Large deletions in nef have been detected in some long-term survivors of HIV-1 infection [1–3], and it is commonly accepted that functional nef genes are important for viral pathogenicity . Nef downregulates CD4, MHC-I and II molecules from the cell surface, upregulates the invariant chain (Ii) associated with immature MHC-II complexes, alters signal transduction pathways, and enhances viral replication and infectivity in vitro [4–6]. Some Nef functions thus allow HIV-1 to evade the antiviral immune response, whereas others enhance virus spread more directly.
Studies of nef alleles derived from HIV-1-infected individuals showing diverse rates of disease progression have shown that some non/slow progressing HIV-1-infected individuals harbour viruses containing nef alleles unable to downmodulate CD4 cells and to stimulate viral replication but fully functional in MHC-I downmodulation [7–9]. Differences in Nef function can thus be associated with different rates of adult HIV-1 disease progression. Results obtained with nef alleles derived from infected adults may not necessarily apply to perinatal HIV-1 infection because children and adults show several immunological and pathological differences. For example, the course of HIV-1 infection is often more accelerated in children, probably because of the immaturity of the immune system . Approximately one third of HIV-1-infected infants become symptomatic within the first months of life and are considered ‘rapid progressors’. A minority of vertically infected children, however, develop little if any evidence of disease progression for more than a decade . The reasons for these highly divergent rates of disease progression are currently poorly understood. It has recently been shown that nef alleles from perinatally infected children with slow/non-progressive infection show a higher frequency of structural defects and are therefore on average less active in downmodulating CD4 and MHC-I cells than those derived from children with rapid disease progression [11,12]. Expanding these previous studies, we analysed a large subset of these nef alleles for their ability to downmodulate MHC-II and to upregulate Ii. Unexpectedly, we found that nef alleles from non-progressors were particularly efficient in these activities, suggesting that in HIV-1-infected children impaired MHC-II function may favour slow/non-progressive infection.
Plasmids and proviral constructs
The cloning, sequence analysis, expression and functional activity of the primary nef alleles in the downmodulation of CD4 and MHC-I cells has been described [11,12]. Bicistronic expression vectors or proviral HIV-1 constructs co-expressing 27 nef alleles derived from 13 HIV-1-infected children and enhanced green fluorescence protein (eGFP) were generated as described elsewhere [13,14].
Cells and virus stocks
Jurkat T and HeLa class II transactivator (CIITA) cells were cultured and transfected with Nef expression constructs as described previously [14,15]. The human monocytic THP-1 cell line  was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics. To isolate monocyte-derived macrophages (MDM), freshly isolated peripheral blood mononuclear cells were incubated in 24-well dishes in RPMI 1640 medium overnight. After extensive washing with phosphate-buffered saline to get rid of the non-adherent cell fraction RPMI 1640 medium supplemented with 10% fetal calf serum and 10 ng/ml macrophage colony-stimulating factor (Strathmann Biotec, Hamburg, Germany) was added for another 6 days. CD4 T cells were isolated by using the CD4 T-cell isolation kit RosetteSep (StemCell Technologies, Vancouver, Canada) as recommended by the manufacturer. Viral particles were generated and Jurkat T cells, CD4 T cells, THP-1 cells and macrophages were transduced with vesicular stomatitis virus (VSV) G-pseudotyped NL4-3 based internal ribosome entry site (IRES)-eGFP reporter viruses and analysed by flow cytometry as described previously [14,17].
CD4, MHC-I in Jurkat T cells and MHC-II, Ii in HeLa CIITA cells transfected with bicistronic Nef expression vectors was measured as described previously . Also cell surface expression of Ii, MHC-II and MHC-I in CD4 T cells, THP-1 cells or macrophages transduced with VSV G-pseudotyped HIV-1 reporter viruses co-expressing Nef and eGFP were measured, and the efficiency of Nef-mediated receptor modulation was calculated as described previously [17,18].
The activities of nef alleles derived from HIV-1-infected children showing no, slow or rapid disease progression were compared using a two-tailed Student's t-test. The PRISM package version 4.0 (Abacus Concepts, Berkeley, California, USA) was used for all calculations.
The role of Nef-mediated downmodulation of CD4 and MHC-I cells in paediatric AIDS progression has previously been investigated [11,12]. It remains to be determined, however, whether nef alleles from perinatally infected children showing different rates of disease progression might differ in other aspects of Nef function, such as the modulation of MHC-II antigen presentation and surface expression. To address this question we cloned 27 nef alleles derived at various timepoints from six non-progressors (NP1-2, NP1-9, NP1-15, NP2-1, NP2-20, NP3-2, NP3-11, NP4-2, NP4-6, NP5-1, NP6-7), three slow progressors (SP1-3, SP1-11, SP2-1, SP2-10, SP3-1, SP3-10) and four rapid progressors (RP1-3, RP1-11, RP1-27, RP2-1, RP2-11, RP3-2, RP3-10, RP4-1, RP4-11, RP4-12) [11,12] into a bicistronic vector co-expressing Nef and green fluorescence protein (GFP) and analysed the modulation of CD4, MHC-I, MHC-II and Ii surface expression in transiently transfected Jurkat and HeLa CIITA cells [14,15]. We selected Nef alleles that were closely related or identical to the respective patient-specific consensus Nef sequence for functional studies. Notably, nef genes containing premature termination codons or other obvious defects were not included in this analysis. All nef alleles downmodulated mature MHC-II molecules and upregulated the surface expression of the MHC-II-associated invariant chain, albeit with differential efficiency (Fig. 1a). For quantitative evaluation the mean intensities of red MHC-II or Ii fluorescence obtained for cells expressing GFP only were divided by the corresponding numbers obtained for cells co-expressing Nef and GFP to calculate n-fold up or downmodulation, respectively. As expected from previous studies [11,12], the subsets of nef alleles analysed did not differ significantly in their abilities to downmodulate CD4 and MHC-I cells (Fig. 1b). Unexpectedly, however, we found that nef alleles from non-progressors (n = 11) were significantly more active in upregulating Ii (8.7 ± 0.7 versus 4.6 ± 1.0; P = 0.0028) and downmodulating MHC-II (9.4 ± 0.8 versus 5.1 ± 1.1; P = 0.014) compared with those derived from rapid progressors (n = 10; Fig. 1b).
Our findings suggested that nef alleles from perinatally infected children with non-progressive HIV-1 infection may impair MHC-II function more severely than those derived from rapid progressors. To verify these functional differences in virally infected THP-1 cells, we cloned five representative nef alleles from each non-progressor and rapid progressor into a replication-competent HIV-1 NL4-3 based vector designed to co-express Nef and eGFP [14,17]. Notably, the human monocytic leukemia THP-1 cell line shares many properties with human MDM , and co-expresses high levels of both MHC-I and MHC-II (Fig. 2a). The relative activities of the 10 nef alleles in modulating Ii and MHC-II surface expression on THP-1 cells correlated well with the results obtained using the HeLa CIITA cell line (Fig. 2b,c). Although the upregulation of Ii was even more dramatic in THP-1 cells (16.6 to 28.8-fold), the effects on MHC-II were weak (less than threefold; Fig. 2a). In contrast, MHC-I was efficiently downmodulated, although the latter function is known to require relatively high levels of Nef expression . Notably, the results obtained using THP-1 cells confirmed that nef alleles derived from paediatric non-progressors are more active in modulating Ii and MHC-II than those derived from rapid progressors (Fig. 2d, left and data not shown).
To evalute the effects of Nef on Ii and MHC-II cell expression in infected primary human cells, we transduced MDM and CD4 T cells with VSV-G pseudotyped HIV-1 NL4-3 particles containing the various nef alleles. As previously reported , Nef also increased the surface expression of Ii in primary CD4 T cells (Fig. 2d, middle panel). Most interestingly, we could demonstrate a marked upregulation of Ii on MDM (Fig. 2d, right panel). In comparison, we observed only marginal effects of Nef on MHC-II expression on macrophages (data not shown). The relative activities of the nef alleles in both primary cell types correlated well with each other (R2 = 0.68, P = 0.0033) and with the results obtained using THP-1 cells (R2 = 0.73, P = 0.0016 and R2 = 0.47, P = 0.029, respectively). Importantly, the results obtained with primary CD4 T cells and MDM further confirmed that the non-progressor-derived nef alleles are significantly more active in upregulating Ii than those isolated from rapid progressors (Fig. 2d).
Our results show that nef alleles from non-progressive children perinatally infected with HIV-1 upregulate Ii and downregulate MHC-II cell surface expression more effectively than those derived from rapid progressors. The dileucine motif and several charged residues in the C-proximal flexible loop of Nef, known to be important for increased Ii expression [14,15], were highly conserved. It thus remains to be determined which sequence variations account for the observed differences in Nef function. Notably, nef alleles derived from adult HIV-1-infected individuals with different rates of disease progression, i.e. 17 non-progressors, 15 slow progressors and 17 rapid progressors, did not differ significantly in their abilities to modulate Ii and MHC-II (data not shown). Similarly to the Nef-mediated downmodulation of MHC-I , the effects of Nef on MHC-II and Ii were cell-type dependent. Marked (MDM, CD4 T cells) to highly efficient (Hela CIITA and THP-1 cells) upregulation of Ii surface expression was readily detectable even at low levels of Nef expression. Importantly, we show for the first time that Nef upregulates Ii on primary antigen-presenting cells (APC) and should thus impair MHC-II antigen presentation [22,23]. In comparison, significant effects of Nef on MHC-II surface levels were only observed in HeLa CIITA cells and required high levels of Nef expression, suggesting that this Nef function may not play an important role in the pathogenesis of HIV-1.
The stable surface expression of Ii prevents peptide presentation , and MHC-II associated with Ii is non-functional in stimulating CD4 T cells . Accordingly, in non-progressive paediatric HIV-1 infection the capacitiy of infected APC to stimulate CD4 T cells should be reduced. Why could this be beneficial for the clinical course of infection? One possible explanation is that reduced MHC-II antigen presentation of infected APC might, to some extent, contribute to the relatively low levels of CD4 T-cell activation, proliferation and apoptotic death typically observed in non-progressive HIV-1 infection. It has been reported that chronically high T-cell activation is the strongest predictor of progression to AIDS in HIV-1-infected individuals [24,25]. More recently, it has been shown that nef alleles from the great majority of primate lentiviruses downmodulate TCR-CD3 from the surface of infected T cells, thereby suppressing their responsiveness to activation  and possibly allowing efficient viral persistence in the context of an intact host immune system. Moreover, it has been proposed that the low responsiveness of chimpanzee T cells to activation might explain why HIV-1 and SIVcpz infection usually does not cause disease in this primate species [26,27]. It is tempting to speculate that primate lentiviral Nef proteins might affect immune activation and the rates of CD4 T-cell depletion by both directly modulating the responsiveness of virally infected T cells to activation and, probably to a lesser extent, indirectly by reducing the capacity of APC to stimulate CD4 T cells. Further studies are required to challenge these hypotheses and to explore the importance of the Nef-mediated modulation of TCR-CD3 and the MHC-II associated invariant chain in the pathogenesis of AIDS in adequate animal models.
The authors would like to thank Thomas Mertens for support and Ingrid Bennett for critical reading of the manuscript.
Sponsorship: This work was supported by grants from the DFG, the Wilhelm-Sander Foundation, and NIH grant 1R01AI067057-01A2 to F.K.
The first two authors contributed equally to this work.
1. Kirchhoff F, Greenough TC, Brettler DB, Sullivan JL, Desrosiers RC. Absence of intact nef
sequences in a long-term, nonprogressing survivor of HIV-1 infection. N Engl J Med 1995; 332:228–232.
2. Deacon NJ, Tsykin A, Solomon A, Smith K, Ludford-Menting M, Hooker DJ, et al
. Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science 1995; 270:988–991.
3. Salvi R, Garbuglia AR, Di Caro A, Pulciani S, Montella F, Benedetto A. Grossly defective nef gene sequences in a human immunodeficiency virus type 1-seropositive long-term nonprogressor. J Virol 1998; 72:3646–3657.
4. Wei BL, Arora VK, Foster JL, Sodora DL, Garcia JV. In vivo analysis of Nef function. Curr HIV Res 2003; 1:41–50.
5. Johnson WE, Desrosiers RC. Viral persistance: HIV's strategies of immune system evasion. Annu Rev Med 2003; 53:499–518.
6. Skowronski J, Greenberg ME, Lock M, Mariani R, Salghetti S, Swigut T, Iafrate AJ. HIV and SIV Nef modulate signal transduction and protein sorting in T cells. Cold Spring Harb Symp Quant Biol 1999; 64:453–463.
7. Carl S, Daniels R, Iafrate AJ, Easterbrook P, Greenough TC, Skowronski J, Kirchhoff F. Partial “repair” of defective nef
genes in a long-term nonprogressor with HIV-1 infection. J Infect Dis 2000; 181:132–140.
8. Mariani R, Kirchhoff F, Greenough TC, Sullivan JL, Desrosiers RC, Skowronski J. High frequency of defective nef-alleles in a long-term survivor with nonprogressive HIV-1 infection. J Virol 1996; 70:7752–7764.
9. Tobiume M, Takahoko M, Yamada T, Tatsumi M, Iwamoto A, Matsuda M. Inefficient enhancement of viral infectivity and CD4 downregulation by human immunodeficiency virus type 1 Nef from Japanese long-term nonprogressors. J Virol 2002; 76:5959–5965.
10. Wilfert CM, Wilson C, Luzuriaga K, Epstein L. Pathogenesis of pediatric human immunodeficiency virus type 1 infection. J Infect Dis 1994; 170:286–292.
11. Casartelli N, Di Matteo G, Potesta M, Rossi P, Doria M. CD4 and major histocompatibility complex class I downregulation by the human immunodeficiency virus type 1 nef protein in pediatric AIDS progression. J Virol 2003; 77:11536–11545.
12. Casartelli N, Di Matteo G, Argentini C, Cancrini C, Bernardi S, Castelli G, et al
. Structural defects and variations in the HIV-1 nef gene from rapid, slow and non-progressor children. AIDS 2003; 17:1291–1301.
13. Greenberg ME, Iafrate AJ, Skowronski J. The SH3 domain-binding surface and an acidic motif in HIV-1 Nef regulate trafficking of class I MHC complexes. EMBO J 1998; 17:2777–2789.
14. Schindler M, Würfl S, Benaroch P, Greenough TC, Daniels RC, Easterbrook P, et al
. Down-modulation of mature MHC class II and up-regulation of invariant chain cell surface expression are well conserved functions of HIV and SIV nef-alleles. J Virol 2003; 77:10548–10556.
15. Stumptner-Cuvelette P, Morchoisne S, Dugast M, Le Gall S, Raposo G, Schwartz O, Benaroch P. HIV-1 Nef impairs MHC class II antigen presentation and surface expression. Proc Natl Acad Sci U S A 2001; 98:12144–12149.
16. Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K. Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer 1980; 26:171–176.
17. Schindler M, Munch J, Kutsch O, Li H, Santiago ML, Bibollet-Ruche F, et al
. Nef-mediated suppression of T cell activation was lost in a lentiviral lineage that gave rise to HIV-1. Cell 2006; 125:1055–1067.
18. Carl S, Greenough TC, Krumbiegel M, Easterbrook P, Daniels R, Greenberg M, et al
. Modulation of HIV-1 Nef activities during AIDS progression. J Virol 2001; 74:3657–3665.
19. Liu X, Schrager JA, Lange GD, Marsh JW. HIV Nef-mediated cellular phenotypes are differentially expressed as a function of intracellular Nef concentrations. J Biol Chem 2001; 276:32763–32770.
20. Keppler OT, Tibroni N, Venzke S, Rauch S, Fackler OT. Modulation of specific surface receptors and activation sensitization in primary resting CD4+ T lymphocytes by the Nef protein of HIV-1. J Leukoc Biol 2006; 79:616–627.
21. Kasper MR, Collins KL. Nef-mediated disruption of HLA-A2 transport to the cell surface in T cells. J Virol 2003; 77:3041–3049.
22. Roche PA, Teletski CL, Karp DR, Pinet V, Bakke O, Long EO. Stable surface expression of invariant chain prevents peptide presentation by HLA-DR. EMBO J 1992; 11:2841–2847.
23. Stumptner-Cuvelette P, Benaroch P. Multiple roles of the invariant chain in MHC class II function. Biochim Biophys Acta 2002; 1542:1–13.
24. Giorgi JV, Hultin LE, McKeating JA, Johnson TD, Owens B, Jacobson LP, et al
. Shorter survival in advanced human immunodeficiency virus type 1 infection is more closely associated with T lymphocyte activation than with plasma virus burden or virus chemokine coreceptor usage. J Infect Dis 1999; 179:859–870.
25. Sousa AE, Carneiro J, Meier-Schellersheim M, Grossman Z, Victorino RM. CD4 T cell depletion is linked directly to immune activation in the pathogenesis of HIV-1 and HIV-2 but only indirectly to the viral load. J Immunol 2002; 169:3400–3406.
26. Nguyen DH, Hurtado-Ziola N, Gagneux P, Varki A. Loss of Siglec expression on T lymphocytes during human evolution. Proc Natl Acad Sci U S A 2006; 103:7765–7770.
27. Cohen J. Immunology. Differences in immune cell “brakes” may explain chimp–human split on AIDS. Science 2006; 312:672–673.