Abstract
Individuals infected with human immunodeficiency virus type-1 (HIV-1) show metabolic alterations of CD4+ T cells through unclear mechanisms with undefined consequences. We analyzed the transcriptome of CD4+ T cells from patients with HIV-1 and revealed that the elevated oxidative phosphorylation (OXPHOS) pathway is associated with poor outcomes. Inhibition of OXPHOS by the US Food and Drug Administration–approved drug metformin, which targets mitochondrial respiratory chain complex-I, suppresses HIV-1 replication in human CD4+ T cells and humanized mice. In patients, HIV-1 peak viremia positively correlates with the expression of NLRX1, a mitochondrial innate immune receptor. Quantitative proteomics and metabolic analyses reveal that NLRX1 enhances OXPHOS and glycolysis during HIV-1-infection of CD4+ T cells to promote viral replication. At the mechanistic level, HIV infection induces the association of NLRX1 with the mitochondrial protein FASTKD5 to promote expression of mitochondrial respiratory complex components. This study uncovers the OXPHOS pathway in CD4+ T cells as a target for HIV-1 therapy.
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Data availability
The authors declare that the data supporting the findings of this study are available with the paper and its supplementary information files. Source data for all figures are provided with the paper. The RV217 CD4+ T cell transcriptome data have been uploaded to the Gene Expression Omnibus (accession number GSE165841). The mass spectrometry proteomics data have been deposited to the ProteomeXchange with identifier PXD023565. The public databases used include UniProt human protein sequence database https://www.uniprot.org/proteomes/UP000005640; KEGG pathway database https://www.genome.jp/kegg/pathway.html; Hallmark gene set https://www.gsea-msigdb.org/gsea/msigdb/genesets.jsp?collection=H; and Reactome Pathway database https://reactome.org/. Source data are provided with this paper.
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Acknowledgements
We thank the UNC Flow Cytometry Core Facility, supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center, for assistance with flow cytometry. We thank S. K. Chanda at Sanford Burnham Prebys Medical Discovery Institute for providing the pNL4-3.EGFP.E- construct. This work was supported by NIH grants R01-AI029564 (J.P.-Y.T.), U19AI109965 (J.P.-Y.T.), AI127346 (L.S.) and DK119937 (L.S.); and UNC CFAR Award P30 AI50410 (H.G.). The RV217 study was supported by a cooperative agreement (W81XWH-18-2-0040) between the Henry M. Jackson Foundation for the Advancement of Military Medicine and the US Department of Defense.
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H.G., L.S. and J.P.-Y.T. designed the experiments; H.G., Q.W., L.W., K.G., E.R., E.H.G., L.C., M.L.R. and L.A.E. conducted the studies; K.G. performed the data and statistical analyses of the RV217 study. X.C. and R.-P.S. assisted with the experiments and provided intellectual input; D.M.M. and C.G. provided critical reagents; J.P.-Y.T. supervised the study; H.G., Q.W., L.S. and J.P.-Y.T. interpreted the data and wrote the manuscript.
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The authors declare no competing interests. The views expressed are those of the authors and should not be construed to represent the positions of the US Army, the Department of Defense or the Henry M. Jackson Foundation.
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Peer review information Nature Immunology thanks Xiao-Ning Xu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Extended data
Extended Data Fig. 1 The network of leading edge genes in the OXPHOS pathway associated with set-point viral load.
a, Cytoscape was used to plot a network highlighting the top genes contributing to enrichment. The ClueGO app was used to infer network connections. Nodes represent genes, and edges reflect the association between these genes. b, STRING was used to plot a network highlighting the top genes contributing to enrichment. Nodes represent genes, and edges reflect the association between these genes.
Extended Data Fig. 2 Metabolic inhibitors suppress HIV-1 replication in CD4 T cells and NRG-hu CD4 mice.
a, The effect of OXPHOS inhibitors on virus replication in VSV-G-NL4-3-Luc-infected Jurkat cells. Rotenone (0.2 µM or 1 µM), metformin (1 mM or 5 mM, mitochondrial complex I inhibitor) and antimycin A (0.2 µM or 1 µM, mitochondrial complex III inhibitor). Ethanol is the vehicle control for rotenone and antimycin A, and sterile water is the vehicle control for metformin. n = 3 cell cultures per experiment. b, The oxygen consumption rate (OCR) was measured in Jurkat cells infected with VSV-G-NL4-3-Luc or left uninfected (mock) in the presence of metformin or the vehicle control (H2O). n = 4 cell cultures per experiment. c, The basal and maximal OCR and reserved respiratory capacity of Jurkat cells infected with VSV-G-NL4-3-Luc or left uninfected (mock) in the presence of metformin or vehicle control (H2O). n = 12 cell cultures per experiment. d, The effect of 2-DG (5 mM or 10 mM), a glycolysis inhibitor, on virus replication in VSV-G-NL4-3-Luc infected Jurkat cells. H2O was used as vehicle control. n = 3 cell cultures per experiment. e, Representative FACS plots of human CD4 T cells from peripheral blood stained with the indicated markers. Cells were obtained from mock or HIV-1 R3A infected NRG-hu CD4 mice with or without metformin treatment. Data are representative of three independent experiments shown as the mean ± s.e.m. Statistical significance was tested by one-way ANOVA (a,d) or two-way ANOVA followed by Tukey’s multiple comparisons test (c).
Extended Data Fig. 3 NLRX1 is upregulated in HIV-1 infected CD4 T cells.
a, Jurkat cells were infected with VSV-G-pseudotyped HIV NL4-3-EGFP virus (MOI = 0.5). After 48 hours post-infection (hpi), cells were stained with an anti-NLRX1 antibody and subjected to ImageStreamX MkII imaging flow cytometry. GFP positive cells indicate HIV-1 infection. b, The mean fluorescence intensity (MFI) of NLRX1 staining in GFP negative (n = 532 cells) and positive cells (n = 222 cells). P value was calculated by two-tailed unpaired Student’s t-test. c, The linear relationship between the expression of NLRX1 and GFP. R, Pearson’s correlation coefficient; Correlation is significant at P < 0.0001. d, Jurkat cells were infected by VSV-G-NL4-3-EGFP pseudovirus (MOI = 1). At 48 hpi, NLRX1 transcripts were assessed by qPCR. n = 3 cell cultures per experiment. P value was calculated by two-tailed unpaired Student’s t-test. e, NLRX1 transcripts were assessed in human primary CD4 T cells infected by three different HIV-1 clinical isolates at 48 hpi by qPCR. n = 3 cell cultures per experiment. Significance was tested by one-way ANOVA followed by Dunnett’s multiple comparisons test. All data are representative of three independent experiments. Error bar is s.e.m.
Extended Data Fig. 4 The effect of metformin on NLRX1 expression in CD4 T cells.
a–d, CD3/CD28 antibody-activated human primary CD4 T cells were left uninfected (a) or infected by three different HIV-1 clinical isolates, #409 (b), #413 (c), and #414 (d) in the presence of 5 mM metformin or the vehicle control, sterile water. NLRX1 transcripts were assessed at 48 hpi by qPCR. Representative data of three independent experiments are shown as the mean ± standard deviation (s.d). n = 4 cell cultures per experiment. Statistical significance was tested by two-tailed unpaired Student’s t-test.
Extended Data Fig. 5 Silencing NLRX1 protects HIV-1 caused human CD4 T cell depletion in the NRG-hu CD4 mouse model.
a, Representative fluorescence-activated cell sorting (FACS) plot of human CD4 T cells expressing the indicated markers in peripheral blood from mock or HIV-1 R3A infected NRG-hu CD4 mice at 1 and 2 wpi. b, Spleens from mock or HIV-1 R3A infected NRG-hu CD4 mice at 3 wpi. n = 6 mice per group. c, Splenocyte numbers of mock or HIV-1 R3A infected NRG-hu CD4 mice at 3 wpi. The data are a pool of two independent experiments. n = 8 mice for mock groups; n = 9 mice for HIV-1 infection groups. Data are presented as the mean ± s.e.m. Statistical significance was tested by two-way ANOVA followed by Sidak’s multiple comparisons test.
Extended Data Fig. 6 NLRX1 promotion of HIV-1 replication in CD4 T cells is independent of IFN-I, autophagy, or ER stress.
a, RLU in Jurkat sh-Ctr or sh-NLRX1 cells infected with VSV-G-NL4-3-Luc (MOI=1) or left uninfected in the presence or absence of JAK1/2 inhibitor Ruxolitinib at 24 hpi. n = 3 cell cultures per experiment. b, Ruxolitinib suppressed VSV-G-NL4-3-Luc infection-induced phosphorylation of STAT1 at 24 hpi. β-actin was used as the loading control. c, Similar to a except for using autophagy inhibitor 3-Methyladenine (3-MA) to treat cells. Numbers on top of the bar are the fold differences. n = 3 cell cultures per experiment. d, Similar to a except for using ER stress inhibitor sodium tauroursodeoxycholate (TUDCA) to treat cells. Luciferase activities were determined at 48 hpi. n = 3 cell cultures per experiment. Representative data of three independent experiments are presented as the mean ± s.e.m. Two-way ANOVA followed by Sidak’s multiple comparisons test.
Extended Data Fig. 7 Expression of FASTKD5 in Jurkat cells and primary human CD4 T cells.
a, Candidates of NLRX1 interacting proteins identified by immunoprecipitation (IP)-mass spectrometry (MS) using overexpressed NLRX1 as the bait in a previous publication. b, Jurkat-sh-Ctr and Jurkat-sh-NLRX1 cells were left uninfected (mock) or infected by VSV-G-NL4-3-Luc pseudovirus (MOI = 1). FASTKD5 transcripts were assessed at 24 hpi by qPCR. Data are shown as the mean ± s.e.m. n = 4 cell cultures per experiment. Statistical significance was tested by two-way ANOVA followed by Sidak’s multiple comparisons test. c, CD3/CD28 antibody-activated human primary CD4 T cells were infected by three different HIV-1 clinical isolates (10 ng p24), and FASTKD5 transcripts were assessed at 24 and 48 hpi by qPCR. Data are shown as the mean ± s.e.m. n = 4 cell cultures per experiment. Statistical significance was tested by one-way ANOVA followed by Dunnett’s multiple comparisons test. d, e, Doxycycline-induced silencing of FASTKD5 (d) and PRDX3 (e) in Jurkat cells transduced by 2 different shRNA containing lentiviruses. β-actin was used as the loading control. Data (b–e) are representative of three independent experiments.
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Guo, H., Wang, Q., Ghneim, K. et al. Multi-omics analyses reveal that HIV-1 alters CD4+ T cell immunometabolism to fuel virus replication. Nat Immunol 22, 423–433 (2021). https://doi.org/10.1038/s41590-021-00898-1
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DOI: https://doi.org/10.1038/s41590-021-00898-1
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