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Immunodeficiency Viruses: Not enough sans Nef
DAN R. LITTMAN
IMMUNODEFICIENCY VIRUSES
Not enough sans Nef The nefgenes of HIV and SIV are dispensable in vitro, but are essential for viral spread and disease progression in vivo. Nef-induced down-regulation of CD4, the viral receptor, may be the key to this requirement. Most pathogens have very different requirements for growth in cell culture systems than for growth in the host organism. Eukaryotic viruses, in particular, have acquired sophisticated tools to evade host defenses and to exploit normal host cellular mechanisms. These functions are often difficult to discern when viruses are studied in tissue culture. This principle is clearly exemplified by the primate lentiviruses, which include the human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV). These viruses have a number of genes that are dispensable for viral replication in various tissue culture systems (Fig. 1). One of these genes, nef, is found only in this class of retroviruses and encodes a cytoplasmic protein that appears to be essential for maintenance of a viral load in vivo and for disease progression. The mechanism of action of Nef within the infected host remains poorly understood. Several recent studies show that Nef expression results in down-regulation of the CD4 glycoprotein, both in transfected cells and in transgenic mice. As primate CD4 serves as the receptor for these retroviruses and is also involved in signal transduction in T lymphocytes, these observations may provide important clues as to why Nef is required in vivo. Nef is encoded by an open reading frame that overlaps the 3' long terminal repeat (LTR) in the genomes of primate lentiviruses. The 27kD protein is myristylated at its amino terminus and is associated with cellular membranes, although the exact subcellular localization has not yet been determined. The analysis of multiple HIV sequences obtained directly from AIDS patients has shown that the amino-terminal amino acids that follow the myristylated glycine in Nef are highly conserved; these have been suggested to serve as a signal that targets the protein to a specific membrane compartment or to other membraneassociated proteins [1]. There are few other clues as to functional domains within Nef, with the possible exception of a potential protein kinase C phosphorylation site that is highly conserved in the human and simian viruses. Early studies of Nef function led to conflicting conclusions about whether Nef affected viral replication positively, negatively, or not at all. It is now apparent that the early discrepancies resulted from the use of different virus strains and tissue culture conditions. The different studies agreed, however, that viruses lacking an intact nef open reading frame could replicate well in vitro. Renewed interest in Nef function was aroused by the startling finding that productive infection of macaques with SIV required an intact nef open reading frame 12]. 618
Animals infected with a strain of SIV containing an intact nef gene had a high viral load and developed immunodeficiency several months later. In contrast, when an equivalent dose of virus with a deletion of the nef gene was administered, the animals remained healthy and infectious virus could not be readily recovered from peripheral blood mononuclear cells. When animals were infected with virus having a premature stop codon in the nef gene, there was rapid reversion to a complete open reading frame and disease progression was observed. These results indicate that in infected animals there is strong selective pressure for replication of virus with an intact nef gene. Remarkably, animals inoculated with virus having a deletion in nef became resistant to subsequent infection with pathogenic strains of SIV, suggesting that they had developed long-term (more than two years) protective immunity [3]. No pathogenic viral sequences could be detected after viral challenge of these animals, suggesting that the attenuated virus can serve as a very potent vaccine. The macaque studies have led to a recent re-evaluation of Nef function in cell-culture systems. Under conditions of low multiplicity of infection in cultures of primary human T cells, several groups have observed that HIV-1 with an intact nef gene replicates to higher titers than isogenic virus in which the nef coding sequence is interrupted [4-6]. This difference was accentuated when resting T cells were infected and subsequently stimulated with mitogens [5,6]. Interestingly, in a single-round infectivity assay, virus produced either in transfected kidney epithelial (COS) cells or in T-cell lines had higher infectious titers per particle when the nef gene was intact [5]. Thus, even when virus was assembled in COS cells, which lack CD4, Nef influenced infectivity of the
Fig. 1. Schematic representation of the HIV-1 genome, with the accessory or 'non-essential' genes highlighted by red lines.
© Current Biology 1994, Vol 4 No 7
DISPATCH Fig. 2. A model for the effect of Nef on CD4 and Lck, a CD4-associated tyrosine kinase. Directly or indirectly, Nef may expose the dileucine motif in CD4 to the endocytic machinery. The same motif may also become exposed following phosphorylation of the adjacent serine by protein kinase C. This is thought to be accompanied by dissociation of Lck from the CD4 cytoplasmic domain.
released virions, possibly by contributing to a decrease in the proportion of defective particles produced. How this occurs is difficult to understand at present, but does not appear to involve Nef incorporation into virions. The relevance of these observations to the in vivo results with SIV remains to be determined. An understanding of the function of Nef at the cellular and molecular level is needed to elucidate the role of this molecule in pathogenesis. In T cells, myristylated Nef down-regulates cell surface CD4 by a post-translational mechanism [7,8]. This down-regulation requires only the cytoplasmic domain of CD4, and can occur in nonlymphoid cells transfected with plasmids encoding CD4 and Nef. Aiken et al. [8] have recently demonstrated that Nef-induced down-regulation involves an increase in the rate of CD4 endocytosis and, consequently, lysosomal degradation. Grafting of the CD4 cytoplasmic domain onto the CD8 glycoprotein renders CD8 sensitive to the down-regulatory effect of Nef. Targeting of CD4 or CD8/CD4 chimeras to endosomes requires the presence of a dileucine motif within the cytoplasmic domain [8]. This sequence is also required for phorbol-ester-induced CD4 internalization, which involves protein-kinase-Cmediated phosphorylation of an adjacent serine residue (Fig. 2) and results in association of CD4 with clathrincoated pits [8,9]. A similar dileucine motif had been previously shown to target endocytosis and lysosomal degradation of CD3 polypeptides. It is hence likely that the dileucine motif in CD4 mediates association with clathrin-coated pits; this motif may normally be masked and may become accessible to the endocytic machinery
upon phosphorylation of the adjacent serine or after interaction of CD4 with, or modification by, Nef. Analysis of transgenic mice expressing Nef specifically in T cells has provided data that are consistent with the results with transfected cells and additionally suggest that Nef may influence signal transduction. Expression of nef transgenes under the control of either CD38, CD2 or T-cell receptor 13-chain regulatory sequences results in down-regulation of cell-surface CD4 on thymocytes and in a block of CD4+ cell development [10-12]. Interestingly, Nef is expressed at high levels in the thymus but not in the periphery, suggesting that a reduction in transgene expression is required for cells to complete maturation [10]. Moreover, thymocytes in these animals are hyper-responsive to stimulation with anti-CD3 antibodies [10]. The T cells that complete development have a phenotype resembling that of memory cells, suggesting that they may have been aberrantly activated [12]. Taken together, these results support the notion that Nef lowers the threshold for thymocyte and T-cell activation. The results with the transgenic mice may be related to the function of the lymphocyte-specific protein tyrosine kinase p5 61k (Lck), which binds to the cytoplasmic domain of CD4 and has a critical role in T-cell activation and development. Two cysteines in CD4 are involved in this interaction, but they are not required for either Nefor phorbol-induced down-regulation [8]. Following treatment with phorbol esters, Lck is released from CD4 prior to CD4 endocytosis [9,13]; Nef may similarly force a dissociation of Lck from CD4, but this has not yet
619
620
Current Biology 1994, Vol 4 No 7 been reported. Based on studies with murine T-cell clones, it has been proposed that sequestration of Lck by CD4 may regulate the sensitivity of the T-cell antigen receptor complex to stimulation by antigen [14]; dissociation of Lck from CD4 may thus increase accessibility of Lck to the T-cell receptor complex, lowering the threshold for stimulation (Fig. 2). Alternatively, Nef may directly activate signaling pathways in T cells, perhaps via a direct or indirect interaction with Lck or with components of the T-cell receptor complex. In this regard, it will be interesting to determine whether elevated T-cell receptor signaling can be observed in the absence of CD4. Little is known of the biochemical basis for Nef-mediated CD4 down-regulation and signal enhancement. Nef immunoprecipitates from human T-cell lines contain serine kinase activity and proteins of 62 and 72 kD that are phosphorylated in vitro [15]. The physiological significance of this association is not yet known, nor is it clear if Nef directly interacts with the cytoplasmic domain of CD4. The different studies suggest several ways in which Nef may influence viral replication. The first possibility is that the primary function of Nef is to down-regulate CD4. It is highly unlikely that this effect of Nef is a coincidence, especially when one considers that HIV-1 has two additional, independent mechanisms to achieve this aim: the HIV-1 envelope glycoprotein, gpl20/gp41, binds to CD4 within the endoplasmic reticulum (ER) and prevents its transport; and HIV-1 protein Vpu induces degradation of ER-localized CD4 by a mechanism that also involves the CD4 cytoplasmic domain. Nef-mediated down-regulation of CD4 precedes that induced by the envelope glycoprotein, presumably because of the earlier synthesis of Nef in the viral life cycle [8]. Early CD4 down-regulation may thus be important for preventing superinfection of HIV-infected cells. Indeed, Nef-expressing cell lines are resistant to infection with SIV and to HIV envelope-mediated fusion. Alternatively, loss of the viral receptor may primarily ensure that newly formed viral particles can be efficiently released from infected cells. A third possibility is that, by preventing a cytopathic effect thought to occur upon binding of newly synthesized envelope glycoprotein to CD4, Nef may promote survival of infected cells, thus contributing to an increased viral load. Finally, the other potential function of Nef, as discussed above, would be to increase signal transduction initiated from the T-cell receptor complex and, as a consequence, to raise the level of virus production. This may involve release of Lck upon CD4 down-regulation or CD4-independent activation by Nef of the signal-transduction pathway. In either case, increased signaling would result in higher levels of NFKB, a transcriptional activator that enhances expression of the integrated HIV provirus. To determine how Nef influences viral replication in the infected host, it will next be necessary to couple in vitro
structure/function analyses with studies in animal model systems. For example, it will be important to learn whether the essential in vivo function of Nef is strictly dependent on the down-regulation of CD4. The macaque model that has served as the stimulus for the current excitement in this area is unlikely to be amenable to such approaches. A recent report indicates that the nef gene contributes significantly to HIV-1 replication and pathogenesis in human lymphoid organs implanted in immunodeficient mice, raising the possibility that this model, known as the SCIDhu mouse, can be harnessed for future studies [16]. Experiments with this system may provide clues to the in vivo mechanism of Nef and other HIV-1 gene products, such as Vpr and Vpu, and may thus create new opportunities for interfering with the viral life cycle and for developing an effective vaccine against HIV-1. References 1. 2. 3.
4.
5.
6.
7. 8. 9.
10.
11.
12.
13. 14.
15.
16.
Shugars DC, Smith MS, Glueck DH, Nantermet PV, Seillier-Moiseiwitsch F, Swanstrom R: Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo. Virol 1993, 67:4639-4650. Kestler HW, Ringler DJ, Mori K, Panicali DL, Sehgal PK, Daniel MD, Desrosiers RC: Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 1991, 65:651-662. Daniel MD, Kirchoff F, Czajak SC, Sehgal PK, Desrosiers RC: Protective effects of a live attenuated SIV vaccine with a deletion of the nef gene. Science 1992,258:1938-1941. De Ronde A, Klaver B, Keulen W, Smit L, Goudsmit : Natural HIV-1 NEF accelerates virus replication in primary human lymphocytes. Virology 1992, 188:391-395. Miller MD, Warmerdam MT, Gaston , Greene WC, Feinberg MB: The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages. I Exp Med 1994, 179:101-113. Spina CA, Kwoh TI, Chowers MY, Guatelli IC, Richman DD: The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes. Exp Med 1994, 179:115-123. Garcia IV, Miller AD: Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature 1991, 350:508-511. Aiken C, Konner J, Landau NR, Lenburg ME, Trono D: Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell 1994, 76:853-864. Pelchen-Matthews A, Parsons 11,March M: Phorbol ester-induced downregulation of CD4 is a multistep process involving dissociation from p5 6 Ick,increased association with clathrin-coated pits, and altered endosomal sorting. J Exp Med 1993,178:1209-1222. Skowronski , Parks D, Mariani R: Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene. EMBO 1 1993, 12:703-713. Brady HI, Pennington Dl, Miles CG, Dzierzak EA: CD4 cell surface downregulation in HIV-1 Nef transgenic mice is a consequence of intracellular sequestration. EMBO J 1993,12:4923-32. Lindemann D, Wilhelm R, Renard P, Althage A, Zinker-Nagel R, Mous : Severe immunodeficiency associated with a human immunodeficiency virus 1 NEF /3'-long terminal repeat transgene. I Exp Med 1994, 179:797-807. Sleckman BP, Shin , Igras VE, Collins TL, Strominger JL, Burakoff SJ: Disruption of the CD4-p56k complex is required for rapid internalization of CD4. Proc Natl Acad Sci U S A 1992, 89:7566-7570. Haughn L, Gratton S, Caron L, Sekaly RP, Veillette A, Julius M: 1' Association of tyrosine kinase p56 d with CD4 inhibits the induction of growth through the alpha beta T-cell receptor. Nature 1992, 358:328-31. Sawai ET, Baur A, Struble H, Peterlin BM, Levy A, Cheng-Mayer C: Human immunodeficiency virus type 1 Nef associates with a cellular serine kinase in T lymphocytes. Proc Natl Acad Sci U S A 1994, 91:1539-1543. Jamieson BD, Aldrovandi GM, Planelles V, owett IBM, Gao L, Bloch LM, Chen ISY, Zack A: Requirement of human immunodeficiency virus type 1 nef for in vivo replication and pathogenicity. Virol 1994, 68:3478-3485.
Dan R. Littman, Howard Hughes Medical Institute, Departments of Microbiology and Immunology and of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0414, USA.
IMMUNODEFICIENCY VIRUSES
Not enough sans Nef The nefgenes of HIV and SIV are dispensable in vitro, but are essential for viral spread and disease progression in vivo. Nef-induced down-regulation of CD4, the viral receptor, may be the key to this requirement. Most pathogens have very different requirements for growth in cell culture systems than for growth in the host organism. Eukaryotic viruses, in particular, have acquired sophisticated tools to evade host defenses and to exploit normal host cellular mechanisms. These functions are often difficult to discern when viruses are studied in tissue culture. This principle is clearly exemplified by the primate lentiviruses, which include the human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV). These viruses have a number of genes that are dispensable for viral replication in various tissue culture systems (Fig. 1). One of these genes, nef, is found only in this class of retroviruses and encodes a cytoplasmic protein that appears to be essential for maintenance of a viral load in vivo and for disease progression. The mechanism of action of Nef within the infected host remains poorly understood. Several recent studies show that Nef expression results in down-regulation of the CD4 glycoprotein, both in transfected cells and in transgenic mice. As primate CD4 serves as the receptor for these retroviruses and is also involved in signal transduction in T lymphocytes, these observations may provide important clues as to why Nef is required in vivo. Nef is encoded by an open reading frame that overlaps the 3' long terminal repeat (LTR) in the genomes of primate lentiviruses. The 27kD protein is myristylated at its amino terminus and is associated with cellular membranes, although the exact subcellular localization has not yet been determined. The analysis of multiple HIV sequences obtained directly from AIDS patients has shown that the amino-terminal amino acids that follow the myristylated glycine in Nef are highly conserved; these have been suggested to serve as a signal that targets the protein to a specific membrane compartment or to other membraneassociated proteins [1]. There are few other clues as to functional domains within Nef, with the possible exception of a potential protein kinase C phosphorylation site that is highly conserved in the human and simian viruses. Early studies of Nef function led to conflicting conclusions about whether Nef affected viral replication positively, negatively, or not at all. It is now apparent that the early discrepancies resulted from the use of different virus strains and tissue culture conditions. The different studies agreed, however, that viruses lacking an intact nef open reading frame could replicate well in vitro. Renewed interest in Nef function was aroused by the startling finding that productive infection of macaques with SIV required an intact nef open reading frame 12]. 618
Animals infected with a strain of SIV containing an intact nef gene had a high viral load and developed immunodeficiency several months later. In contrast, when an equivalent dose of virus with a deletion of the nef gene was administered, the animals remained healthy and infectious virus could not be readily recovered from peripheral blood mononuclear cells. When animals were infected with virus having a premature stop codon in the nef gene, there was rapid reversion to a complete open reading frame and disease progression was observed. These results indicate that in infected animals there is strong selective pressure for replication of virus with an intact nef gene. Remarkably, animals inoculated with virus having a deletion in nef became resistant to subsequent infection with pathogenic strains of SIV, suggesting that they had developed long-term (more than two years) protective immunity [3]. No pathogenic viral sequences could be detected after viral challenge of these animals, suggesting that the attenuated virus can serve as a very potent vaccine. The macaque studies have led to a recent re-evaluation of Nef function in cell-culture systems. Under conditions of low multiplicity of infection in cultures of primary human T cells, several groups have observed that HIV-1 with an intact nef gene replicates to higher titers than isogenic virus in which the nef coding sequence is interrupted [4-6]. This difference was accentuated when resting T cells were infected and subsequently stimulated with mitogens [5,6]. Interestingly, in a single-round infectivity assay, virus produced either in transfected kidney epithelial (COS) cells or in T-cell lines had higher infectious titers per particle when the nef gene was intact [5]. Thus, even when virus was assembled in COS cells, which lack CD4, Nef influenced infectivity of the
Fig. 1. Schematic representation of the HIV-1 genome, with the accessory or 'non-essential' genes highlighted by red lines.
© Current Biology 1994, Vol 4 No 7
DISPATCH Fig. 2. A model for the effect of Nef on CD4 and Lck, a CD4-associated tyrosine kinase. Directly or indirectly, Nef may expose the dileucine motif in CD4 to the endocytic machinery. The same motif may also become exposed following phosphorylation of the adjacent serine by protein kinase C. This is thought to be accompanied by dissociation of Lck from the CD4 cytoplasmic domain.
released virions, possibly by contributing to a decrease in the proportion of defective particles produced. How this occurs is difficult to understand at present, but does not appear to involve Nef incorporation into virions. The relevance of these observations to the in vivo results with SIV remains to be determined. An understanding of the function of Nef at the cellular and molecular level is needed to elucidate the role of this molecule in pathogenesis. In T cells, myristylated Nef down-regulates cell surface CD4 by a post-translational mechanism [7,8]. This down-regulation requires only the cytoplasmic domain of CD4, and can occur in nonlymphoid cells transfected with plasmids encoding CD4 and Nef. Aiken et al. [8] have recently demonstrated that Nef-induced down-regulation involves an increase in the rate of CD4 endocytosis and, consequently, lysosomal degradation. Grafting of the CD4 cytoplasmic domain onto the CD8 glycoprotein renders CD8 sensitive to the down-regulatory effect of Nef. Targeting of CD4 or CD8/CD4 chimeras to endosomes requires the presence of a dileucine motif within the cytoplasmic domain [8]. This sequence is also required for phorbol-ester-induced CD4 internalization, which involves protein-kinase-Cmediated phosphorylation of an adjacent serine residue (Fig. 2) and results in association of CD4 with clathrincoated pits [8,9]. A similar dileucine motif had been previously shown to target endocytosis and lysosomal degradation of CD3 polypeptides. It is hence likely that the dileucine motif in CD4 mediates association with clathrin-coated pits; this motif may normally be masked and may become accessible to the endocytic machinery
upon phosphorylation of the adjacent serine or after interaction of CD4 with, or modification by, Nef. Analysis of transgenic mice expressing Nef specifically in T cells has provided data that are consistent with the results with transfected cells and additionally suggest that Nef may influence signal transduction. Expression of nef transgenes under the control of either CD38, CD2 or T-cell receptor 13-chain regulatory sequences results in down-regulation of cell-surface CD4 on thymocytes and in a block of CD4+ cell development [10-12]. Interestingly, Nef is expressed at high levels in the thymus but not in the periphery, suggesting that a reduction in transgene expression is required for cells to complete maturation [10]. Moreover, thymocytes in these animals are hyper-responsive to stimulation with anti-CD3 antibodies [10]. The T cells that complete development have a phenotype resembling that of memory cells, suggesting that they may have been aberrantly activated [12]. Taken together, these results support the notion that Nef lowers the threshold for thymocyte and T-cell activation. The results with the transgenic mice may be related to the function of the lymphocyte-specific protein tyrosine kinase p5 61k (Lck), which binds to the cytoplasmic domain of CD4 and has a critical role in T-cell activation and development. Two cysteines in CD4 are involved in this interaction, but they are not required for either Nefor phorbol-induced down-regulation [8]. Following treatment with phorbol esters, Lck is released from CD4 prior to CD4 endocytosis [9,13]; Nef may similarly force a dissociation of Lck from CD4, but this has not yet
619
620
Current Biology 1994, Vol 4 No 7 been reported. Based on studies with murine T-cell clones, it has been proposed that sequestration of Lck by CD4 may regulate the sensitivity of the T-cell antigen receptor complex to stimulation by antigen [14]; dissociation of Lck from CD4 may thus increase accessibility of Lck to the T-cell receptor complex, lowering the threshold for stimulation (Fig. 2). Alternatively, Nef may directly activate signaling pathways in T cells, perhaps via a direct or indirect interaction with Lck or with components of the T-cell receptor complex. In this regard, it will be interesting to determine whether elevated T-cell receptor signaling can be observed in the absence of CD4. Little is known of the biochemical basis for Nef-mediated CD4 down-regulation and signal enhancement. Nef immunoprecipitates from human T-cell lines contain serine kinase activity and proteins of 62 and 72 kD that are phosphorylated in vitro [15]. The physiological significance of this association is not yet known, nor is it clear if Nef directly interacts with the cytoplasmic domain of CD4. The different studies suggest several ways in which Nef may influence viral replication. The first possibility is that the primary function of Nef is to down-regulate CD4. It is highly unlikely that this effect of Nef is a coincidence, especially when one considers that HIV-1 has two additional, independent mechanisms to achieve this aim: the HIV-1 envelope glycoprotein, gpl20/gp41, binds to CD4 within the endoplasmic reticulum (ER) and prevents its transport; and HIV-1 protein Vpu induces degradation of ER-localized CD4 by a mechanism that also involves the CD4 cytoplasmic domain. Nef-mediated down-regulation of CD4 precedes that induced by the envelope glycoprotein, presumably because of the earlier synthesis of Nef in the viral life cycle [8]. Early CD4 down-regulation may thus be important for preventing superinfection of HIV-infected cells. Indeed, Nef-expressing cell lines are resistant to infection with SIV and to HIV envelope-mediated fusion. Alternatively, loss of the viral receptor may primarily ensure that newly formed viral particles can be efficiently released from infected cells. A third possibility is that, by preventing a cytopathic effect thought to occur upon binding of newly synthesized envelope glycoprotein to CD4, Nef may promote survival of infected cells, thus contributing to an increased viral load. Finally, the other potential function of Nef, as discussed above, would be to increase signal transduction initiated from the T-cell receptor complex and, as a consequence, to raise the level of virus production. This may involve release of Lck upon CD4 down-regulation or CD4-independent activation by Nef of the signal-transduction pathway. In either case, increased signaling would result in higher levels of NFKB, a transcriptional activator that enhances expression of the integrated HIV provirus. To determine how Nef influences viral replication in the infected host, it will next be necessary to couple in vitro
structure/function analyses with studies in animal model systems. For example, it will be important to learn whether the essential in vivo function of Nef is strictly dependent on the down-regulation of CD4. The macaque model that has served as the stimulus for the current excitement in this area is unlikely to be amenable to such approaches. A recent report indicates that the nef gene contributes significantly to HIV-1 replication and pathogenesis in human lymphoid organs implanted in immunodeficient mice, raising the possibility that this model, known as the SCIDhu mouse, can be harnessed for future studies [16]. Experiments with this system may provide clues to the in vivo mechanism of Nef and other HIV-1 gene products, such as Vpr and Vpu, and may thus create new opportunities for interfering with the viral life cycle and for developing an effective vaccine against HIV-1. References 1. 2. 3.
4.
5.
6.
7. 8. 9.
10.
11.
12.
13. 14.
15.
16.
Shugars DC, Smith MS, Glueck DH, Nantermet PV, Seillier-Moiseiwitsch F, Swanstrom R: Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo. Virol 1993, 67:4639-4650. Kestler HW, Ringler DJ, Mori K, Panicali DL, Sehgal PK, Daniel MD, Desrosiers RC: Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 1991, 65:651-662. Daniel MD, Kirchoff F, Czajak SC, Sehgal PK, Desrosiers RC: Protective effects of a live attenuated SIV vaccine with a deletion of the nef gene. Science 1992,258:1938-1941. De Ronde A, Klaver B, Keulen W, Smit L, Goudsmit : Natural HIV-1 NEF accelerates virus replication in primary human lymphocytes. Virology 1992, 188:391-395. Miller MD, Warmerdam MT, Gaston , Greene WC, Feinberg MB: The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages. I Exp Med 1994, 179:101-113. Spina CA, Kwoh TI, Chowers MY, Guatelli IC, Richman DD: The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes. Exp Med 1994, 179:115-123. Garcia IV, Miller AD: Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature 1991, 350:508-511. Aiken C, Konner J, Landau NR, Lenburg ME, Trono D: Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell 1994, 76:853-864. Pelchen-Matthews A, Parsons 11,March M: Phorbol ester-induced downregulation of CD4 is a multistep process involving dissociation from p5 6 Ick,increased association with clathrin-coated pits, and altered endosomal sorting. J Exp Med 1993,178:1209-1222. Skowronski , Parks D, Mariani R: Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene. EMBO 1 1993, 12:703-713. Brady HI, Pennington Dl, Miles CG, Dzierzak EA: CD4 cell surface downregulation in HIV-1 Nef transgenic mice is a consequence of intracellular sequestration. EMBO J 1993,12:4923-32. Lindemann D, Wilhelm R, Renard P, Althage A, Zinker-Nagel R, Mous : Severe immunodeficiency associated with a human immunodeficiency virus 1 NEF /3'-long terminal repeat transgene. I Exp Med 1994, 179:797-807. Sleckman BP, Shin , Igras VE, Collins TL, Strominger JL, Burakoff SJ: Disruption of the CD4-p56k complex is required for rapid internalization of CD4. Proc Natl Acad Sci U S A 1992, 89:7566-7570. Haughn L, Gratton S, Caron L, Sekaly RP, Veillette A, Julius M: 1' Association of tyrosine kinase p56 d with CD4 inhibits the induction of growth through the alpha beta T-cell receptor. Nature 1992, 358:328-31. Sawai ET, Baur A, Struble H, Peterlin BM, Levy A, Cheng-Mayer C: Human immunodeficiency virus type 1 Nef associates with a cellular serine kinase in T lymphocytes. Proc Natl Acad Sci U S A 1994, 91:1539-1543. Jamieson BD, Aldrovandi GM, Planelles V, owett IBM, Gao L, Bloch LM, Chen ISY, Zack A: Requirement of human immunodeficiency virus type 1 nef for in vivo replication and pathogenicity. Virol 1994, 68:3478-3485.
Dan R. Littman, Howard Hughes Medical Institute, Departments of Microbiology and Immunology and of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0414, USA.