Mutagenesis

2 – 3

2

GG → AA: 70% loss of infectivity. Ref.3

Mutagenesis

20

1

M → A: Amost complete loss of Nef-induced MHC-I down-regulation.

Mutagenesis

20

1

M → R: Amost complete loss of Nef-induced MHC-I down-regulation.

Mutagenesis

71

1

T → R: Increases infectivity by a factor of three. Ref.3 Ref.14

Mutagenesis

72

1

P → A: Complete loss of binding to HCK SH3 domain and altered viral growth; when associated with P-76. No effect on Nef-induced CD4 down-regulation. Ref.3 Ref.5

Mutagenesis

75

1

P → A: Complete loss of binding to HCK SH3 domain and altered viral growth; when associated with P-73. No effect on Nef-induced CD4 down-regulation. Ref.3 Ref.5

Mutagenesis

147

1

P → A: Complete loss of binding to HCK SH3 domain and altered viral growth. No effect on Nef-induced CD4 down-modulation. Ref.3 Ref.5

Mutagenesis

150

1

P → A: No effect. Ref.3 Ref.5

Mutagenesis

164 – 165

2

LL → AA: Loss of interaction with AP-2 complex.

Mutagenesis

174 – 175

2

DD → AA: Loss of interaction with AP-2 complex.

Secondary structure

1

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

206

Helix Strand Turn

Details...

Helix

81 – 93

13

Turn

97 – 99

3

Helix

104 – 117

14

Beta strand

143 – 147

5

Beta strand

181 – 185

5

Helix

187 – 190

4

Helix

194 – 197

4

Helix

200 – 202

3

Sequences

	Sequence		Length	Mass (Da)
	P03406-1 [UniParc]. Last modified January 23, 2007. Version 3. Checksum: 77453FC80B6004F2	FASTA	206	23,342
	10 20 30 40 50 60 MGGKWSKSSV VGWPTVRERM RRAEPAADGV GAASRDLEKH GAITSSNTAA TNAACAWLEA 70 80 90 100 110 120 QEEEEVGFPV TPQVPLRPMT YKAAVDLSHF LKEKGGLEGL IHSQRRQDIL DLWIYHTQGY 130 140 150 160 170 180 FPDWQNYTPG PGVRYPLTFG WCYKLVPVEP DKVEEANKGE NTSLLHPVSL HGMDDPEREV 190 200 LEWRFDSRLA FHHVARELHP EYFKNC

References

[1]	"Nucleotide sequence of the AIDS virus, LAV." Wain-Hobson S., Sonigo P., Danos O., Cole S., Alizon M. Cell 40:9-17(1985) [PubMed: 2981635] [Abstract] Cited for: NUCLEOTIDE SEQUENCE.
[2]	Buckler C.E., Buckler-White A.J., Willey R.L., McCoy J. Submitted (JUN-1988) to the EMBL/GenBank/DDBJ databases Cited for: NUCLEOTIDE SEQUENCE. Strain: Clone pNL4-3.
[3]	"Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene." Chowers M.Y., Spina C.A., Kwoh T.J., Fitch N.J.S., Richman D.D., Guatelli J.C. J. Virol. 68:2906-2914(1994) [PubMed: 8151761] [Abstract] Cited for: MYRISTOYLATION AT GLY-2, FUNCTION IN VIRULENCE, MUTAGENESIS OF 2-GLY-GLY-3. Strain: Clone pNL4-3.
[4]	"Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain." Aiken C., Konner J., Landau N.R., Lenburg M.E., Trono D. Cell 76:853-864(1994) [PubMed: 8124721] [Abstract] Cited for: FUNCTION, DI-LEUCINE MOTIF.
[5]	"Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4." Saksela K., Cheng G., Baltimore D. EMBO J. 14:484-491(1995) [PubMed: 7859737] [Abstract] Cited for: SH3-BINDING SITE, MUTAGENESIS OF PRO-72; PRO-75; PRO-147 AND PRO-150.
[6]	"Cleavage of recombinant and cell derived human immunodeficiency virus 1 (HIV-1) Nef protein by HIV-1 protease." Gaedigk-Nitschko K., Schoen A., Wachinger G., Erfle V., Kohleisen B. FEBS Lett. 357:275-278(1995) [PubMed: 7835426] [Abstract] Cited for: CLEAVAGE BY VIRAL PROTEASE.
[7]	"Human immunodeficiency virus type 1 Nef binds directly to LCK and mitogen-activated protein kinase, inhibiting kinase activity." Greenway A.L., Azad A., Mills J., McPhee D.A. J. Virol. 70:6701-6708(1996) [PubMed: 8794306] [Abstract] Cited for: INTERACTION WITH HUMAN LCK AND HUMAN MAPK3.
[8]	"Human immunodeficiency virus type 1 Nef protein is incorporated into virus particles and specifically cleaved by the viral proteinase." Welker R., Kottler H., Kalbitzer H.R., Kraeusslich H.-G. Virology 219:228-236(1996) [PubMed: 8623533] [Abstract] Cited for: SUBCELLULAR LOCATION, CLEAVAGE BY VIRAL PROTEASE. Strain: Clone pNL4-3.
[9]	"Virion incorporation of human immunodeficiency virus type 1 Nef is mediated by a bipartite membrane-targeting signal: analysis of its role in enhancement of viral infectivity." Welker R., Harris M., Cardel B., Kraeusslich H.-G. J. Virol. 72:8833-8840(1998) [PubMed: 9765428] [Abstract] Cited for: SUBCELLULAR LOCATION, N-TERMINAL DOMAIN. Strain: Clone pNL4-3.
[10]	"Nef-induced major histocompatibility complex class I down-regulation is functionally dissociated from its virion incorporation, enhancement of viral infectivity, and CD4 down-regulation." Akari H., Arold S., Fukumori T., Okazaki T., Strebel K., Adachi A. J. Virol. 74:2907-2912(2000) [PubMed: 10684310] [Abstract] Cited for: FUNCTION, MUTAGENESIS OF MET-20. Strain: clone pNL-432.
[11]	"Lentivirus Nef specifically activates Pak2." Arora V.K., Molina R.P., Foster J.L., Blakemore J.L., Chernoff J., Fredericksen B.L., Garcia J.V. J. Virol. 74:11081-11087(2000) [PubMed: 11070003] [Abstract] Cited for: FUNCTION, INTERACTION WITH HUMAN PAK2.
[12]	"Mechanism for down-regulation of CD28 by Nef." Swigut T., Shohdy N., Skowronski J. EMBO J. 20:1593-1604(2001) [PubMed: 11285224] [Abstract] Cited for: FUNCTION. Strain: Clone pNL4-3.
[13]	"HIV-1 Nef inhibits ASK1-dependent death signalling providing a potential mechanism for protecting the infected host cell." Geleziunas R., Xu W., Takeda K., Ichijo H., Greene W.C. Nature 410:834-838(2001) [PubMed: 11298454] [Abstract] Cited for: FUNCTION, INTERACTION WITH HUMAN MAP3K5.
[14]	"A natural variability in the proline-rich motif of Nef modulates HIV-1 replication in primary T cells." Fackler O.T., Wolf D., Weber H.O., Laffert B., D'Aloja P., Schuler-Thurner B., Geffin R., Saksela K., Geyer M., Peterlin B.M., Schuler G., Baur A.S. Curr. Biol. 11:1294-1299(2001) [PubMed: 11525746] [Abstract] Cited for: MUTAGENESIS OF THR-71. Strain: Clone pNL4-3.
[15]	"Human immunodeficiency virus type 1 Nef binds to tumor suppressor p53 and protects cells against p53-mediated apoptosis." Greenway A.L., McPhee D.A., Allen K., Johnstone R., Holloway G., Mills J., Azad A., Sankovich S., Lambert P. J. Virol. 76:2692-2702(2002) [PubMed: 11861836] [Abstract] Cited for: FUNCTION. Strain: Clone pNL4-3.
[16]	"HIV Nef-mediated major histocompatibility complex class I down-modulation is independent of Arf6 activity." Larsen J.E., Massol R.H., Nieland T.J.F., Kirchhausen T. Mol. Biol. Cell 15:323-331(2004) [PubMed: 14617802] [Abstract] Cited for: FUNCTION. Strain: Clone pNL4-3.
[17]	"The Nef protein of human immunodeficiency virus establishes superinfection immunity by a dual strategy to downregulate cell-surface CCR5 and CD4." Michel N., Allespach I., Venzke S., Fackler O.T., Keppler O.T. Curr. Biol. 15:714-723(2005) [PubMed: 15854903] [Abstract] Cited for: FUNCTION. Strain: Clone pNL4-3.
[18]	"Expression of Nef downregulates CXCR4, the major coreceptor of human immunodeficiency virus, from the surfaces of target cells and thereby enhances resistance to superinfection." Venzke S., Michel N., Allespach I., Fackler O.T., Keppler O.T. J. Virol. 80:11141-11152(2006) [PubMed: 16928758] [Abstract] Cited for: FUNCTION. Strain: Clone pNL4-3.
[19]	"HIV-1 Nef assembles a Src family kinase-ZAP-70/Syk-PI3K cascade to downregulate cell-surface MHC-I." Hung C.H., Thomas L., Ruby C.E., Atkins K.M., Morris N.P., Knight Z.A., Scholz I., Barklis E., Weinberg A.D., Shokat K.M., Thomas G. Cell Host Microbe 1:121-133(2007) [PubMed: 18005690] [Abstract] Cited for: FUNCTION. Strain: Clone pNL4-3.
[20]	"A diacidic motif in human immunodeficiency virus type 1 Nef is a novel determinant of binding to AP-2." Lindwasser O.W., Smith W.J., Chaudhuri R., Yang P., Hurley J.H., Bonifacino J.S. J. Virol. 82:1166-1174(2008) [PubMed: 18032517] [Abstract] Cited for: DIACIDIC MOTIF, MUTAGENESIS OF 164-LEU-LEU-165 AND 174-ASP-ASP-175, INTERACTION WITH HOST AP-2 COMPLEX.
[21]	"A basic patch on alpha-adaptin is required for binding of human immunodeficiency virus type 1 Nef and cooperative assembly of a CD4-Nef-AP-2 complex." Chaudhuri R., Mattera R., Lindwasser O.W., Robinson M.S., Bonifacino J.S. J. Virol. 83:2518-2530(2009) [PubMed: 19129443] [Abstract] Cited for: IDENTIFICATION IN A CD4-NEF-AP2 COMPLEX. Strain: Clone pNL4-3.
[22]	"HIV-1 Nef dimerization is required for Nef-mediated receptor down-regulation and viral replication." Poe J.A., Smithgall T.E. J. Mol. Biol. 394:329-342(2009) [PubMed: 19781555] [Abstract] Cited for: SUBUNIT. Strain: Clone pNL4-3.
[23]	"Crystal structure of the conserved core of HIV-1 Nef complexed with a Src family SH3 domain." Lee C.H., Saksela K., Mirza U.A., Chait B.T., Kuriyan J. Cell 85:931-942(1996) [PubMed: 8681387] [Abstract] Cited for: X-RAY CRYSTALLOGRAPHY (2.5 ANGSTROMS) OF 54-205 IN COMPLEX WITH A SRC FAMILY SH3 DOMAIN.
+	Additional computationally mapped references.

Cross-references

Sequence databases

EMBL
GenBank
DDBJ

K02013 Genomic RNA. Translation: AAB59752.1.
M19921 Genomic RNA. Translation: AAA44993.1.
A04321 Unassigned RNA. Translation: CAA00353.1.

PIR

ASLJFV. A04008.

3D structure databases

PDBe
RCSB PDB
PDBj

Entry	Method	Resolution (Å)	Chain	Positions	PDBsum
1AVV	X-ray	3.00	A	58-206	[»]
1AVZ	X-ray	3.00	A/B	58-206	[»]
1EFN	X-ray	2.50	B/D	54-205	[»]

SMR

P03406. Positions 2-57.

ModBase

Search...

Protein-protein interaction databases

DIP

DIP-29968N.

Enzyme and pathway databases

Reactome

REACT_6185. HIV Infection.

Family and domain databases

InterPro

IPR001558. HIV_Nef.
[Graphical view]

Gene3D

G3DSA:3.30.62.10. HIV_Nef. 1 hit.

Pfam

PF00469. F-protein. 1 hit.
[Graphical view]

ProtoNet

Search...

Other Resources

BindingDB

P03406.

Entry information

Entry name

NEF_HV1BR

Accession

Primary (citable) accession number: P03406

Entry history

Integrated into UniProtKB/Swiss-Prot:	July 21, 1986
Last sequence update:	January 23, 2007
Last modified:	February 9, 2010
This is version 99 of the entry and version 3 of the sequence. [Complete history]

Entry status

Reviewed (UniProtKB/Swiss-Prot)

Annotation project

Virus (Virus annotation project)