- Short report
- Open Access
Effects of naturally-arising HIV Nef mutations on cytotoxic T lymphocyte recognition and Nef's functionality in primary macrophages
© Mwimanzi et al; licensee BioMed Central Ltd. 2011
- Received: 11 March 2011
- Accepted: 22 June 2011
- Published: 22 June 2011
Although HIV can infect several cellular subsets, such as CD4+ T lymphocytes and macrophages, it remains unclear whether an HIV infection in macrophages supports cytotoxic T lymphocyte (CTL) escape. Here, we tested two naturally-arising mutations located in the well-conserved polyproline region of Nef for their effects on CTL recognition, Nef's functionality, and viral replication capacity in macrophages. These mutations were selected because they are known to cause CTL escape in the context of T lymphocytes.
Monocyte-derived macrophages (MDMs) infected with the wild-type virus, but not with variant viruses, were efficiently killed by CTL clones targeting Nef epitopes, VY8 (VPLRPMTY) and RY11 (RPQVPLRPMTY). The CTL-escape mutation, Arg75Thr, or Arg75Thr/Tyr85Phe double mutation, reduced the HLA class I down-regulation activity and, interestingly, increased the susceptibility of virus-infected MDMs to recognition by CTLs targeting a different epitope. The same mutations reduced the CCR5, but not CD4, down-regulation activity. Moreover, the Nef variants were impaired for Hck activation and enhancement of viral replication in MDMs.
These results suggest that HIV-infected MDMs are killed by CTLs targeting Nef epitopes, contributing to selection and adaptation of CTL-escape viral variants.
- Variant Virus
- PxxP Motif
Several different cellular subsets such as CD4+ T lymphocytes, macrophages, and dendritic cells can be targets for an HIV infection; although they differentially support HIV replication and persistence in vivo [1–3]. Macrophages may be the early target of HIV, but are highly resistant to the cytopathic effects of an HIV infection and continuously produce infectious virions for a long period of time [4, 5]. It is thought that the differences in fitness of viral replication among the different cellular environments could influence the selection and adaptation of viral quasispecies in these cells. The HLA class I-restricted CD8+ cytotoxic T lymphocyte (CTL) response is thought to play an important role in controlling HIV replication [6–8] and to mediate a major selective force for the emergence of viral variants [9, 10]. Certain CTL escape mutations, in well-conserved regions of Gag and Nef, have been reported to impose functional constraints on these proteins and to modulate viral replication in the context of T lymphocytes [11–13]. However, in the context of macrophages, the selection of CTL escape variants and functional adaptation of viral proteins are not yet fully understood. We previously showed that the HLA-B35-restricted CTL responses toward a well-conserved proline-rich region in Nef results in the emergence of a CTL escape mutation, either Arg75Thr or Tyr85Phe, from phylogenetically different viral quasispecies even within an HIV-infected host . These mutations constrain some of the important Nef functions in CD4+ T cells . Here we tested whether an HIV-1 infection in macrophages would have any influence on CTL recognition and escape as well as Nef's functionality and adaptation in the infected macrophages.
Susceptibility of HIV-infected macrophages to recognition by the cognate CTLs
We previously reported that in HIV-infected patients with HLA-B35, the Nef protein elicits dominant CD8 T cell responses , with the short epitope VY8 (Nef78-85; VPLRPMTY) being the early epitope, which subsequently shifts to the amino terminal-extended longer epitope RY11 (Nef75-85; RPQVPLRPMTY) . Autologous virus sequence analysis revealed that the mutations Tyr85 to Phe (85F) and Arg75 to Thr (75T) are associated with the early and chronic phase of an HIV infection, respectively, in HIV-infected individuals with HLA-B35 but that these 85F and 75T mutations are derived from phylogenetically different lineages .
Effects of the Nef mutations on Hck activation
Effects of the Nef mutations on HLA class I down-regulation
Susceptibility of HIV-infected MDMs to recognition by CTLs of another specificity
Next, we also determined CTL cytotoxic activity toward MDMs infected with 75T, 85F, and TF variant viruses. The A24-Nef CTLs showed the most potent activity toward MDMs infected with either the 75T or TF variant viruses; whereas their cytotoxic activity was less potent toward MDMs infected with either the wt or the 85F mutant virus (Figure 4). These data suggest that the diminished HLA-I down-regulation (i.e., increased level of cell-surface HLA-I) in MDMs infected with the 75T and the TF mutant viruses (Figure 3) resulted in increased susceptibility to killing by CTLs of another specificity (Figure 4), leading to a possible selective disadvantage for the variant viruses under anti-HIV CTL responses.
Effects of the Nef mutations on down-regulation of viral receptors
Effects of the Nef mutations on viral replication
Although the Nef protein is thought to have very high mutational plasticity, we showed here that the naturally-arising CTL escape variants in the well-conserved PxxP region in Nef alone or in combination can modulate some pathogenic functions of Nef in the context of human primary macrophages infected with a CCR5-tropic virus. There are 2 different aspects of CTL-mediated functional constraints on the PxxP-dependent Nef activities in MDMs reported here, one through immune evasion activity (HLA-I down-regulation activity) and the other acting on the intrinsic capacity to boost viral replication and persistence (Hck activation, viral co-receptor down-regulation activity, and enhancement of viral replication). In particular, one of the single mutants, 75T, impaired these Nef activities in MDMs infected with a CCR5-tropic virus. This is in line with the previous report showing that 75T mutation modulated Nef-stimulated viral replication in immature dendritic cell/T cell cocultures infected with a CCR5-tropic virus  although this mutation alone had virtually no influence on the same Nef activities in primary CD4+ T cells infected with a CXCR4-tropic virus in the previous study . In addition, the 75T mutation, located outside the VY8 epitope, reduced the cytolytic activity of VY8-specific CTLs in the context of CD4+ T cells , but did not affect their cytolytic activity in the context of MDMs (Figure 1), suggesting the differential intracellular processing of the VY8 peptide between CD4+ T cells and MDMs. This observation is in line with the previous report showing a substantial difference in intracellular processing of antigenic HIV peptides between monocytes and lymphocytes . Overall, these results suggest that an antigenic variation of viruses can differentially influence viral replication and persistence between cellular subsets because of their different effects on the intracellular antigen-processing machinery, the susceptibility to CTL killing, as well as the fitness cost to viral replication.
Of particular interest are the data showing that the CTL-escape Nef mutation, 75T, impaired HLA-I down-regulation activity by Nef and rendered the HIV-infected MDMs more susceptible to killing by CTLs with another specificity. Such phenomenon was also observed in the context of CD4+ T cells in our previous study . However, wt-virus-infected cells, regardless of CD4+ T cells or MDMs, could be killed to some extent by CTLs, suggesting that the Nef-mediated HLA-I down-regulation is insufficient for HIV to escape from CTL recognition and that, CTL-escape variant viruses are selected and emerged. Conversely, Swigut et al.,  reported that monkeys infected with SIV containing nef mutations that selectively eliminated MHC down-regulation activity exhibited higher level of SIV-specific CD8 T cell responses. In any event, an important question remains to be addressed which is how significant is Nef-mediated HLA-I down-regulation activity for HIV replication and persistence in HIV-infected humans.
Although HLA-B*35-restricted CTLs targeting PxxP region of Nef can impose functional constraints in viral replication in this study, we did not find any beneficial effects on clinical parameters (such as CD4 count and viral load) in HIV-infected patients with HLA-B*35 as well as those with HLA-B*35 and HLA-A*24 in our cohort to date (data not shown). Functional impairment in Nef induced by CTL-escape variants could be compensated later by mutations at secondary sites in Nef. For example, an inverse dose-response relationship has been observed between the number of CTL-escape mutations in Nef and CD4 counts in patients in a large population study . Therefore, only some CTL-escape variants may play a role in modulating Nef functions in vivo, such as in the case of HLA-B57+ elite suppressors . Further studies using a large number of clinically-isolated nef alleles are needed to extend this observation, such as how Nef-specific CTL responses, Nef functions, and clinical outcome of HIV-infected individuals are related to each other at the population level.
We thank Dr. M Fujiwara and Ms. S. Doki for their great help. This research was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan and by a grant-in-aid for AIDS research from the Ministry of Health, Labor, and Welfare of Japan.
- Eckstein DA, Penn ML, Korin YD, Scripture-Adams DD, Zack JA, Kreisberg JF, Roederer M, Sherman MP, Chin PS, Goldsmith MA: HIV-1 actively replicates in naive CD4+ T cells residing within human lymphoid tissues. Immunity. 2001, 15: 671-682. 10.1016/S1074-7613(01)00217-5.View ArticlePubMedGoogle Scholar
- Keele BF, Tazi L, Gartner S, Liu Y, Burgon TB, Estes JD, Thacker TC, Crandall KA, McArthur JC, Burton GF: Characterization of the follicular dendritic cell reservoir of human immunodeficiency virus type 1. J Virol. 2008, 82: 5548-5561. 10.1128/JVI.00124-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhu T, Muthui D, Holte S, Nickle D, Feng F, Brodie S, Hwangbo Y, Mullins JI, Corey L: Evidence for human immunodeficiency virus type 1 replication in vivo in CD14+ monocytes and its potential role as a source of virus in patients on highly active antiretroviral therapy. J Virol. 2002, 76: 707-716. 10.1128/JVI.76.2.707-716.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Aquaro S, Bagnarelli P, Guenci T, De Luca A, Clementi M, Balestra E, Caliò R, Perno CF: Long-term survival and virus production in human primary macrophages infected by human immunodeficiency virus. J Med Virol. 2002, 68: 479-488. 10.1002/jmv.10245.View ArticlePubMedGoogle Scholar
- Brown A, Zhang H, Lopez P, Pardo CA, Gartner S: In vitro modeling of the HIV-macrophage reservoir. J Leu Biol. 2006, 80: 1127-1135. 10.1189/jlb.0206126.View ArticleGoogle Scholar
- Borrow PH, Lewicki BH, Hahn GM, Shaw MB, Oldstone : Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection. J Virol. 1994, 68: 6103-6110.PubMed CentralPubMedGoogle Scholar
- Koup RA, Safrit JT, Cao Y, Andrews CA, McLeod G, Borkowsky W, Farthing C, Ho DD: Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J Virol. 1994, 68: 4650-4655.PubMed CentralPubMedGoogle Scholar
- Ogg GS, Jin X, Bonhoeffer S, Dunbar PR, Nowak MA, Monard S, Segal JP, Cao Y, Rowland-Jones SL, Cerundolo V, et al: Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science. 1998, 279: 2103-10.1126/science.279.5359.2103.View ArticlePubMedGoogle Scholar
- Goulder PJR, Watkins DI: HIV and SIV CTL escape: implications for vaccine design. Nat Rev Immunol. 2004, 4: 630-640. 10.1038/nri1417.View ArticlePubMedGoogle Scholar
- Motozono C, Mwimanzi P, Ueno T: Dynamic interplay between viral adaptation and immune recognition during HIV-1 infection. Protein & Cell. 2010, 1: 514-519.View ArticleGoogle Scholar
- Brockman MA, Schneidewind A, Lahaie M, Schmidt A, Miura T, DeSouza I, Ryvkin F, Derdeyn CA, Allen S, Hunter E, et al: Escape and compensation from early HLA-B57-mediated cytotoxic T-lymphocyte pressure on human immunodeficiency virus type 1 Gag alter capsid interactions with cyclophilin A. J Virol. 2007, 81: 12608-12618. 10.1128/JVI.01369-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Schneidewind A, Brockman MA, Sidney J, Wang YE, Chen H, Suscovich TJ, Li B, Adam RI, Allgaier RL, Mothe BR, et al: Structural and functional constraints limit options for cytotoxic T-lymphocyte escape in the immunodominant HLA-B27-restricted epitope in human immunodeficiency virus type 1 capsid. J Virol. 2008, 82: 5594-5605. 10.1128/JVI.02356-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Ueno T, Motozono C, Dohki S, Mwimanzi P, Rauch S, Fackler OT, Oka S, Takiguchi M: CTL-mediated selective pressure influences dynamic evolution and pathogenic functions of HIV-1 Nef. J Immunol. 2008, 180: 1107-1116.View ArticlePubMedGoogle Scholar
- Ueno T, Idegami Y, Motozono C, Oka S, Takiguchi M: Altering effects of antigenic variations in HIV-1 on antiviral effectiveness of HIV-specific CTLs. J Immunol. 2007, 178: 5513-5523.View ArticlePubMedGoogle Scholar
- Fujiwara M, Takiguchi M: HIV-1-specific CTLs effectively suppress replication of HIV-1 in HIV-1-infected macrophages. Blood. 2007, 109: 4832-4838. 10.1182/blood-2006-07-037481.View ArticlePubMedGoogle Scholar
- Motozono C, Yanaka S, Tsumoto K, Takiguchi M, Ueno T: Impact of intrinsic cooperative thermodynamics of peptide-MHC complexes on antiviral activity of HIV-specific CTL. J Immunol. 2009, 182: 5528-5536. 10.4049/jimmunol.0803471.View ArticlePubMedGoogle Scholar
- Briggs SD, Sharkey M, Stevenson M, Smithgall TE: SH3-mediated Hck tyrosine kinase activation and fibroblast transformation by the Nef protein of HIV-1. J Biol Chem. 1997, 272: 17899-17902. 10.1074/jbc.272.29.17899.View ArticlePubMedGoogle Scholar
- Trible RP, Emert-Sedlak L, Smithgall TE: HIV-1 Nef selectively activates Src family kinases Hck, Lyn, and c-Src through direct SH3 domain interaction. J Biol Chem. 2006, 281: 27029-27038. 10.1074/jbc.M601128200.PubMed CentralView ArticlePubMedGoogle Scholar
- Hassan R, Suzu S, Hiyoshi M, Takahashi-Makise N, Ueno T, Agatsuma T, Akari H, Komano J, Takebe Y, Motoyoshi K, Okada S: Dys-regulated activation of a Src tyrosine kinase Hck at the Golgi disturbs N-glycosylation of a cytokine receptor Fms. J Cell Physiol. 2009, 221: 458-468. 10.1002/jcp.21878.View ArticlePubMedGoogle Scholar
- Collins KL, Chen BK, Kalams SA, Walker BD, Baltimore D: HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature. 1998, 391: 397-401. 10.1038/34929.View ArticlePubMedGoogle Scholar
- Saksela K, Cheng G, Baltimore D: 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. 1995, 14: 484-491.PubMed CentralPubMedGoogle Scholar
- Akari H, Arold S, Fukumori T, Okazaki T, Strebel K, Adachi A: Nef-induced major histocompatibility complex class I down-regulation is functionally dissociated from its virion incorporation, enhancement of viral infectivity, and CD4 down-regulation. J Virol. 2000, 74: 2907-2912. 10.1128/JVI.74.6.2907-2912.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Yamada T, Kaji N, Odawara T, Chiba J, Iwamoto A, Kitamura Y: Proline 78 is crucial for human immunodeficiency virus type 1 Nef to down-regulate class I human leukocyte antigen. J Virol. 2003, 77: 1589-1594. 10.1128/JVI.77.2.1589-1594.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Miller MD, Warmerdam MT, Gaston I, Greene WC, Feinberg MB: The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages. J Exp Med. 1994, 179: 101-113. 10.1084/jem.179.1.101.View ArticlePubMedGoogle Scholar
- Fackler OT, Wolf D, Weber HO, Laffert B, D'Aloja P, Schuler-Thurner B, Geffin R, Saksela K, Geyer M, Peterlin BM: A natural variability in the proline-rich motif of Nef modulates HIV-1 replication in primary T cells. Current Biology. 2001, 11: 1294-1299. 10.1016/S0960-9822(01)00373-6.View ArticlePubMedGoogle Scholar
- Lazaro E, Godfrey SB, Stamegna P, Ogbechie T, Kerrigan C, Zhang M, Walker BD, Le Gall S: Differential HIV epitope processing in monocytes and CD4 T cells affects cytotoxic T lymphocyte recognition. J Infect Dis. 2009, 200: 236-243. 10.1086/599837.PubMed CentralView ArticlePubMedGoogle Scholar
- Swigut T, Alexander L, Morgan J, Lifson J, Mansfield KG, Lang S, Johnson RP, Skowronski J, Desrosiers R: Impact of Nef-mediated downregulation of major histocompatibility complex class I on immune response to simian immunodeficiency virus. J Virol. 2004, 78: 13335-13344. 10.1128/JVI.78.23.13335-13344.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Brumme ZL, Brumme CJ, Heckerman D, Korber BT, Daniels M, Carlson J, Kadie C, Bhattacharya T, Chui C, Szinger J, et al: Evidence of differential HLA class I-mediated viral evolution in functional and accessory/regulatory genes of HIV-1. PLoS Pathogens. 2007, 3: e94-10.1371/journal.ppat.0030094.PubMed CentralView ArticlePubMedGoogle Scholar
- Bailey JR, Brennan TP, O'Connell KA, Siliciano RF, Blankson JN: Evidence of CD8+ T-cell-mediated selective pressure on human immunodeficiency virus type 1 nef in HLA-B*57+ elite suppressors. J Virol. 2009, 83: 88-97. 10.1128/JVI.01958-08.PubMed CentralView ArticlePubMedGoogle Scholar
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