Viremic long-term nonprogressive HIV-1 infection is not associated with abnormalities in known Nef functions
© Heigele et al.; licensee BioMed Central Ltd. 2014
Received: 1 October 2013
Accepted: 26 January 2014
Published: 4 February 2014
A small minority of HIV-1-infected individuals show low levels of immune activation and do not develop immunodeficiency despite high viral loads. Since the accessory viral Nef protein modulates T cell activation and plays a key role in the pathogenesis of AIDS, we investigated whether specific properties of Nef may be associated with this highly unusual clinical outcome of HIV-1 infection.
Comprehensive functional analyses of sequential HIV-1 strains from three viremic long-term non-progressors (VNP) showed that they encode full-length Nef proteins that are capable of modulating CD4, CD28, CD8ß, MHC-I and CD74 cell surface expression. Similar to Nef proteins from HIV-1-infected individuals with progressive infection (P-Nefs) and unlike Nefs from simian immunodeficiency viruses (SIVs) that do not cause chronic immune activation and disease in their natural simian hosts, VNP-Nefs were generally unable to down-modulate TCR-CD3 cell surface expression to block T cell activation and apoptosis. On average, VNP-Nefs suppressed NF-AT activation less effectively than P-Nefs and were slightly less active in enhancing NF-κB activity. Finally, we found that VNP-Nefs increased virion infectivity and enhanced HIV-1 replication and cytopathicity in primary human cells and in ex vivo infected lymphoid tissues.
Our results show that nef alleles from VNPs and progressors of HIV-1 infection show only modest differences in established functions. Thus, the lack of chronic immune activation and disease progression in HIV-1-infected VNPs is apparently not associated with unusual functional properties of the accessory viral Nef protein.
High viral loads are almost invariably associated with chronic inflammation and progression to AIDS in HIV-1-infected individuals. In contrast, some non-human primates (NHPs) that are naturally infected with SIVs, such as sooty mangabeys (SMs) or African green monkeys (AGMs), show low levels of immune activation and do not develop disease despite high levels of viral replication [1, 2]. Recent data show that a small minority (<1%) of highly viremic HIV-1-infected individuals, so called viremic long-term non-progressors (VNP), show a similar phenotype and remain asymptomatic with low levels of inflammation and high CD4+ T cell counts .
It is poorly understood why VNPs can tolerate high levels of HIV-1 replication. It has been reported, however, that they show lower levels of proliferating and activated T cells than progressing individuals with similar viral loads . Here, we examined whether specific functional properties of the accessory viral Nef protein may contribute to the low levels of T cell activation and the lack of disease progression in VNPs. Nef is a multi-functional manipulator of the viral host cell that facilitates viral immune evasion and is critical for efficient viral replication and disease progression in HIV-1-infected individuals [4, 5]. Primate lentiviral Nef proteins generally down-modulate CD4 and MHC-I from the cell surface and enhance viral infectivity and replication [4, 5]. They differ fundamentally, however, in their effect on the responsiveness of virally infected T cells to stimulation. HIV-1 Nef proteins may render infected T cells hyper-responsive to stimulation and promote the induction of cellular transcription factors, activation markers and inflammatory cytokines [6–9]. In contrast, most primate lentiviral Nefs block T cell activation by down-modulation of TCR-CD3 and CD28 from the cell surface . These latter Nef functions are highly conserved in sooty mangabeys that are naturally infected with SIVsmm  and may play a protective role in vivo since efficient modulation of TCR-CD3 and CD28 correlates with high and stable CD4+ T cell counts in natural SIVsmm infection  and in viremic HIV-2-infected individuals .
In addition to reducing the lysis of HIV-1-infected cells by cytotoxic T lymphocytes (CTL) by removing MHC-I from the cell surface, Nef is also able to impair CTL function directly by down-modulation of CD8β [15, 16]. Analyses performed in CEM cells stably expressing A2-CD8β fusions  confirmed that CD8β down-modulation is conserved between nef alleles derived from both groups of HIV-1-infected individuals (Figures 2E and Additional file 1: FigureS2). Unexpectedly, HIV-2, SIVsmm and SIVmac Nefs that all belong to the same lineage of primate lentiviruses were significantly more active than the HIV-1 control Nefs (Figure 2E). Nef may also impair MHC-II-dependent antigen-presentation by up-regulating surface expression of the invariant chain (CD74) [17, 18]. Potentially, inefficient activation of T cells by antigen-presenting cells (APCs) due to potent up-modulation of CD74 may reduce the levels of immune activation. We found, however, that VNP- and P-Nefs did not differ significantly in their ability to enhance CD74 cell surface expression in infected THP-1 cells (Figure 2F). Up-modulation of CD74 was reduced for nef alleles obtained later during infection of VNPs 68 and 337, whereas the ability of VNP-Nefs to modulate other receptors did not change significantly throughout the course of infection (Additional file 1: Figure S3). Whether or not the ability of Nef to suppress MHC-II antigen presentation by up-regulation of CD74 may be modulated throughout the course of HIV-1 infection and if this Nef function plays a role in the pathogenesis of AIDS remains to be analyzed in larger patient cohorts.
It has been reported that HIV-1 Nef may increase activation of the nuclear factors of activated T cells and kappa B (NF-AT and NF-κB), respectively . Both of these transcription factors play key roles in innate immunity and inflammatory responses [22, 23] and may thus affect the levels of infection-associated immune activation. Measurements of the impact of Nef on NF-AT-dependent luciferase expression in stably transfected Jurkat T cells  showed that HIV-1 Nefs were generally associated with higher levels of NF-AT activation than HIV-2 and SIV Nefs (Figure 3I, J). While this was expected from published data , it came as a surprise that P-Nefs suppressed NF-AT activation more efficiently than VNP-Nefs. In contrast, P-Nefs trended towards association with higher levels of NF-κB activation than VNP-Nefs, although this difference failed to reach significance (Figure 3K). Notably, we observed that nef alleles from the three T cell line adapted molecular clones of HIV-1 were associated with higher levels of CD69 (p = 0.0013) and CD25 (p = 0.0012) expression, apoptosis (p = 0.0002) and NF-AT activation (p = 0.0111) than primary patient-derived nef genes (Figure 3). It is also noteworthy that HIV-2 and SIV Nefs efficiently suppressed induction of NF-AT but were particularly active in stimulating NF-κB (Figure 3I, K). Thus, these primate lentiviruses may efficiently activate the viral LTR via its NF-κB containing core enhancer element but avoid NF-AT dependent induction of immune response genes.
In conclusion, low levels of chronic inflammation and lack of disease progression in VNPs are not due to efficient Nef-mediated suppression of T cell activation. The possible relevance and significance of the modest differences in modulation of NF-AT and NF-κB by VNP- and P-Nefs observed in the present study needs to be further examined in larger patient cohorts. Notably, our data suggest that nef alleles derived from primary HIV-1 strains but not those of T cell line adapted molecular clones of HIV-1 may suppress T cell activation, albeit much less efficiently than HIV-2 and SIV nef genes that down-modulate TCR-CD3. Thus, our results further underline the necessity to use primary nef alleles to avoid in vitro artifacts. Finally, our findings suggest that host factors rather than specific viral properties may allow VNPs to avoid harmful chronic immune activation by HIV-1 replication.
The authors thank Kerstin Regensburger, Martha Meyer, Birgit Ott and Daniela Krnavek for excellent technical assistance, Dre van der Merwe and Daniel Sauter for critical reading, and Heinz Maier from the Military Hospital Ulm and Thomas Hoffmann from the Department of Ear-Nose and Throat, Ulm University Medical Center for providing tonsillary tissues. Further we thank Bernd Baumann, University Ulm for providing the NF-κB firefly luciferase reporter plasmid. HIV-infected progressor (P) samples were sampled from the BRESCIA cohorts and kindly provided by Carlo Torti. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) and the European Research Council (ERC).
The Amsterdam Cohort Studies on HIV infection and AIDS, a collaboration between the Amsterdam Health Service, the Academic Medical Center of the University of Amsterdam, Sanquin Blood Supply Foundation, the University Medical Center Utrecht, and the Jan van Goyen Clinic are part of the Netherlands HIV Monitoring Foundation and financially supported by the Center for Infectious Disease Control of the Netherlands National Institute for Public Health and the Environment. All experiments reported in this study and bllod sampling were performed with the approval of the local ethics committees. We are indebted to all participants for their continuous participation in the study.
- Sodora DL, Allan JS, Apetrei C, Brenchley JM, Douek DC, Else JG, Estes JD, Hahn BH, Hirsch VM, Kaur A, Kirchhoff F, Muller-Trutwin M, Pandrea I, Schmitz JE, Silvestri G: Toward an AIDS vaccine: lessons from natural simian immunodeficiency virus infections of African nonhuman primate hosts. Nat Med. 2009, 15: 861-865.PubMed CentralView ArticlePubMedGoogle Scholar
- Chahroudi A, Bosinger SE, Vanderford TH, Paiardini M, Silvestri G: Natural SIV hosts: showing AIDS the door. Science. 2012, 335: 1188-1193.View ArticlePubMedGoogle Scholar
- Choudhary SK, Vrisekoop N, Jansen CA, Otto SA, Schuitemaker H, Miedema F, Camerini D: Low immune activation despite high levels of pathogenic human immunodeficiency virus type 1 results in long-term asymptomatic disease. J Virol. 2007, 81: 8838-8842.PubMed CentralView ArticlePubMedGoogle Scholar
- Kirchhoff F: Role of Nef in primate lentiviral immunopathogenesis. Cell Mol Life Sci. 2008, 65: 2621-2636.View ArticlePubMedGoogle Scholar
- Arien KK, Verhasselt B: HIV Nef: role in pathogenesis and viral fitness. Curr HIV Res. 2008, 6: 200-208.View ArticlePubMedGoogle Scholar
- Skowronski J, Parks D, Mariani R: Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene. EMBO. 1993, 12: 703-713.Google Scholar
- Wang JK, Kiyokawa E, Verdin E, Trono D: The Nef protein of HIV-1 associates with rafts and primes T cells for activation. Proc Natl Acad Sci USA. 2000, 97: 394-399.PubMed CentralView ArticlePubMedGoogle Scholar
- Fenard D, Yonemoto W, de Noronha C, Cavrois M, Williams SA, Greene WC: Nef is physically recruited into the immunological synapse and potentiates T cell activation early after TCR engagement. J Immunol. 2005, 175: 6050-6057.View ArticlePubMedGoogle Scholar
- Fortin JF, Barat C, Beauséjour Y, Barbeau B, Tremblay MJ: Hyper-responsiveness to stimulation of human immunodeficiency virus-infected CD4+ T cells requires Nef and Tat virus gene products and results from higher NFAT, NF-kappaB, and AP-1 induction. J Biol Chem. 2004, 279: 39520-39531.View ArticlePubMedGoogle Scholar
- Schindler M, Münch J, Kutsch O, Li H, Santiago ML, Bibollet-Ruche F, Müller-Trutwin MC, Novembre FJ, Peeters M, Courgnaud V, Bailes E, Roques R, Sodora DL, Silvestri G, Sharp PM, Hahn BH, Kirchhoff F: Nef-mediated suppression of T cell activation was lost in a lentiviral lineage that gave rise to HIV-1. Cell. 2006, 125: 1055-1067.View ArticlePubMedGoogle Scholar
- Schmökel J, Li H, Bailes E, Schindler M, Silvestri G, Hahn BH, Apetrei C, Kirchhoff F: Conservation of Nef function across highly diverse lineages of SIVsmm. Retrovirology. 2009, 6: 36-PubMed CentralView ArticlePubMedGoogle Scholar
- Schindler M, Schmökel J, Specht A, Li H, Münch J, Khalid M, Sodora DL, Hahn BH, Silvestri G, Kirchhoff F: Inefficient Nef-mediated downmodulation of CD3 and MHC-I correlates with loss of CD4 + T cells in natural SIV infection. PLoS Pathog. 2008, 4: e1000107-PubMed CentralView ArticlePubMedGoogle Scholar
- Khalid M, Yu H, Sauter D, Usmani SM, Schmökel J, Feldman J, Gruters RA, van der Ende ME, Geyer M, Rowland-Jones S, Osterhaus AD, Kirchhoff F: Efficient Nef-mediated downmodulation of TCR-CD3 and CD28 is associated with high CD4+ T cell counts in viremic HIV-2 infection. J Virol. 2012, 86: 4906-4920.PubMed CentralView ArticlePubMedGoogle Scholar
- Münch J, Rajan D, Schindler M, Specht A, Rücker E, Novembre FJ, Nerrienet E, Müller-Trutwin MC, Peeters M, Hahn BH, Kirchhoff F: Nef-mediated enhancement of virion infectivity and stimulation of viral replication are fundamental properties of primate lentiviruses. J Virol. 2007, 81: 13852-13864.PubMed CentralView ArticlePubMedGoogle Scholar
- Leonard JA, Filzen T, Carter CC, Schaefer M, Collins KL: HIV-1 Nef disrupts intracellular trafficking of MHC-I, CD4, CD8, and CD28 by distinct pathways that share common elements. J Virol. 2011, 85: 6867-6881.PubMed CentralView ArticlePubMedGoogle Scholar
- Heigele A, Schindler M, Gnanadurai CW, Leonard JA, Collins KL, Kirchhoff F: Down-modulation of CD8αβ is a fundamental activity of primate lentiviral Nef proteins. J Virol. 2012, 86: 36-48.PubMed CentralView ArticlePubMedGoogle Scholar
- 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.PubMed CentralView ArticlePubMedGoogle Scholar
- Schindler M, Wildum S, Casartelli N, Doria M, Kirchhoff F: Nef alleles from children with non-progressive HIV-1 infection modulate MHC-II expression more efficiently than those from rapid progressors. AIDS. 2007, 21: 1103-1107.View ArticlePubMedGoogle Scholar
- Renkema GH, Saksela K: Interactions of HIV-1 Nef with cellular signal transducing proteins. Front Biosci. 2000, 5: D268-D283.View ArticlePubMedGoogle Scholar
- Greenway AL, Holloway G, McPhee DA, Ellis P, Cornall A, Lidman M: HIV-1 nef control of cell signaling molecules: multiple strategies to promote virus replication. J Biosci. 2003, 28: 323-335.View ArticlePubMedGoogle Scholar
- Hotter D, Kirchhoff F, Sauter D: HIV-1 Vpu does not degrade interferon regulatory factor 3. J Virol. 2013, 87: 7160-7165.PubMed CentralView ArticlePubMedGoogle Scholar
- Fric J, Zelante T, Wong AY, Mertes A, Yu HB, Ricciardi-Castagnoli P: NFAT control of innate immunity. Blood. 2012, 120: 1380-1389.View ArticlePubMedGoogle Scholar
- Pitha PM: Innate antiviral response: role in HIV-1 infection. Viruses. 2011, 3: 1179-1203.PubMed CentralView ArticlePubMedGoogle Scholar
- Charneau PG, Mirambeau P, Roux P, Paulous S, Buc H, Clavel F: HIV-1 reverse transcription. A termination step at the center of the genome. J Mol Biol. 1994, 241: 651-662.View ArticlePubMedGoogle Scholar
- Schindler M, Rajan D, Specht A, Ritter C, Pulkkinen K, Saksela K, Kirchhoff F: Association of Nef with p21-activated kinase 2 is dispensable for efficient human immunodeficiency virus type 1 replication and cytopathicity in ex vivo-infected human lymphoid tissue. J Virol. 2007, 81: 13005-13014.PubMed CentralView ArticlePubMedGoogle Scholar
- Specht A, Telenti A, Martinez R, Fellay J, Bailes E, Evans DT, Carrington M, Hahn BH, Goldstein DB, Kirchhoff F: Counteraction of HLA-C-mediated immune control of HIV-1 by Nef. J Virol. 2010, 84: 7300-7311.PubMed CentralView ArticlePubMedGoogle Scholar
- Glushakova S, Grivel JC, Suryanarayana K, Meylan P, Lifson JD, Desrosiers R, Margolis L: Nef enhances human immunodeficiency virus replication and responsiveness to interleukin-2 in human lymphoid tissue ex vivo. J Virol. 1999, 73: 3968-3974.PubMed CentralPubMedGoogle Scholar
- Papkalla A, Münch J, Otto C, Kirchhoff F: Nef enhances human immunodeficiency virus type 1 infectivity and replication independently of viral coreceptor tropism. J Virol. 2002, 76: 8455-8459.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.