HIV Nef: The Mother of All Evil?

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1 Immunity, Vol. 9, , October, 1998, Copyright 1998 by Cell Press HIV Nef: The Mother of All Evil? Minireview Frank Peter* Nef downregulates CD4 by accelerating its endocyto- Institute of Molecular and Cell Biology sis but has no detectable effect on CD4 synthesis or 30 Medical Drive transport through the exocytotic pathway (Aiken et al., Singapore ; Rhee and Marsh, 1994; Sanfridson et al., 1994), Republic of Singapore although an as yet physiologically unexplained binding to -COP has been reported (Benichou et al., 1994). Endocytosed CD4 molecules are then accumulated in HIV-1 encodes three signatory retroviral genes (gag, pol, early endosomes and subsequently degraded in the lyand env) and seven regulatory genes (tat, rev, tev, vup, sosomes (Rhee and Marsh, 1994; Sanfridson et al., 1994; vpr, vif, and nef). tat, rev, and nef are transcribed early Schwartz et al., 1995). Inhibition of CD4 degradation by in viral replication as multiply spliced mrnas. The nef lysosomotropic agents did not reconstitute the normal gene of human and simian immunodeficiency viruses CD4 cell surface levels (Sanfridson et al., 1994). In order (HIV-1, HIV-2, and SIV) is highly conserved and seems to be endocytosed, membrane-associated receptors to be a substantial virulence factor and critical for the like CD4 commonly contain specific motifs within their development of AIDS. It is an important determinant cytoplasmic tail. These serve as recognition motifs for of viral pathogenicity in humans and monkeys and is clathrin-coated pits (CCP), which then in turn endocyresponsible for high titer virus replication in vivo (Kestler tose the receptor. CCPs appear on both the plasma et al., 1991). This importance is highlighted by the obser- membrane and the trans-golgi and are assembled from vations that nef-deleted variant strains of SIV lead to cytosolic clathrin and adaptors (reviewed in Robinson, low viral loads and AIDS does not subsequently develop 1994). Three different classes of adaptor complexes (AP) in monkeys. Moreover, long-time survivors of HIV infec- are found in a cell, two of which are involved in protein tion show a lack of disease progression, commonly as- sorting at the trans-golgi and plasma membrane (AP-1 sociated with deletions in the nef gene (Deacon et al., and AP-2), while the third (AP-3) was recently shown to 1995; Kirchhoff et al., 1995) or defective nef alleles (Mari- take part in cargo-selective transport from the Golgi to ani et al., 1996). Nef is a phosphorylated, N-myristoy- the vacuole in yeast (Cowles et al., 1997). Adaptors are lated membrane-associated cytoplasmic protein of 27 heterotetramers made of two large subunits of 100 kda in HIV-1 or 34 kda in HIV-2 and SIV. It forms homo- kda, one medium subunit of 50 kda (called the meric oligomers and intramolecular disulfide bonds (Guy chain), and one small chain of 20 kda. Clathrin is reet al., 1987), is highly conserved in primate lentiviruses, cruited by the adaptor complexes to assemble CCPs. and is synthesized at the earliest stage of viral gene APs are also able to recruit membrane receptors that expression. Although its function in vivo is still unknown, display an internalization motif in their cytoplasmic tail. well established in vitro studies demonstrate that it has Generally, these sequences are short, containing only great implications in cell function. Nef mainly interferes 4 6 amino acids, and display either a dileucine repeat or with three important pathways in the cell: downregula- an important tyrosine residue (Letourneur and Klausner, tion of CD4 as well as MHC class I expression on the 1992; Trowbridge et al., 1993). The AP subunit responsicell surface and perturbation of the cellular signal trans- ble for the recognition of these binding motifs has not duction pathways. been determined for most cases, but recent work has shown that the medium ( ) chains of AP-1 and AP-2 Nef-Induced CD4 Downregulation specifically interact with certain tyrosine-based motifs CD4 is a 55 kda cell surface glycoprotein and a compo- on the cytoplasmic tail of the receptor (Ohno et al., nent of the T cell receptor on MHC class II restricted T 1995, 1996; Rapoport et al., 1997). CD4 contains four cells (i.e., helper T-lymphocytes). It also serves as the hydrophobic amino acids, including two leucine resicellular receptor for HIV and SIV (Littman, 1996). The dues in the membrane proximal region of its cytoplasmic significance of the downregulation of CD4 by Nef in tail that are necessary for its endocytosis (Aiken et al., vivo is currently not understood, but in transgenic mice 1994; Salghetti et al., 1995). This binding site is usually expressing HIV-1 Nef a significant decrease in CD4 ex- associated with a regulatory member of the Src kinase pression on the surface of immature double-positive family, the T cell specific protein tyrosine kinase p56 lck. thymocytes is associated with compromised positive Binding of p56 lck disables the connection between CD4 selection of CD4 T lymphocytes (Skowronski et al., and the APs and thereby prevents endocytosis (Sal- 1993). The well-established roles of CD4 in immuno- ghetti et al., 1995). Upon stimulation by phosphorylation deficiency virus infection suggest that through the CD4 of Ser-408 of the receptor, the CD4-p56 lck complex disdownregulating effect Nef could enhance viral particle sociates and CD4 becomes internalized. Nef also conrelease by preventing the sequestration of the viral enve- tains a binding site for the CD4 receptor that recognizes lope by the CD4 receptor or influence other aspects the presence of this dileucin motif. Changing the Nef related to the assembly and entry of viral particles. It amino acid Asp-204 to lysine (D204K-mutant) in this could also prevent superinfection, which could lead to binding site completely abolishes binding to CD4, and the killing of the host cell. in the presence of this mutated Nef, endocytosis of CD4 occurs at a normal rate (Iafrate et al., 1997). Additionally, * mcbpfj@imcb.nus.edu.sg. Nef itself contains a critical dileucine motif necessary

2 Immunity 434 for binding to the adaptor complex. It was shown re- complexes to be very rapidly endocytosed and to be cently that Nef recruits AP-2 via the AP- chain (Greenberg accumulated in endosomal vesicles, from where they et al., 1997; Piguet et al., 1998) and that mutations are subsequently degraded (Schwartz et al., 1996). In in the Nef critical tyrosines Tyr-28 and Tyr-39 prevented nef-transfected fibroblasts, MHC class I complexes are this interaction. In contrast, binding of the Nef D204K- internalized and accumulate in vesicles at the transmutant to was unaffected (Piguet et al., 1998). Taken Golgi. From there they are probably targeted for degra- together, Nef acts as a connector between CD4 and the dation (Greenberg et al., 1998). The downregulation of CCP via. This ability to act as a bridge between cell MHC class I cell surface expression is dependent on an surface receptors and CCPs has recently been shown intact SH3 domain binding motif and a cluster of acidic for several cellular proteins as well. Eps15 serves as a amino acids within the Nef protein. This cluster is remi- connector between the EGF receptor and adaptin (van niscent of a retention motif for trans-golgi proteins such Delft et al., 1997), whereas -arrestin bridges the -2 as the AP-1 adaptor complexes. Mutations in the binding adrenergic receptor directly to clathrin (Goodman et al., site for the adaptor chain that disable the downregulation 1996). It is worthwhile to point out that the recruitment of CD4 as mentioned above do not affect the down- of APs to the cell surface is not sufficient to form CCPs; regulation of MHC class I expression. On the other hand, they have to first be activated to bind to clathrin triskelions mutations in the SH3 binding domain and in the acidic and to initiate the formation of clathrin lattices (Keen cluster, which abolish rapid MHC class I internalization, et al., 1991; Gallussen and Kirchhausen, 1993). Whether have no effect on CD4 downregulation (Greenberg et Nef can trigger this activation by binding to adaptin al., 1998). They are, however, required to block CD3- is as yet unclear. initiated signal transduction (see below). In contrast to By displacing the regulatory component p56 lck, Nef CD4, downregulation of MHC class I complexes is not overrides the internal control for the stimulated internalization due to a direct interaction between Nef and the complex. of CD4 and thereby reduces the CD4 steady state It has been speculated that alterations in the signal surface expression. The association of CD4 with the T transduction pathway, particularly via Src kinase family cell specific protein tyrosine kinase p56 lck is essential members, could cause phosphorylation of a critical tyrosine for T cell activation. The Nef protein may affect T cell in MHC class I molecules. This in turn could lead activation by preventing the expression of CD4 p56 lck to an enhanced endocytosis of the complex. This hypothesis complexes at the cell surface, reducing the surface expression is very intriguing, since it suggests that the level of CD4, binding directly to p56 lck, and Nef protein is able to link the endocytotic and signal disrupting the interaction of CD4 with p56 lck, thus providing transduction machinery to regulate MHC class I cell a mechanism for the virus to control the activation surface expression (Greenberg et al., 1998). state of its host cell. It is noteworthy to mention that Nef can bind efficiently to the SH2 and SH3 domains in p56 lck via a proline-rich Nef Induces Alterations in Common Signal motive, but this binding has no effect on the downregula- Transduction Pathways tion of CD4 (Saksela et al., 1995; Collette et al., 1996a). Nef Blocks IP 3 -Induced Calcium Release However, it was proposed that this interaction reduces Nef shows some amino acid similarity to the family of both the enzymatic activity of p56 lck and its affinity for GTP-binding proteins. Since GTPases are involved in tyrosine-phosphorylated substrates. This in turn might intracellular signal transduction processes, it has been promote cellular factors needed for viral replication (Colpathways speculated that Nef might be able to interfere with these lette et al., 1996a). (Guy et al., 1987) and thereby alter cell func- tion. One major signal transduction pathway in the cell is growth factor induced cell proliferation. Bombesin Nef-Induced Downregulation of MHC Class I and platelet-derived growth factor (PDGF) mediate cell Heterotrimeric functional MHC class I complexes con- proliferation via a G protein coupled receptor by activating sist of the MHC class I heavy chain, 2 -microglobulin, phospholipase C (PLC) in an energy-dependent and a small peptide. They are assembled in the ER and manner. PLC subsequently hydrolyzes phosphatidylinositol follow the exocytotic pathway to the cell surface. There 4,5-bisphosphate to the second messengers diacfollow the peptide is presented to cytotoxic T lymphocytes, ylglycerol (DAG) and inositoltrisphosphate (IP 3 ). IP 3 binds thereby allowing the destruction of cells displaying foreign to a specific receptor on the ER and induces the release antigens. This mechanism is the basis of acquired of stored calcium ions from vesicular pools via a Ca 2 anti-viral immune response (reviewed in Kagi et al., ATPase, thereby stimulating cell proliferation and growth. 1996). By interfering with this process, Nef efficiently In NIH 3T3 cells transfected with nef, this growth factor protects infected primary cells from being killed by cytotoxic stimulated calcium release is blocked. IP 3 formation and T lymphocytes (Collins et al., 1998). This could Ca 2 ATPase activity are not compromised, and it was clearly ensure the survival of HIV- and SIV-infected cells implied that Nef disables the IP 3 receptor (Graziani et in vivo. In T cells, the surface expression of MHC class al., 1996). This finding is of considerable interest since I is fairly transient, since the complex is constitutively it shows that Nef can modulate molecular alterations in endocytosed via CCPs (reviewed in York and Rock, a universal cellular pathway. The essential pathogenic 1996). After internalization the complexes are either recycled effect of Nef is therefore not limited to T cells. to the cell surface or degraded. In Nef-expressing Nef Blocks CD3 Signaling in T Cells cells, Nef does not seem to interfere with the synthesis CD4 T helper cells bind antigen with the help of the and the trafficking of the complexes to the plasma mem- major histocompatibility class II complex (MHC class brane. In lymphoid cells Nef causes the MHC class I II) and thereby induce the transcriptional activation of

3 Minireview 435 several cytokine genes, including the interleukin 2 (IL-2) Nef Is Phosphorylated and the interferon- (INF ) genes. Cytokines are necessary Nef was found to be phosphorylated in vitro (Guy et al., for the clonal expansion of antigen-reactive T cells 1987) and in vivo (Coates et al., 1997) on serine and, to and for the efficient recruitment of other hemopoetic a lesser extent, threonine. Studies with selective protein cells. Upon stimulation of CD3-initiated signaling, nor- kinase inhibitors identified protein kinase C (PKC) as the mal Jurkat T cells exhibited prompt response in IL-2 kinase responsible. Myristoylation of Nef is a prerequisite mrna induction. In contrast, nef-transfected Jurkat for phosphorylation, possibly to ensure close proximity cells did not show this induction, although the IL-2 gene to the activated PKC localized to the plasma membrane. is still intact (Luria et al., 1991). Expression of Nef down- However, both cytosolic and plasma membrane attached regulated the synthesis of both IL-2 and INF in a dosedependent Nef pools showed a similar extent of phosphorylation, manner, but not that of other cytokines (Col- indicating that membrane attachment of Nef is only a lette et al., 1996b). This effect has been attributed to a transient event (Coates et al., 1997). The biological role specific interaction of Nef with the transcription machin- of Nef phosphorylation remains as yet elusive. ery of IL-2 and INF. Interestingly, a critical region in Nef Binds to Another Member of the Src Family the INF promoter shares homology with a region of the The protein tyrosine kinase Hck is part of the Src family IL-2 promoter (Collette et al., 1996b) but not with the and is predominantly expressed in monocytes. As for sequences of other cytokines. Nef could interact with Lck, the proline-rich region of Nef binds to the SH3 one or more cellular factors specific for transcription of domain of Hck. The binding of Nef to Hck is required this particular cytokine pathway (Collette et al., 1996a, for enhanced growth of nef viruses in monocytes (Shugars 1996b). A second feature of TCR stimulation is the formation et al., 1993; Saksela et al., 1995). Enhanced infectiv- of high-affinity IL-2 receptors (IL-2R) and the ity is also observed after Nef is incorporated into the release of intracellular calcium. nef-transfected cells, virus particle and cleaved by the viral protease to Nef, despite not being able to induce IL-2 mrna in response although the biochemical mechanism for this observation to stimulation, exhibited normal induction of IL-2R and is unknown (Goldsmith et al., 1995; Welker et al., 1996). calcium mobilization. Taken together, these data suggest that only some TCR complex induced signal trans- Conclusion duction pathways are affected by Nef (Luria et al., 1991; HIV infection initially leads to a rapid virus replication and Collette et al., 1996a) and that the ability to downregulate dissemination, particularly within the lymphoid system. CD4 surface expression (see above) is separable from The outstanding pathological consequence of HIV infecthe ability to block CD3 signaling in T cells (Iafrate et tion is the dramatic quantitative and qualitative depleal., 1997). tion of CD4 T cells, resulting in a complete dysfunction of the immune system. But it is only after a prolonged Nef Interacts with Several Other Proteins quiescent stage that a decline in CD4 T cells is ob- Nef Associates with a Member of the p21-activated served and AIDS develops. What causes the onset is Kinase Family currently not known. Although several other HIV-encoded Nef has been reported to associate with a serine kinase proteins show some immunosuppressive effect, it is in virally infected T cells (Sawai et al., 1994). Immunopremost likely the nef gene product that causes the severe cipitation experiments led to the identification of a 65 immunodeficiency leading from HIV infection to AIDS. kda protein (p65) that is serologically and functionally Even today, the actual consequences of Nef expression related to the p21-activated kinases (PAKs) (Nunn and in vivo are only poorly understood, but in vitro studies Marsh, 1996). P65 is capable of autophosphorylation clearly show that Nef is responsible for compromising and has histone H4 as a preferred substrate. Moreover, CD4 and MHC class I cell surface expression and for the GTP-bound forms of Rac and Cdc42 positively reguserious interruptions in different signal transduction late its kinase activity. All of the above characteristics pathways. In fact, recent studies in vivo showed that are reminiscent for members of the PAK family (Manser mutations in the either of the two PXXP domains of et al., 1994, 1995; Bagrodia et al., 1995). PAKs are ex- Nef do not affect pathogenicity, suggesting that some pressed in a variety if tissues and play integral roles in functions described for Nef in vitro might not be relevant cellular responses to exogenous stimuli. PAKs initiate in vivo after all (Aldrovandi et al., 1998). Only after fully the kinase cascade leading to the activation of c-jun understanding each and every aspect of Nef function N-terminal kinase, JNK, and c-jun transcriptional activwill researchers be able to transfer their knowledge from ity (Minden et al., 1995). Despite p65 being a member in vitro to in vivo conditions. Ultimately, the devastating of the PAK family, any influence in transcriptional regulaeffects of Nef can and will be tackled. This will of course tion has not been identified. A proposed function of p65 not eliminate HIV, but preventing the infection from deis the phosphorylation of the HIV matrix protein, which veloping into AIDS would be a major step, potentially could lead to the Nef-induced enhanced infectivity of saving the lives of millions of people already carrying viral particles. the virus. Nef Binds to a Thioesterase Several other interactions of Nef with various proteins have been reported. A human homolog of the E. coli References thioesterase TE II, hte, was identified using the yeast Aiken, C., Konner, J., Landau, N.R., Lenburg, M.E., and Trono, D. two hybrid system. Not only could hte and Nef be coim- (1994). Nef induces CD4 endocytosis: requirement for a critical dileumunoprecipitated, but Nef mutants ineffective to down- cine motif in the membrane-proximal CD4 cytoplasmic domain. Cell regulate CD4 were also unable to bind to hte (Liu et al., 76, ). Aldrovandi, G.M., Gao, L., Bristol, G., and Zack, J.A. (1998). Regions

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