SGM Journals
Research Article

The Pro78 residue regulates the capacity of the human immunodeficiency virus type 1 Nef protein to inhibit recycling of major histocompatibility complex class I molecules in an SH3-independent manner

Casartelli, Nicoletta, Giolo, Giorgia, Neri, Francesca, Haller, Claudia, Potesta, Marina, Rossi, Paolo, Fackler, Oliver T., Doria, Margherita

,†, Giorgia Giolo1, Francesca Neri1, Claudia Haller2, Marina Potestà1, Paolo Rossi1,3, Oliver T. Fackler2 and Margherita Doria1

1 Division of Immunology and Infectious Disease, Children's Hospital Bambino Gesù, Piazza S. Onofrio 4, 00165 Rome, Italy
2 Department of Virology, Universitätsklinikum Heidelberg, 69120 Heidelberg, Germany
3 Department of Pediatrics, University Tor Vergata, 00133 Rome, Italy

Correspondence
Margherita Doria
doria{at}uniroma2.it

Journal of General Virology 2006; 87(8):2291 · https://doi.org/10.1099/vir.0.81775-0

View at publisher PubMed

Abstract

The Nef protein is a crucial pathogenicity factor of human immunodeficiency virus type 1 (HIV-1) that contains a proline-rich motif consisting of four conserved prolines: Pro69 (P69), P72, P75 and P78. P72 and P75 were shown to bind Src homology domains 3 (SH3) and have been implicated in many biological functions of Nef, including downmodulation of cell-surface major histocompatibility complex class I (MHC-I). P78 is involved together with P69 in positioning of the NefSH3 complex and it has been shown to be essential for Nef activity of MHC-I downmodulation. It is shown here that alteration of P78 affects recycling of MHC-I molecules to the cell surface, but does not interfere with SH3 binding. In addition, it is demonstrated that P72 and P75, and thus the SH3-binding capacity, are fully dispensable for Nef activity on MHC-I.
We thank Matthias Geyer for critical reading. This work was supported by the V National Program of AIDS Research, Istituto Superiore di Sanità (40F.35) and Ricerche Correnti of Children's Hospital Bambino Gesù to M. D., a short-term EMBO fellowship (ASTF 156-2004) to N. C. and by grants from the Deutsche Forschungsgemeinschaft (SFB 638 project A11, TR 7013 project C4) and the CHS Stiftung to O. T. F.

Footnotes

†Present address: Virus and Immunity Group, Department of Virology, Institut Pasteur, 75724 Paris Cedex 15, France.



The Nef protein of human immunodeficiency virus type 1 (HIV-1) is a critical factor for high virus replication and disease progression in humans and animal models (Deacon et al., 1995; Hanna et al., 1998; Kirchhoff et al., 1995). Nef has several activities: downmodulation of cell-surface molecules such as CD4 and major histocompatibility complex class I (MHC-I), alteration of cellular signalling pathways and stimulation of HIV-1 infectivity and replication (reviewed by Fackler & Baur, 2002; Federico, 2004; Peterlin & Trono, 2003). Conserved amino acid residues and motifs in Nef have been characterized for their role in specific activities and interactions (Arold & Baur, 2001; Geyer et al., 2001), but it is still unclear which one(s) of these ultimately mediate(s) the pathogenic potential of Nef. A conserved proline-rich motif (PxxP) of Nef was accredited as a Src homology domain 3 (SH3)-binding site, with its central prolines, Pro72 (P72) and P75, contacting two hydrophobic pockets in the SH3 domain, whereas P69 and P78 assist the PxxP positioning (Arold et al., 1997; Lee et al., 1996). Through PxxP, Nef binds SH3-containing signalling molecules such as Hck (Saksela et al., 1995) and Vav (Fackler et al., 1999). Although the PxxP motif has been involved in most Nef functions (Greenberg et al., 1998; Hanna et al., 2001; Iafrate et al., 1997; Mangasarian et al., 1999; Saksela et al., 1995), some studies have shown that it is dispensable for Nef activity on MHC-I (Riggs et al., 1999) or CD4 (Saksela et al., 1995) and HIV-1 replication and pathogenesis (Kawano et al., 1997). In addition, it is unclear whether the role of the PxxP motif in the various biological functions of Nef is invariably mediated by its binding to SH3-containing proteins. As for MHC-I downmodulation, P78 rather than P69/P72/P75 was shown to be crucial (Yamada et al., 2003). Besides, conflicting reports exist on the putative role of PI3-K (Blagoveshchenskaya et al., 2002; Kasper & Collins, 2003) and Hck (Chang et al., 2001; Greenberg et al., 1998; Mangasarian et al., 1999) as relevant SH3 ligands for Nef activity on MHC-I.

To further investigate the role of the PxxP motif of Nef, we performed various analyses based on an in vivo-selected mutation. We showed previously that two Nef proteins, NP5-7 and NP5-8, derived from a non-progressor patient (Casartelli et al., 2003a), were impaired in MHC-I downmodulation and partially defective in CD4 downregulation (Casartelli et al., 2003b). As both proteins contained a leucine at position 78, we restored P78 in NP5-8 by mutagenesis and tested the resulting NP5-8P78 protein for both CD4 and MHC-I downregulation activities by means of a retrovirus-based transduction system in RMAS-A2 (MHC-I), HeLa-CD4 (CD4) and Jurkat (CD4 and MHC-I) cells, followed by two-colour flow cytometry (Casartelli et al., 2003b). Nef proteins derived from the NL4-3 viral strain, either wild type (NEF), with P78 mutated into a leucine (NEF-L78) or mutated at residues M20 and EEEE65 [shown previously to be required for MHC-I downmodulation (reviewed by Arold & Baur, 2001; Geyer et al., 2001)] (NEF-A20 and NEF-4E4Q, respectively; shown only for Jurkat; Fig. 1a) were also tested. As shown in Fig. 1(a), the L78P substitution restored NP5-8 activity on both CD4 and MHC-I fully. The detrimental effect of L78 was unrelated to the allelic background, as NEF-L78 was also impaired (Fig. 1a, b), and was not caused by altered steady-state protein expression (Fig. 1c) or aberrant subcellular distribution (data not shown). However, in line with a previous report (Yamada et al., 2003), a Nef mutant in which P78 was substituted with alanine (NEF-A78) was defective for MHC-I downregulation, but was active on CD4, similarly to NEF-A20 and NEF-4E4Q. It is likely that, although P78 has no direct role in CD4 downmodulation, selected amino acid substitutions induce conformational changes resulting in a Nef protein defective for this activity.



(42K):

 		Fig. 1. Role of P78 in Nef activity of MHC-I and CD4 downmodulation. (a) Downregulation of cell-surface MHC-I and CD4 by wild-type and mutated Nef proteins. Cells were infected as described previously (Casartelli et al., 2003b) with empty retrovirus (Pinco) or with recombinant Pinco retroviruses expressing NL4-3-derived Nef (NEF), NP5-8 or the indicated mutants. Specific mutations have been introduced into NEF and NP5-8 by standard site-directed mutagenesis based on recombinant PCR. After 48 h, cells were analysed by two-colour flow cytometry for expression of GFP encoded by the Pinco vector and of cell-surface MHC-I (Jurkat cells, top panels), human leukocyte antigen A2 molecules (HLA-A2; RMAS-A2 cells) or CD4 (HeLa-CD4 cells; Jurkat, bottom panels). HLA-A2 and CD4 stainings were performed as described previously (Casartelli et al., 2003b). For MHC-I staining, a phycoerythrin (PE)-conjugated anti-HLA-A/B/C antibody (Becton Dickinson) was used. (b) Jurkat cells were infected and analysed for expression of MHC-I (top) and CD4 (bottom) as described in (a). The geometric mean fluorescence intensities (MFI) specific for MHC-I or CD4 were evaluated in cells expressing medium levels of GFP fluorescence [gated in the R1 region in (a)]. Values are expressed as a percentage of Nef activity (empty bars) of MHC-I (filled bars) and CD4 (shaded bars) downregulation. Reported values are the means±SD of three independent experiments. (c) Immunoblotting analysis of NEF, NP5-8 and their mutants. Lysates of Phoenix cells transfected with the indicated clones were immunoblotted with an anti-Nef antibody (upper panel; ARP3026; MRC AIDS Reagent Project) as described previously (Casartelli et al., 2003b). The arrow indicates the Nef protein bands. A cross-reacting, slower-migrating band present in the lysates is also visible. Due to their slower migration (Casartelli et al., 2003b), NP5-8 and NP5-8P78 co-migrate with the cross-reacting band. The blot was then stripped and reprobed with an anti-GFP antibody (Clontech) to evaluate transfection efficiency (lower panel). Similar results were obtained with lysates of cells shown in (a) (not shown).

As P78 may participate in the formation of a NefSH3 complex (Arold et al., 1997; Lee et al., 1996), functional defects of L78 mutants might be associated with an altered SH3-binding capacity. We therefore investigated the contribution of the SH3-binding property to Nef activities by analysing two mutants in which either the central two or all four prolines of PxxP were replaced by alanines (NEF-A72A75 and NEF-AAAA, respectively). Fig. 1(a, b) shows that NEF-A72A75 was fully active on CD4 and partially impaired in MHC-I downmodulation. Of note, the steady-state expression level of the mutated protein was lower than that of the wild type (Figs 1c, 2a), in line with previous studies showing NEF-A72A75 protein instability (Craig et al., 1999; Iafrate et al., 1997). As MHC-I downmodulation requires significantly higher intracellular Nef concentrations than does CD4 downmodulation (Liu et al., 2001), low protein amounts should affect Nef activity on MHC-I preferentially. Consistently, NEF-AAAA, which was barely detectable by immunoblotting analysis (Fig. 1c), lost its activity on CD4 and MHC-I partly and completely, respectively. Thus, NEF-AAAA was disregarded for further analysis. To test whether the NEF-A72A75 defect in MHC-I downregulation was due to reduced protein expression, we titrated the retroviral particles expressing NEF or NEF-A72A75 by one to four spin-infection cycles (Fig. 2a, b). We found that cells infected with the same amount of virus displayed lower MHC-I downmodulation, as well as lower Nef protein amounts, when NEF-A72A75 was expressed in place of NEF, as expected for an unstable protein. However, in cells expressing similar levels of NEF and NEF-A72A75 following two and three spin infections, respectively, the extent of MHC-I downmodulation was equivalent (Fig. 2b). Thus, NEF and NEF-A72A75 display identical MHC-I downmodulation activities, for which the SH3-binding facet of the PxxP motif is fully dispensable, at least in this experimental system. In CD4+ T cells infected with HIV-1 carrying the NEF-A72A75 mutant, MHC-I downregulation was reduced (78 % of the activity of wild-type virus), concomitant with a lower Nef protein expression (36 % of the wild-type NEF; unpublished data), suggesting that, also in primary HIV-1-infected lymphocytes, the SH3-binding surface of Nef is important for protein stability, but not for its activity on MHC-I.



(48K):

 		Fig. 2. The MHC-I downmodulation activity of Nef is independent of its SH3-binding capacity. (a, b) Jurkat cells were infected with one to four spin-infection cycles with retroviruses expressing NEF or NEF-A72A75. (a) The steady-state expression of Nef proteins was evaluated by immunoblotting cell lysates with the anti-Nef antibody mAb 158 (Fackler et al., 1997) (upper panel). Next, the blot was stripped and probed with anti-GFP antibody (lower panel). (b) An aliquot of cells was employed to assess MHC-I downregulation (columns) as described in Fig. 1(b). The downregulation efficiencies were calculated by considering as 100 % the activity of NEF in cells infected by four infection cycles. Grey columns indicate similar MHC-I downmodulation activities. Grey lines indicate Nef protein levels as quantified by densitometry of blots shown in (a). Arbitrary densitometric units for Nef-specific bands were calculated by considering as 1 unit the value corresponding to the NEF-specific band in 1x-infected cells. (c) In vitro binding of Hck to NEF-R, NP5-8 and their derivatives. The upper panel shows proteins bound to GST-fusion proteins revealed by immunoblotting with an anti-Hck antibody (sc-72; Santa Cruz Biotechnology). Coomassie staining (lower panel) was used to evaluate sample loading. The value corresponding to Hck was normalized for that of GST-fusion protein in the same sample, both measured by densitometry and expressed as a percentage of the Hck protein bound to GSTNEF-R. Representative data from one of three independent experiments are shown.

We then analysed the SH3-binding capacity of NP5-8 and NP5-8P78 by testing their ability to form a complex with Hck. NEF, NEF-A72A75 and NEF-L78 were mutated by T71R substitution to optimize NefSH3 interaction (Saksela et al., 1995), generating NEF-R, NEF-RA72A75 and NEF-RL78. The T71R substitution did not alter CD4 or MHC-I downregulation activities (data not shown). All variants were expressed as glutathione S-transferase (GST)-fusion proteins and tested for their capacity to bind Hck from U937 cellular lysates [as described by Lee et al. (1995)]. NP5-8 and NEF-R did not differ from the corresponding mutated variants, NP5-8P78 and NEF-RL78, respectively, in their relative Hck-binding capacity (Fig. 2c). The binding specificity was confirmed by the absence of Hck associated with GSTNEF-A72A75 or GST alone. Measurement of the capacity to associate with p21-activated kinase (Krautkrämer et al., 2004), which depends strictly on the SH3-binding capacity of Nef (Manninen et al., 1998), confirmed that P78 has no role in NefSH3 interactions (data not shown).

As the clathrin adaptor-protein complex AP-1 is required for Nef-mediated MHC-I downmodulation (Le Gall et al., 1998; Roeth et al., 2004), we tested the ability of NP5-8 to form a complex with AP-1 by an in vitro binding assay (Fig. 3a). No difference in association with AP-1 was detected for NEF, NP5-8 or NP5-8P78. Thus, the functional defect induced by L78 cannot be attributed to inefficient in vitro AP-1 binding, although we cannot exclude the possibility that L78 might interfere with the in vivo interaction between Nef and AP-1 that is relevant for MHC-I downmodulation.



(23K):

 		Fig. 3. Role of P78 in Nef capacity to bind AP-1 (a) and regulate MHC-I trafficking (bd). (a) In vitro binding of AP-1 to GSTNef fusion proteins. GST alone, GSTNEF, GSTNP5-8 and GSTNP5-8P78 were produced and tested for their capacity to bind AP-1 from Jurkat cell lysates as described previously (Casartelli et al., 2003b). Relative AP-1-binding activity was calculated as the amount of AP-1 γ subunit detected by immunoblotting (top panel) normalized for the amount of GST-fusion protein detected by Coomassie staining (bottom panel) and expressed as a percentage of the value measured for GSTNEF. (b) Rates of MHC-I internalization. Jurkat cells were infected with retroviruses expressing NEF, NEF-L78, NEF-4E4Q or with the Pinco empty virus. At 48 h, MHC-I endocytosis was analysed at the indicated time points as described by Kasper & Collins (2003), but with W6/32 anti-MHC-I mAb. The relative amount of cell-surface MHC-I was expressed by considering as100 % the initial MHC-I expression of each sample. (c) Transport of newly synthesized MHC-I to the cell surface. Jurkat cells transduced as in (b) were treated or not with cycloheximide and stripped of MHC-I molecules by low-pH treatment as described by Kasper & Collins (2003). Then, cells were incubated at 37 °C and 5 % CO2 for the indicated time and stained with PE-conjugated anti-HLA-A/B/C mAb. The amount of MHC-I transported to the cell surface was determined by subtracting from each sample the corresponding MHC-I staining remaining after stripping, then subtracting the MFI of the cycloheximide-treated cells from the MFI of the untreated cells. (d) Recycling of MHC-I to the cell surface. Transduced Jurkat cells were treated as described in (c). The amount of recycled MHC-I was determined by subtracting the corresponding MFI remaining after stripping from the MFI of the cycloheximide-treated cells at each time point. Reported values are the means±SD of duplicates from one representative experiment out of three.

To gain insights into the mechanism by which P78 regulates Nef activity on MHC-I, we examined MHC-I trafficking in Jurkat cells infected with retroviruses expressing NEF, NEF-L78 and the NEF-4E4Q mutant by a method described by Kasper & Collins (2003). In agreement with previous reports (Kasper & Collins, 2003; Larsen et al., 2004), we observed that NEF stimulated MHC-I internalization from the cell surface slightly (Fig. 3b) and reduced both transport of newly synthesized class I molecules (Fig. 3c) and recycling of internalized MHC-I to the cell surface (Fig. 3d). Interestingly, NEF-L78 displayed the same phenotype as NEF-4E4Q, being unable to reduce MHC-I recycling.

Here, we provide evidence that P78 is required for Nef activity of MHC-I downmodulation without contributing to Nef interactions with SH3-containing proteins. Besides, we demonstrate that P78 mediates the capacity of Nef to inhibit recycling of MHC-I to the cell surface. Further studies are needed to investigate the role of P78 in Nef interactions with cellular cofactors regulating MHC-I retention. Of note, our results also demonstrate that the SH3-binding capacity of Nef is fully dispensable for downmodulating MHC-I. Previous studies performed with NEF-A72A75 (Blagoveshchenskaya et al., 2002; Greenberg et al., 1998; Mangasarian et al., 1999) or with dominant-negative Hck (Chang et al., 2001) suggested a role for SH3 interactions in Nef activity on MHC-I. Our results suggest that the reduced capacity of NEF-A72A75 to downmodulate MHC-I should be ascribed to a low protein amount rather than defective SH3 binding. It is likely that the association of dominant-negative Hck with the SH3-binding site of Nef inhibits Nef activity on MHC-I indirectly as a consequence of steric hindrance and/or allosteric effect. The uncoupling of Nef activity on MHC-I from SH3-binding capacity will have implications for our understanding of the cellular pathways exploited by the viral protein and for attempts to interfere therapeutically with its pathogenic functions.

References

Arold, S. T. & Baur, A. S. (2001). Dynamic Nef and Nef dynamics: how structure could explain the complex activities of this small HIV protein. Trends Biochem Sci 26, 356363.[CrossRef][Medline]

Arold, S., Franken, P., Strub, M. P., Hoh, F., Benichou, S., Benarous, R. & Dumas, C. (1997). The crystal structure of HIV-1 Nef protein bound to the Fyn kinase SH3 domain suggests a role for this complex in altered T cell receptor signaling. Structure 5, 13611372.[Medline]

Blagoveshchenskaya, A. D., Thomas, L., Feliciangeli, S. F., Hung, C.-H. & Thomas, G. (2002). HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6 endocytic pathway. Cell 111, 853866.[CrossRef][Medline]

Casartelli, N., Di Matteo, G., Argentini, C. & 7 other authors (2003a). Structural defects and variations in the HIV-1 nef gene from rapid, slow and non-progressor children. AIDS 17, 12911301.[CrossRef][Medline]

Casartelli, N., Di Matteo, G., Potestà, M., Rossi, P. & Doria, M. (2003b). CD4 and major histocompatibility complex class I downregulation by the human immunodeficiency virus type 1 Nef protein in pediatric AIDS progression. J Virol 77, 1153611545.[Abstract/Free Full Text]

Chang, A. H., O'Shaughnessy, M. V. & Jirik, F. R. (2001). Hck SH3 domain-dependent abrogation of Nef-induced class 1 MHC down-regulation. Eur J Immunol 31, 23822387.[CrossRef][Medline]

Craig, H. M., Pandori, M. W., Riggs, N. L., Richman, D. D. & Guatelli, J. C. (1999). Analysis of the SH3-binding region of HIV-1 Nef: partial functional defects introduced by mutations in the polyproline helix and the hydrophobic pocket. Virology 262, 5563.[CrossRef][Medline]

Deacon, N. J., Tsykin, A., Solomon, A. & 17 other authors (1995). Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science 270, 988991.[Abstract/Free Full Text]

Fackler, O. T. & Baur, A. S. (2002). Live and let die: Nef functions beyond HIV replication. Immunity 16, 493497.[CrossRef][Medline]

Fackler, O. T., Kienzle, N., Kremmer, E., Boese, A., Schramm, B., Klimkait, T., Kucherer, C. & Mueller-Lantzsch, N. (1997). Association of human immunodeficiency virus Nef protein with actin is myristoylation dependent and influences its subcellular localization. Eur J Biochem 247, 843851.[Medline]

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. Mol Cell 3, 729739.[CrossRef][Medline]

Federico, M. (2004). Targeting the Nef induced increase of HIV infectivity. Curr Drug Targets Immune Endocr Metabol Disord 4, 321326.[CrossRef][Medline]

Geyer, M., Fackler, O. T. & Peterlin, B. M. (2001). Structurefunction relationships in HIV-1 Nef. EMBO Rep 2, 580585.[CrossRef][Medline]

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 J 17, 27772789.[CrossRef][Medline]

Hanna, Z., Kay, D. G., Rebai, N., Guimond, A., Jothy, S. & Jolicoeur, P. (1998). Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell 95, 163175.[CrossRef][Medline]

Hanna, Z., Weng, X., Kay, D. G., Poudrier, J., Lowell, C. & Jolicoeur, P. (2001). The pathogenicity of human immunodeficiency virus (HIV) type 1 Nef in CD4C/HIV transgenic mice is abolished by mutation of its SH3-binding domain, and disease development is delayed in the absence of Hck. J Virol 75, 93789392.[Abstract/Free Full Text]

Iafrate, A. J., Bronson, S. & Skowronski, J. (1997). Separable functions of Nef disrupt two aspects of T cell receptor machinery: CD4 expression and CD3 signaling. EMBO J 16, 673684.[CrossRef][Medline]

Kasper, M. R. & Collins, K. L. (2003). Nef-mediated disruption of HLA-A2 transport to the cell surface in T cells. J Virol 77, 30413049.[Abstract/Free Full Text]

Kawano, Y., Tanaka, Y., Misawa, N. & 10 other authors (1997). Mutational analysis of human immunodeficiency virus type 1 (HIV-1) accessory genes: requirement of a site in the nef gene for HIV-1 replication in activated CD4+ T cells in vitro and in vivo. J Virol 71, 84568466.[Abstract]

Kirchhoff, F., Greenough, T. C., Brettler, D. B., Sullivan, J. L. & Desrosiers, R. C. (1995). Absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection. N Engl J Med 332, 228232.[Free Full Text]

Krautkrämer, E., Giese, S. I., Gasteier, J. E., Muranyi, W. & Fackler, O. T. (2004). Human immunodeficiency virus type 1 Nef activates p21-activated kinase via recruitment into lipid rafts. J Virol 78, 40854097.[Abstract/Free Full Text]

Larsen, J. E., Massol, R. H., Nieland, T. J. F. & Kirchhausen, T. (2004). HIV Nef-mediated major histocompatibility complex class I down-modulation is independent of Arf6 activity. Mol Biol Cell 15, 323331.[Abstract/Free Full Text]

Lee, C.-H., Leung, B., Lemmon, M. A., Zheng, J., Cowburn, D., Kuriyan, J. & Saksela, K. (1995). A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein. EMBO J 14, 50065015.[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, 931942.[CrossRef][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 µ subunit of clathrin adaptor complexes and reveals a cryptic sorting signal in MHC I molecules. Immunity 8, 483495.[CrossRef][Medline]

Liu, X., Schrager, J. A., Lange, G. D. & Marsh, J. W. (2001). HIV Nef-mediated cellular phenotypes are differentially expressed as a function of intracellular Nef concentrations. J Biol Chem 276, 3276332770.[Abstract/Free Full Text]

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 alpha helix and proline repeat of Nef selectively regulate MHC-I trafficking. J Virol 73, 19641973.[Abstract/Free Full Text]

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, 273282.[CrossRef][Medline]

Peterlin, B. M. & Trono, D. (2003). Hide, shield and strike back: how HIV-infected cells avoid immune eradication. Nat Rev Immunol 3, 97107.[CrossRef][Medline]

Riggs, N. L., Craig, H. M., Pandori, M. W. & Guatelli, J. C. (1999). The dileucine-based sorting motif in HIV-1 Nef is not required for down-regulation of class I MHC. Virology 258, 203207.[CrossRef][Medline]

Roeth, J. F., Williams, M., Kasper, M. R., Filzen, T. M. & Collins, K. L. (2004). HIV-1 Nef disrupts MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail. J Cell Biol 167, 903913.[Abstract/Free Full Text]

Saksela, K., Cheng, G. & Baltimore, D. (1995). 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. EMBO J 14, 484491.[Medline]

Yamada, T., Kaji, N., Odawara, T., Chiba, J., Iwamoto, A. & Kitamura, Y. (2003). Proline 78 is crucial for human immunodeficiency virus type 1 Nef to down-regulate class I human leukocyte antigen. J Virol 77, 15891594.[Medline]

Received 17 December 2005; accepted 27 March 2006.