Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy1
University of Erlangen, Department of Dermatology, Erlangen, Germany2
Department of Biochemistry and Molecular Biology, University College London, London, UK3
Author for correspondence: Maurizio Federico. Fax +39 06 49903002. e-mail federico{at}iss.it
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
F12-HIVNef is a primary Nef mutant characterized by three rare amino acid substitutions, G140E, V153L and E177G (Carlini et al., 1992 ). We reported recently that the in cis expression of F12-HIVNef transforms the highly productive NL4-3 HIV-1 into a replication-defective strain (Olivetta et al., 2000
). We were interested in defining Nef functions involved in the mechanism of the F12-HIVNef antiviral effect. We have already established that (i) the expression of F12-HIVNef fails to down-regulate CD4 (DAloja et al., 1998
), (ii) the block of virus replication occurs at the level of virus assembly and/or release (DAloja et al., 1998
; Olivetta et al., 2000
), and (iii) both CD4 and HIV-1 gp41 Env intracytoplasmic tails are essential for this antiviral effect (Olivetta et al., 2000
). Here, we report that the lack of activation of the p62 Nef-associated kinase (p62NAK), a serine/threonine kinase with a molecular mass of 62 kDa, which is typically phosphorylated upon wild-type (wt) Nef expression (Sawai et al., 1994
), correlates with the F12-HIVNef antiviral phenotype. In addition, we demonstrate that the concomitant lack of both CD4 accelerated endocytosis and an, as yet, still unidentified function are required for the inhibitory effect of F12-HIVNef.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HIV-1 infections and protein detection.
Supernatants from transiently transfected 293 cells were the source of NL4-3 strain HIV-1 and derivatives thereof. Infections were performed by adsorbing the virus inoculum onto either cell monolayers (for adherent cells) or cell pellets (for non-adherent cells) for 1 h at 37 °C with occasional shaking. Cells were then washed extensively and re-fed. Detection of virus infection in supernatants was performed by a reverse transcriptase (RT) assay (Rossi et al., 1987 ). RT activity was measured as c.p.m./ml and normalized for 106 cells after background subtraction. Virus titrations were carried out either by scoring the number of syncytia in C8166 cells at 5 days after challenge (Federico et al., 1993
) or by evaluating the number of blue cells at 2 days after the infection of HeLaCD4-LTR-
gal cells (Bryant et al., 1991
). To determine Nef expression from chimeric viruses expressing the F12-HIVNef back mutants, Western blot analyses on 293 cells transiently transfected with the different HIV-1 molecular clones were performed by means of the enhanced chemiluminescence method (Amersham), as described previously (DAloja et al., 1998
). The stability of CD8Nef chimeric proteins was assessed either by Western blot or by anti-CD8 immunoprecipitation of lysates from 35S-labelled cells (DAloja et al., 1998
) followed by 12% PAGE.
Mutagenesis, cloning and subcloning.
To introduce site-specific mutations into the F12-HIVnef allele, the overlapping PCR technique was carried out as described previously (Taddeo et al., 1996 ). A first amplification step using F12-HIVnef as the template served to generate two half fragments of nef. Internal primer couples carried the back mutations. The external primers contained HindIII (5') and EcoRI (3') restriction enzyme sites. Full-length nef fragments were generated by second-round PCR using only the external primers. The PCR mixture contained 20 mM TrisHCl (pH 8·8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0·2 mM dNTP, 0·6 µg of each primer, 0·5 µg of DNA template and 2 U of Vent DNA polymerase (New England Biolabs) in a final volume of 50 µl. Reaction mixtures were subjected to 15 cycles of 95 °C for 1 min, 58 °C for 1 min and 72 °C for 2 min. Finally, full-length nef fragments were digested with HindIII/EcoRI and inserted into the homologous sites of the pcDNA3 vector.
Construction of the pNL4-3 molecular clone expressing F12-HIVNef was described previously (Olivetta et al., 2000 ). pNL4-3 clones expressing each F12-HIVNef back mutant were obtained in a similar way. Briefly, nef mutants were amplified by PCR using the pcDNA3nef constructs as a template and a pair of oligoprimers carrying the MluI (5' end) and ClaI (3' end) restriction sites, which overlapped the nef initiation and stop codons, respectively. Amplified nef genes were then inserted into a derivative of the pNL4-3 plasmid, where the MluI (whose restriction site was present in a linker inserted between the env stop and nef start codons) and ClaI (downstream to the nef stop codon) sites had been created previously. The infectious molecular clone expressing the NL4-3 HIV-1 genome defective in nef expression has been described previously (Chowers et al., 1994
).
CD8Nef-expressing vectors were obtained by inserting in-frame nef mutants obtained by PCR using oligoprimers carrying BamHI (forward) and SalI (reverse) restriction sites into the homologous unique sites of the pEF-BOS-CD8T vector (Lu et al., 1996 , 1998
). The vector expressing the green fluorescence protein (GFP) has been described previously (Palm et al., 1997
). The pCMX/CD4LL413AA construct expressing a CD4 molecule defective in the spontaneous internalization process was a generous gift from C. Aiken.
All of the sequences obtained by PCR amplification were rigorously checked by the dideoxy chain-termination method using the Sequenase II kit (US Biochemicals).
Analysis of the steady-state levels, internalization and recycling of CD4.
Steady-state levels of CD4 were assayed by membrane immunolabelling performed, as described previously (Olivetta et al., 2000 ), using phycoerythrin (PE)-conjugated Leu3A monoclonal antibody (MAb) (Becton Dickinson) on 293/CD4 cells co-transfected with both GFP- and Nef-expressing vectors (molar ratio 1:5). Labelled cells were assessed by FACS analysis (Becton Dickinson). Assays for CD4 internalization and recycling were performed, as reported previously (Mangasarian et al., 1997
; Piguet et al., 1998
), on transfected 293/CD4 cells. Briefly, cells harvested 48 h after transfection were labelled with PE-conjugated anti-CD4 MAb for 30 min at 4 °C and then washed extensively. Afterwards, cells were incubated at 37 °C and, after 15 and 30 min (the time-points at which the triggering of accelerated CD4 internalization by Nef is more clearly distinguishable) (Mangasarian et al., 1997
; Piguet et al., 1998
, 1999
), washed with a 7-fold excess of cold PBS (pH 2). Fractions of internalized CD4 were calculated as the product of the mean fluorescence intensity (MFI) value recorded after the pulse at 37 °C and acid washes, subtracted of the background MFI value recorded after incubation at 4 °C and acid washes, all divided by the total (i.e. surface plus internalized) MFI value recorded after the incubation of cells at 37 °C and before acid washes. Rates of internalization of CD8Nef chimeras were measured in the same way, except that the Leu3A PE-conjugated anti-CD8 MAb was used. To measure the rate of CD4 recycling (Piguet et al., 1998
; Mangasarian et al., 1999
), fractions of cells from the endocytosis assay were warmed for 15 and 30 min and washed again in cold, acidic PBS. The remaining fluorescence was measured by FACS analysis. Percentages of recycled CD4 were calculated as 1-(MFI rew/MFI fp), where the numerator refers to the MFI values after the re-warming and acid washes and the denominator refers to the MFI values measured after the first pulse at 37 °C and acid washes. In all of the analyses, dead cells were excluded by means of side and forward scatter parameters during FACS acquisition.
p62NAK activation assay.
293T cells transfected with vectors expressing diverse CD8Nef chimeras were lysed 48 h after transfection in 0·5 ml of extraction buffer containing 50 mM TrisHCl (pH 8·0), 0·5% NP40, 2 mM EDTA, 250 mM NaCl, 10% glycerol, 10 µg/ml aprotinin, 10 µg/ml leupeptin and 1 mM NaV03. Cell lysates were incubated with 100 µl of anti-CD8-coupled beads (Dynal) for 4 h at 4 °C, washed three times with extraction buffer and once with kinase activation buffer (KAB) containing 50 mM HEPES, 150 mM NaCl, 5 mM EDTA, 0·02% Triton X-100 and 10 mM MnCl2. Immunoprecipitated proteins were incubated in 50 µl KAB containing 0·37 MBq of [-32P]ATP for 10 min at room temperature and finally washed three times with lysis buffer. Phosphorylated proteins were resolved by 10% SDSPAGE and detected by autoradiography.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In order to evaluate the effects of the three amino acid substitutions in terms of HIV-1 replication, human lymphoblastoid CEMss cells were infected with virus preparations yielded upon transfection of 293 cells with pNL4-3 molecular clones expressing each of the F12-HIVNef back mutants. As a control, CEMss cells were infected with an equal m.o.i. of either wt (NL4-3) or NL4-3/F12-HIVNef strains. Clearly, each back mutation led the NL4-3 HIV-1 to regain the productive phenotype (Fig. 1A). The apparent stability of diverse Nef proteins was proven by Western blot analyses on lysates of 293 cells transfected with the respective infectious molecular clones (Fig. 1B
). Overall, similar results were obtained by challenging pools of HeLaCD4 cell clones stably expressing each of the F12-HIVNef back mutants (data not shown).
|
F12-HIVNef expression does not induce accelerated CD4 internalization
Cells expressing F12-HIVNef are defective for CD4 down-regulation (DAloja et al., 1998 ; Olivetta et al., 2000
). Nevertheless, F12-HIVNef interacts with the CD4 intracytoplasmic tail, whose presence is required for the antiviral effect (Olivetta et al., 2000
). Considering that high levels of membrane CD4 lead to a decreased production of infectious HIV-1 by means of impaired viral envelope protein incorporation (Lama et al., 1999
; Ross et al., 1999
), it was conceivable that unaltered levels of CD4 in the membrane are indispensable for the antiviral phenotype of F12-HIVNef. The measurement of steady-state CD4 levels does not exhaustively describe the effects of Nef on CD4. This is a receptor that constitutively undergoes internalization and recycling (Marsh & Pelchen-Matthews, 1996
). Two genetically and functionally distinguishable events had been identified as the result of the CD4Nef interaction (Piguet et al., 1998
, 1999
), i.e. the triggering of a strong acceleration in the rate of CD4 internalization and the inhibition of recycling by routing internalized CD4 to the lysosome compartment. Attempting to correlate the mechanics of the F12-HIVNef-induced antiviral action with its effects on CD4, we investigated the functional defect(s) of F12-HIVNef that underlie the lack of CD4 down-regulation. Steady-state CD4 levels were measured in 293/CD4 cells by co-transfecting GFP- and F12-HIVnef-expressing vectors in a 1:5 molar ratio. CD4 FACS analysis of GFP-positive cells carried out 48 h post-transfection demonstrated that, as reported previously (DAloja et al., 1998
; Olivetta et al., 2000
), F12-HIVNef expression did not alter the steady-state exposition of CD4 (Fig. 2A
). Both CD4 internalization and recycling assays were performed by transfecting 293/CD4 cells with the vector expressing pcDNA3/F12-HIVNef. Results were considered exclusively in the presence of transfection efficiencies >70%, as monitored by scoring GFP-positive cells in parallel conditions. As shown in Fig. 2(B)
, F12-HIVNef expression failed to accelerate CD4 internalization, as the endocytosis rate was similar to that detected in cells transfected with the control vector. As expected, results from recycling assays did not increase the significance of the overall CD4 analyses (data not shown). We conclude that F12-HIVNef expression does not induce CD4 down-regulation as a consequence of a defect in the acceleration of CD4 endocytosis.
|
|
|
F12-HIVNef expression does not induce activation of p62NAK and rescue by the G177E back mutation
As the expression of the G177E F12-HIVNef back mutant did not inhibit either CD4 membrane expression or HIV-1 release, we conclude that the F12-HIVNef antiviral effect could not be merely the consequence of unaltered levels of membrane CD4 exposition. For these reasons, additional markers of Nef functions were investigated. Among the cellular proteins known already to associate with and/or to be activated by Nef, the most informative results were obtained by analysing p62NAK activation. This is a serine/threonine kinase capable of auto-phosphorylation (Sawai et al., 1996 ) and is both immunologically and functionally related to the p21-activated kinases (PAK) (Nunn & Marsh, 1996
). Of note, the activity of p62NAK has been correlated positively with the process of HIV-1 release (Lu et al., 1996
), the step where F12-HIVNef acts specifically in blocking HIV-1 replication.
No p62NAK activation was found in F12-HIVNef-expressing cells, where, conversely, the apparent phosphorylation of the, as yet, unidentified protein with a molecular mass of 75 kDa could be appreciated (Fig. 5
). Similarly, both the E140G and the L153V back mutants failed to activate p62NAK, whereas the G177E back mutation was sufficient to rescue p62NAK activation (Fig. 5
). This indicates that the lack of p62NAK activation is not sufficient per se to explain the F12-HIVNef antiviral phenotype. Both the amount and the integrity of CD8Nef chimeric proteins after immunoprecipitation were checked by anti-Nef Western blot analysis (Fig. 5
).
|
F12-HIV Nef antiviral effect depends on both the lack of accelerated CD4 internalization and an, as yet, still unidentified function
We demonstrated that the NL4-3/F12-HIVnef genome regains the infectious phenotype upon reversion of any of the three F12-HIVNef typical amino acid substitutions (Fig. 1). Furthermore, the G177E back mutant does not accelerate the rate of CD4 internalization. Both the E140G and the L153V back mutants are defective for p62NAK activation, whereas parental F12-HIVNef lacks both functions (Table 1
). In order to define more stringently the role of CD4 down-regulation in the F12-HIVNef-inhibitory phenotype, we recovered a 293 cell population that stably expresses a CD4 molecule mutated in the intracytoplasmic dileucine motif (LL413AA). In this way, CD4 becomes defective for its physiological internalization activity, thus resisting the Nef-induced accelerated internalization (Aiken et al., 1994
).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The absolute requirement of the CD4 intracytoplasmic tail for the F12-HIVNef-induced antiviral effect has been demonstrated previously (Olivetta et al., 2000 ). In this paper, we provide data that point out the lack of Nef-induced accelerated CD4 internalization as an additional requisite for the antiviral effect. However, HIV-1 expressing the G177E back mutant is a replication competent virus, despite its inability to accelerate CD4 endocytosis, whereas HIV-1 expressing the E140G back mutant maintains the producer phenotype seen also in Nef-resistant CD4-expressing cells (Table 1
). This indicates that the lack of Nef-induced accelerated CD4 internalization is not sufficient to explain the F12-HIVNef phenotype.
We show that F12-HIVNef does not activate p62NAK and, among the back mutants analysed, only the G177E back mutant re-acquires such a function. p62NAK is a serine/threonine kinase identified recently as PAK-1 (Fackler et al., 2000 ) or PAK-2 (Arora et al., 2000
; Renkema et al., 1999
). It has been demonstrated that the Nef-dependent p62NAK activation induces cytoskeleton rearrangement, which facilitates the virus release process (Fackler et al., 1999
) and, in general, seems to be involved in the progress of AIDS pathogenesis (Khan et al., 1998
; Sawai et al., 1996
).
By matching data from CD4 and p62NAK analyses with infection experiments (Table 1), we conclude that the simultaneous lack of both functions correlates with the Nef-induced block of virus release. However, the productive phenotype resulting from the infection of 293 LL413AA CD4 cells with HIV-1 expressing the E140G back mutant indicates that still unknown factors/functions are also involved. Among the large array of Nef protein-binding partners we analysed, neither the Nef-binding protein-1, identified as the catalytic subunit of the vacuolar ATPase (Lu et al., 1998
), the p85 regulatory subunit of the phosphatidylinositol-3-kinase (Andreas Baur, unpublished observations), nor Vav seemed to play a role in the F12-HIVNef phenotype. In the kinase assay reported here, a protein with a molecular mass of
75 kDa is detectable in lysates from cells expressing F12-HIVNef. Further investigations aimed to establish whether this product represents a novel F12-HIVNef-binding partner are required.
It is conceivable that the strong antiviral effect of F12-HIVNef is not solely the consequence of the lack of Nef-specific functions, but also of an active negative function. The existence of such a function could be deduced by the opposite phenotypes of the E140G and L153V back mutants in LL413AA CD4-expressing cells. This function seems to correlate with the presence of the E140 residue and acts only in the absence of both CD4 accelerated internalization and p62NAK activation.
Our findings allow us to speculate on the structural basis of the F12-HIVNef phenotype. Wt Nef protein has been crystallized, although not in its full-length form (Franken et al., 1997 ; Grzesiek et al., 1997
; Lee et al., 1996
). Thus, we have the possibility to predict the structural consequences of each F12-HIVNef amino acid substitution. At position 140, F12-HIVNef displays an E residue instead of a G residue, which is normally present in replication-competent HIV-1 strains. This position resides in the turn between the B and C
-strands (Fig. 7
). The introduction of the E side chain may cause both steric (lack of space for the long side chain of E) and charge problems (the environment, formed by V85, Y81, F139, W141 and A190, is rather hydrophobic, as opposed to E, which is negatively charged). It is thus conceivable that the G140E mutation could generate a partially unfolded protein. Of note, we observed that such an amino acid substitution effectively co-operates with the V153L mutation in failing to trigger accelerated CD4 endocytosis.
|
The Nef regions specific for p62NAK binding have been mapped in the Nef core domain, in particular, in both the polyproline and the highly conserved arginine motifs (Manninen et al., 1998 ; Sawai et al., 1995
). Here, we show that the amino acid at position 177 is important also for p62NAK activation. This could be deduced considering that both the E140G and the L153V Nef back mutants as well as the parental F12-HIVNef do not activate p62NAK and that only the G177E mutant reverts towards the wt phenotype. This result appears to be consistent with the data from Luo et al. (1997)
, who highlighted the importance of the C-terminal region of Nef in p62NAK activation.
It has been reported already that both accelerated CD4 endocytosis and p62NAK activation favour virus spread (Fackler et al., 1999 ; Lama et al., 1999
; Ross et al., 1999
). Here, we strengthen these observations by describing a complete block of release in HIV-1 expressing a Nef mutant lacking both functions. This is additional evidence to support the fact that interactions of Nef with appropriated cell targets could be critical for virus replication.
We were able to define both the genetic and the functional markers of the antiviral action of F12-HIVNef by the analysis of its back mutants. This system represents a potent tool for gaining even more detailed insight into the antiviral action mechanism of F12-HIVNef and also for revealing Nef functions that are still uncharacterized.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arora, V. K., Molina, R. P., Foster, J. L., Blakemore, J. L., Chernoff, J., Fredericksen, B. L. & Garcia, J. V. (2000). Lentivirus Nef specifically activates Pak2. Journal of Virology 74, 11081-11087.
Bell, I., Ashman, C., Maughan, J., Hooker, E., Cook, F. & Reinhart, T. A. (1998). Association of simian immunodeficiency virus Nef with the T-cell receptor (TCR) chain leads to TCR down-modulation. Journal of General Virology 79, 2717-2727.[Abstract]
Brambilla, A., Turchetto, L., Gatti, A., Bovolenta, C., Veglia, F., Santagostino, E., Gringeri, A., Clementi, M., Poli, G., Bagnarelli, P. & Vicenzi, E. (1999). Defective nef alleles in a cohort of hemophiliacs with progressing and nonprogressing HIV-1 infection. Virology 259, 349-368.[Medline]
Bryant, M. L., Ratner, L., Duronio, R. J., Kishore, N. S., Devadas, B., Adams, S. P. & Grodon, J. I. (1991). Incorporation of 12-methoxydodecanoate into the human immunodeficiency virus 1 Gag polyprotein precursor inhibits its proteolytic processing and virus production in a chronically infected human lymphoid cell line. Proceedings of the National Academy of Sciences, USA 88, 2055-2059.[Abstract]
Carlini, F., Federico, M., Equestre, M., Ricci, S., Ratti, G., Zibai, Q., Verani, P. & Rossi, G. B. (1992). Sequence analysis of an HIV-1 proviral DNA from a non-producer chronically infected Hut-78 cellular clone. Journal of Virological Diseases 1, 40-55.
Chowers, M. Y., Spina, C. A., Kwoh, T. J., Fitch, N. J. S., Richman, D. D. & Guatelli, J. C. (1994). Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene. Journal of Virology 68, 2906-2914.[Abstract]
Cohen, G. B., Gandhi, R. T., Davis, D. M., Mandelboim, O., Chen, B. K., Strominger, J. L. & Baltimore, D. (1999). The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity 10, 661-671.[Medline]
Collins, K. L., Chen, B. K., Kalams, S. A., Walker, B. D. & Baltimore, D. (1998). HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature 391, 397-401.[Medline]
Craig, H. M., Pandori, M. W. & Guatelli, J. C. (1998). Interaction of HIV-1 Nef with the cellular dileucine-based sorting pathway is required for CD4 down-regulation and optimal viral infectivity. Proceedings of the National Academy of Sciences, USA 95, 11229-11234.
DAloja, P., Olivetta, E., Bona, R., Nappi, F., Pedacchia, D., Pugliese, K., Ferrari, G., Verani, P. & Federico, M. (1998). gag, vif, and nef genes contribute to the homologous viral interference induced by a nonproducer human immunodeficiency virus type 1 (HIV-1) variant: identification of novel HIV1-inhibiting viral protein mutants. Journal of Virology 72, 4308-4319.
Fackler, O. T., Luo, W., Geyer, M., Alberts, A. S. & Peterlin, B. M. (1999). Activation of Vav by Nef induces cytoskeletal rearrangements and downstream effector functions. Molecular Cell 3, 729-739.[Medline]
Fackler, O. T., Lu, X., Frost, J. A. A., Geyer, M., Jiang, B., Luo, W., Abo, A., Alberts, A. S. & Peterlin, B. M. (2000). p21-activated kinase 1 plays a critical role in cellular activation by Nef. Molecular and Cellular Biology 20, 2619-2627.
Federico, M., Taddeo, B., Carlini, F., Nappi, F., Verani, P. & Rossi, G. B. (1993). A recombinant retrovirus carrying a non-producer human immunodeficiency virus (HIV) type 1 variant induces resistance to superinfecting HIV. Journal of General Virology 74, 2099-2110.[Abstract]
Franken, P., Arold, S., Padilla, A., Bodeus, M., Hoh, F., Strub, M. P., Boyer, M., Jullien, M., Benarous, R. & Dumas, C. (1997). HIV-1 Nef protein: purification, crystallization, and preliminary X-ray diffraction studies. Protein Science 6, 2681-2683.
Greenberg, M. E., Bronson, S., Lock, M., Neumann, M., Pavlakis, G. N. & Skowronski, J. (1997). Co-localization of HIV-1 Nef with the AP-2 adaptor protein complex correlates with Nef-induced CD4 down-regulation. EMBO Journal 16, 6964-6976.
Greenberg, M. E., Iafrate, A. J. & Skowronski, J. (1998). The SH3 domain-binding surface and an acidic motif in HIV-1 Nef regulate trafficking of class I MHC complexes. EMBO Journal 17, 2777-2789.
Grzesiek, S., Bax, A., Hu, J. S., Kaufman, J., Palmer, I., Stahl, S. J., Tjandra, N. & Wingfield, P. T. (1997). Refined solution structure and backbone dynamics of HIV-1 Nef. Protein Science 6, 1248-1263.
Khan, I. H., Sawai, E. T., Antonio, E., Weber, C. J., Mandell, C. P., Montbriand, P. & Luciw, P. A. (1998). Role of the SH3-ligand domain of simian immunodeficiency virus Nef in interaction with Nef-associated kinase and simian AIDS in rhesus macaques. Journal of Virology 72, 5820-5830.
Lama, J., Mangasarian, A. & Trono, D. (1999). Cell-surface expression of CD4 reduces HIV-1 infectivity by blocking Env incorporation in a Nef- and Vpu-inhibitable manner. Current Biology 9, 622-631.[Medline]
Lee, C. H., Saksela, K., Mirza, U. A., Chait, B. T. & Kuriyan, J. (1996). Crystal structure of the conserved core of HIV-1 Nef complexed with a Src family SH3 domain. Cell 85, 931-942.[Medline]
Le Gall, S., Erdtmann, L., Benichou, S., Berlioz-Torrent, C., Liu, L., Benarous, R., Heard, J. M. & Schwartz, O. (1998). Nef interacts with the mu subunit of clathrin adaptor complexes and reveals a cryptic sorting signal in MHC I molecules. Immunity 8, 483-495.[Medline]
Lu, X., Wu, X., Plemenitas, A., Yu, H., Sawai, E. T., Abo, A. & Peterlin, B. M. (1996). CDC42 and Rac1 are implicated in the activation of the Nef-associated kinase and replication of HIV-1. Current Biology 6, 1677-1684.[Medline]
Lu, X., Yu, H., Brodsky, F. M. & Peterlin, B. M. (1998). Interactions between HIV-1 Nef and vacuolar ATPase facilitate the internalization of CD4. Immunity 8, 647-656.[Medline]
Luo, T., Livingston, R. A. & Garcia, J. V. (1997). Infectivity enhancement by human immunodeficiency virus type 1 Nef is independent of its association with a cellular serine/threonine kinase. Journal of Virology 71, 9524-9530.[Abstract]
Mangasarian, A., Foti, M., Aiken, C., Chin, D., Carpentier, J. L. & Trono, D. (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.[Medline]
Mangasarian, A., Piguet, V., Wang, J. K., Chen, Y. L. & Trono, D. (1999). Nef-induced CD4 and major histocompatibility complex class I (MHC-I) down-regulation are governed by distinct determinants: N-terminal -helix and proline repeat of Nef selectively regulate MHC-I trafficking. Journal of Virology 73, 1964-1973.
Manninen, A., Hiipakka, M., Vihinen, M., Lu, W., Mayer, B. J. & Saksela, K. (1998). SH3-domain binding function of HIV-1 Nef is required for association with a PAK-related kinase. Virology 250, 273-282.[Medline]
Marsh, M. & Pelchen-Matthews, A. (1996). Endocytic and exocytic regulation of CD4 expression and function. Current Topics in Microbiology and Immunology 205, 107-135.[Medline]
Miller, M., Warmerdam, M. T., Gaston, I., Greene, W. C. & Feinberg, M. B. (1994). The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages. Journal of Experimental Medicine 179, 101-113.[Abstract]
Nunn, M. F. & Marsh, J. W. (1996). Human immunodeficiency virus type 1 Nef associates with a member of the p21-activated kinase family. Journal of Virology 70, 6157-6161.[Abstract]
Olivetta, E., Pugliese, K., Bona, R., DAloja, P., Ferrantelli, F., Santarcangelo, A. C., Mattia, G., Verani, P. & Federico, M. (2000). cis expression of the F12 human immunodeficiency virus (HIV) Nef allele transforms the highly productive NL4-3 HIV type 1 to a replication-defective strain: involvement of both Env gp41 and CD4 intracytoplasmic tails. Journal of Virology 74, 483-492.
Palm, G. J., Zdanov, A., Gaitanaris, G. A., Stauber, R., Pavlakis, G. N. & Wlodawer, A. (1997). The structural basis for spectral variation in green fluorescent protein. Nature Structural Biology 4, 361-365.[Medline]
Piguet, V., Chen, Y. L., Mangasarian, A., Foti, M., Carpentier, J. L. & Trono, D. (1998). Mechanism of Nef-induced CD4 endocytosis: Nef connects CD4 with the mu chain of adaptor complexes. EMBO Journal 17, 2472-2481.
Piguet, V., Gu, F., Foti, M., Demaurex, N., Gruenberg, J., Carpentier, J. L. & Trono, D. (1999). Nef-induced CD4 degradation: a diacidic-based motif in Nef functions as a lysosomal targeting signal through the binding of -COP in endosomes. Cell 97, 63-73.[Medline]
Renkema, G. H., Manninen, A., Mann, D. A., Harris, M. & Saksela, K. (1999). Identification of the Nef-associated kinase as p21-activated kinase 2. Current Biology 9, 1407-1410.[Medline]
Ross, T. M., Oran, A. E. & Cullen, B. R. (1999). Inhibition of HIV-1 progeny virion release by cell-surface CD4 is relieved by expression of the viral Nef protein. Current Biology 9, 613-621.[Medline]
Rossi, G. B., Verani, P., Macchi, B., Federico, M., Orecchia, A., Nicoletti, L., Buttò, S., Lazzarin, A., Mariani, G., Ippolito, G. & Manzari, V. (1987). Recovery of HIV-related retroviruses from Italian patients with AIDS or AIDS-related complex and from asymptomatic at-risk individuals. Annals of the New York Academy of Sciences 511, 390-400.[Abstract]
Saksela, K. (1997). HIV-1 Nef and host cell protein kinases. Frontiers of Bioscience 2, 606-618.
Sawai, E. T., Baur, A., Struble, H., Peterlin, B. M., Levy, J. A. & Cheng-Mayer, C. (1994). Human immunodeficiency virus type 1 Nef associates with a cellular serine kinase in T lymphocytes. Proceedings of the National Academy of Sciences, USA 91, 1539-1543.[Abstract]
Sawai, E. T., Baur, A. S., Peterlin, B. M., Levy, J. A. & Cheng-Mayer, C. (1995). A conserved domain and membrane targeting of Nef from HIV and SIV are required for association with a cellular serine kinase activity. Journal of Biological Chemistry 270, 15307-15314.
Sawai, E. T., Khan, I. H., Montbriand, P. M., Peterlin, B. M., Cheng-Mayer, C. & Luciw, P. A. (1996). Activation of PAK by HIV and SIV Nef: importance for AIDS in rhesus macaques. Current Biology 6, 1519-1527.[Medline]
Schwartz, O., Maréchal, V., Le Gall, S., Lemonnier, F. & Heard, J. M. (1996). Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein. Nature Medicine 2, 338-342.[Medline]
Spina, C. A., Kwoh, T. J., Chowers, M. Y., Guatelli, J. C. & Richman, D. D. (1994). The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes. Journal of Experimental Medicine 179, 115-123.[Abstract]
Taddeo, B., Carlini, F., Verani, P. & Engelman, A. (1996). Reversion of a human immunodeficiency virus type 1 integrase mutant at a second site restores enzyme function and virus infectivity. Journal of Virology 70, 8277-8284.[Abstract]
Wigler, M., Sweet, R., Sim, G. K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, S. & Awel, R. (1979). Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell 16, 758-777.
Xu, X. N., Laffert, B., Screaton, G. R., Kraft, M., Wolf, D., Kolanus, W., Mongkolsapai, J., McMichael, A. J. & Baur, A. S. (1999). Induction of Fas ligand expression by HIV involves the interaction of Nef with the T cell receptor chain. Journal of Experimental Medicine 189, 1489-1496.
Received 22 March 2001;
accepted 1 August 2001.