The di-leucine motif in the cytoplasmic tail of CD4 is not required for binding to human immunodeficiency virus type 1 Nef, but is critical for CD4 down-modulation
The human immunodeficiency virus type 1 (HIV-1) nef gene encodes a 205 residue, myristoylated phosphoprotein that has been shown to play a critical role in the replication and pathogenesis of the virus. One of the most studied functions of the Nef protein is the down-modulation of cell surface CD4. Nef has been reported to interact with both the cytoplasmic tail of CD4 and proteins that are components of the endocytic machinery, thereby enhancing the endocytosis of CD4 through clathrin-coated pits. A di-leucine motif in the cytoplasmic tail of CD4 (residues 413/414) was reported to be essential both for Nef mediated down-modulation and for Nef binding. In order to further characterize the involvement of this di-leucine motif in CD4 down-modulation we generated a CD4 mutant in which the leucines were substituted by alanines, termed CD4(LL-AA). We demonstrate here that, contrary to previous data obtained with the cytoplasmic tail of CD4 alone, full-length CD4(LL-AA) bound to Nef both in vivo, in recombinant baculovirus-infected Sf9 cells, and in vitro. In contrast the di-leucine motif was required for both Nef-mediated and phorbol ester-induced CD4 down-modulation, suggesting that the essential requirement for the di-leucine motif in CD4 down-modulation reflects the fact that this motif is needed for the interactions of CD4 with the endocytic machinery, not for the interaction with Nef. We have also exploited the observation that CD4(LL-AA) is refractory to Nef-mediated down-modulation to provide the first experimental evidence for a physical interaction between Nef and CD4 in intact mammalian cells.
AikenC.,
KonnerJ.,
LandauN. R.,
LenburgM. E.,
TronoD.1994; Nef induces CD4 endocytosis: requirement for a critical di-leucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell 76:853–864
AldrovandiG. M.,
ZackJ. A.1996; Replication and pathogenicity of human immunodeficiency virus type 1 accessory gene mutants in SCID-Hu mice. J Virol 70:1505–1511
AndersonS. J.,
LenburgM.,
LandauN. R.,
GarciaJ. V.1994; The cytoplasmic domain of CD4 is sufficient for its down-regulation from the cell surface by human immunodeficiency virus type 1 Nef. J Virol 68:3092–3101
BenkiraneM.,
HirnM.,
CarriereD.,
DevauxC.1995; Functional epitope analysis of the human CD4 molecule: antibodies that inhibit human immunodeficiency virus type 1 gene expression bind to the immunoglobulin CDR3-like region of CD4. J Virol 69:6898–6903
CarlS.,
GreenoughT. C.,
KrumbiegelM.,
GreenbergM.,
SkowronskiJ.,
SullivanJ. L.,
KirchhoffF.2001; Modulation of different human immunodeficiency virus type 1 Nef functions during progression to AIDS. J Virol 75:3657–3665
CookeS. J.,
CoatesK.,
BartonC. H.7 other authors1997; Regulated expression vectors demonstrate cell-type-specific sensitivity to human immunodeficiency virus type 1 Nef-induced cytostasis. J Gen Virol 78:381–392
DeaconN. J.,
TsykinA.,
SolomonA.17 other authors1995; Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science 270:988–991
GlushakovaS.,
MunchJ.,
CarlS.,
GreenoughT. C.,
SullivanJ. L.,
MargolisL.,
KirchhoffF.2001; CD4 down-modulation by human immunodeficiency virus type 1 Nef correlates with the efficiency of viral replication and with CD4+ T-cell depletion in human lymphoid tissue ex vivo
. J Virol 75:10113–10117
GreenbergM. E.,
BronsonS.,
LockM.,
NeumannM.,
PavlakisG. N.,
SkowronskiJ.1997; Co-localization of HIV-1 Nef with the AP-2 adaptor protein complex correlates with Nef-induced CD4 down-regulation. EMBO J 16:6964–6976
GrzesiekS.,
StahlS. J.,
WingfieldP. T.,
BaxA.1996; The CD4 determinant for down-regulation by HIV-1 Nef directly binds to Nef-mapping of the Nef binding surface by NMR. Biochemistry 35:10256–10261
HannaZ.,
KayD. G.,
RebaiN.,
GuimondA.,
JothyS.,
JolicoeurP.1998; Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell 95:163–175
HarrisM.,
CoatesK.1993; Identification of cellular proteins that bind to the human immunodeficiency virus type 1 nef gene product in vitro : a role for myristylation. J Gen Virol 74:1581–1589
KestlerH. W.,
RinglerD. J.,
MoriK.,
PanicaliD. L.,
SehgalP. K.,
DanielM. D.,
DesrosiersR. C.1991; Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 65:651–662
LuX. B.,
YuH. F.,
LiuS. H.,
BrodskyF. M.,
PeterlinB. M.1998; Interactions between HIV1 Nef and vacuolar ATPase facilitate the internalization of CD4. Immunity 8:647–656
MangasarianA.,
FotiM.,
AikenC.,
ChinD.,
CarpentierJ. L.,
TronoD.1997; The HIV-1 Nef protein acts as a connector with sorting pathways in the Golgi and at the plasma membrane. Immunity 6:67–77
Pelchen-MatthewsA.,
ParsonsI. J.,
MarshM.1993; Phorbol ester-induced downregulation of CD4 is a multistep process involving dissociation from p56lck, increased association with clathrin-coated pits, and altered endosomal sorting. J Exp Med 178:1209–1222
PitcherC.,
HoningS.,
FingerhutA.,
BowersK.,
MarshM.1999; Cluster of differentiation antigen 4 (CD4) endocytosis and adaptor complex binding require activation of the CD4 endocytosis signal by serine phosphorylation. Mol Biol Cell 10:677–691
PreusserA.,
BrieseL.,
BaurA. S.,
WillboldD.2001; Direct in vitro binding of full-length human immunodeficiency virus type 1 Nef protein to CD4 cytoplasmic domain. J Virol 75:3960–3964
PreusserA.,
BrieseL.,
WillboldD.2002; Presence of a helix in human CD4 cytoplasmic domain promotes binding to HIV-1 Nef protein. Biochem Biophys Res Commun 292:734–740
StoddartC. A.,
GeleziunasR.,
FerrellS.8 other authors2003; Human immunodeficiency virus type 1 Nef-mediated downregulation of CD4 correlates with Nef enhancement of viral pathogenesis. J Virol 77:2124–2133
WrayV.,
MertinsD.,
KiessM.,
HenkleinP.,
Trowitzsch-KienastW.,
SchubertU.1998; Solution structure of the cytoplasmic domain of the human CD4 glycoprotein by CD and 1H NMR spectroscopy: implications for biological functions. Biochemistry 37:8527–8538
The di-leucine motif in the cytoplasmic tail of CD4 is not required for binding to human immunodeficiency virus type 1 Nef, but is critical for CD4 down-modulation