Article | Published:

CCR5AS lncRNA variation differentially regulates CCR5, influencing HIV disease outcome

Abstract

Multiple genome-wide studies have identified associations between outcome of human immunodeficiency virus (HIV) infection and polymorphisms in and around the gene encoding the HIV co-receptor CCR5, but the functional basis for the strongest of these associations, rs1015164A/G, is unknown. We found that rs1015164 marks variation in an activating transcription factor 1 binding site that controls expression of the antisense long noncoding RNA (lncRNA) CCR5AS. Knockdown or enhancement of CCR5AS expression resulted in a corresponding change in CCR5 expression on CD4+ T cells. CCR5AS interfered with interactions between the RNA-binding protein Raly and the CCR5 3′ untranslated region, protecting CCR5 messenger RNA from Raly-mediated degradation. Reduction in CCR5 expression through inhibition of CCR5AS diminished infection of CD4+ T cells with CCR5-tropic HIV in vitro. These data represent a rare determination of the functional importance of a genome-wide disease association where expression of a lncRNA affects HIV infection and disease progression.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. 1.

    Kopp, F. & Mendell, J. T. Functional classification and experimental dissection of long noncoding RNAs. Cell 172, 393–407 (2018).

  2. 2.

    Qiu, L. et al. Long non-coding RNAs: regulators of viral infection and the interferon antiviral response. Front. Microbiol. 9, 1621 (2018).

  3. 3.

    Wang, P., Xu, J., Wang, Y. & Cao, X. An interferon-independent lncRNA promotes viral replication by modulating cellular metabolism. Science 358, 1051–1055 (2017).

  4. 4.

    Chen, Y. G., Satpathy, A. T. & Chang, H. Y. Gene regulation in the immune system by long noncoding RNAs. Nat. Immunol. 18, 962–972 (2017).

  5. 5.

    Kumar, V. et al. Human disease-associated genetic variation impacts large intergenic non-coding RNA expression. PLoS Genet. 9, e1003201 (2013).

  6. 6.

    Ling, H. et al. CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer. Genome Res. 23, 1446–1461 (2013).

  7. 7.

    Castellanos-Rubio, A. et al. A long noncoding RNA associated with susceptibility to celiac disease. Science 352, 91–95 (2016).

  8. 8.

    Kenyan Bacteraemia Study Group, et al. Polymorphism in a lincRNA Associates with a doubled risk of pneumococcal bacteremia in Kenyan children. Am. J. Hum. Genet. 98, 1092–1100 (2016).

  9. 9.

    Berger, E. A., Murphy, P. M. & Farber, J. M. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu. Rev. Immunol. 17, 657–700 (1999).

  10. 10.

    McLaren, P. J. & Carrington, M. The impact of host genetic variation on infection with HIV-1. Nat. Immunol. 16, 577–583 (2015).

  11. 11.

    Naranbhai, V. & Carrington, M. Host genetic variation and HIV disease: from mapping to mechanism. Immunogenetics 69, 489–498 (2017).

  12. 12.

    Paxton, W. A. et al. Reduced HIV-1 infectability of CD4+lymphocytes from exposed-uninfected individuals: association with low expression of CCR5 and high production of beta-chemokines. Virology 244, 66–73 (1998).

  13. 13.

    Wu, L. et al. CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro. J. Exp. Med. 185, 1681–1691 (1997).

  14. 14.

    Gonzalez, E. et al. Race-specific HIV-1 disease-modifying effects associated with CCR5 haplotypes. Proc. Natl Acad. Sci. USA 96, 12004–12009 (1999).

  15. 15.

    Kawamura, T. et al. R5 HIV productively infects Langerhans cells, and infection levels are regulated by compound CCR5 polymorphisms. Proc. Natl Acad. Sci. USA 100, 8401–8406 (2003).

  16. 16.

    Martin, M. P. et al. Genetic acceleration of AIDS progression by a promoter variant of CCR5. Science 282, 1907–1911 (1998).

  17. 17.

    Mummidi, S. et al. Evolution of human and non-human primate CC chemokine receptor 5 gene and mRNA. Potential roles for haplotype and mRNA diversity, differential haplotype-specific transcriptional activity, and altered transcription factor binding to polymorphic nucleotides in the pathogenesis of HIV-1 and simian immunodeficiency virus. J. Biol. Chem. 275, 18946–18961 (2000).

  18. 18.

    Thomas, S. M. et al. CCR5 expression and duration of high risk sexual activity among HIV-seronegative men who have sex with men. AIDS 20, 1879–1883 (2006).

  19. 19.

    Gervaix, A. et al. Response to treatment and disease progression linked to CD4+T cell surface CC chemokine receptor 5 density in human immunodeficiency virus type 1 vertical infection. J. Infect. Dis. 185, 1055–1061 (2002).

  20. 20.

    Choudhry, V. et al. Increased efficacy of HIV-1 neutralization by antibodies at low CCR5 surface concentration. Biochem. Biophys. Res. Commun. 348, 1107–1115 (2006).

  21. 21.

    Reynes, J., Baillat, V., Portales, P., Clot, J. & Corbeau, P. Relationship between CCR5 density and viral load after discontinuation of antiretroviral therapy. JAMA 291, 46 (2004).

  22. 22.

    Heredia, A. et al. CCR5 density levels on primary CD4 T cells impact the replication and Enfuvirtide susceptibility of R5 HIV-1. AIDS 21, 1317–1322 (2007).

  23. 23.

    Dean, M. et al. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study. Science 273, 1856–1862 (1996).

  24. 24.

    Mummidi, S. et al. Genealogy of the CCR5 locus and chemokine system gene variants associated with altered rates of HIV-1 disease progression. Nat. Med. 4, 786–793 (1998).

  25. 25.

    Smith, M. W. et al. Contrasting genetic influence of CCR2 and CCR5 variants on HIV-1 infection and disease progression. Hemophilia Growth and Development Study (HGDS), Multicenter AIDS Cohort Study (MACS), Multicenter Hemophilia Cohort Study (MHCS), San Francisco City Cohort (SFCC), ALIVE Study. Science 277, 959–965 (1997).

  26. 26.

    McLaren, P. J. et al. Polymorphisms of large effect explain the majority of the host genetic contribution to variation of HIV-1 virus load. Proc. Natl Acad. Sci. USA 112, 14658–14663 (2015).

  27. 27.

    Sciaranghella, G. et al. CCR5 expression levels in HIV-uninfected women receiving hormonal contraception. J. Infect. Dis. 212, 1397–1401 (2015).

  28. 28.

    Joshi, A. et al. CCR5 promoter activity correlates with HIV disease progression by regulating CCR5 cell surface expression and CD4 T cell apoptosis. Sci. Rep. 7, 232 (2017).

  29. 29.

    Picton, A. C., Shalekoff, S., Paximadis, M. & Tiemessen, C. T. Marked differences in CCR5 expression and activation levels in two South African populations. Immunology 136, 397–407 (2012).

  30. 30.

    Zeller, T. et al. Genetics and beyond—the transcriptome of human monocytes and disease susceptibility. PLoS ONE 5, e10693 (2010).

  31. 31.

    Ingolia, N. T., Lareau, L. F. & Weissman, J. S. Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147, 789–802 (2011).

  32. 32.

    Lin, M. F., Jungreis, I. & Kellis, M. PhyloCSF: a comparative genomics method to distinguish protein coding and non-coding regions. Bioinformatics 27, i275–i282 (2011).

  33. 33.

    Hon, C. C. et al. An atlas of human long non-coding RNAs with accurate 5′ ends. Nature 543, 199–204 (2017).

  34. 34.

    Guil, S. & Esteller, M. Cis-acting noncoding RNAs: friends and foes. Nat. Struct. Mol. Biol. 19, 1068–1075 (2012).

  35. 35.

    Lee, B., Sharron, M., Montaner, L. J., Weissman, D. & Doms, R. W. Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages. Proc. Natl Acad. Sci. USA 96, 5215–5220 (1999).

  36. 36.

    Rashid, F., Shah, A. & Shan, G. Long non-coding RNAs in the cytoplasm. Genom. Proteom. Bioinform. 14, 73–80 (2016).

  37. 37.

    Yoon, J. H. et al. LincRNA-p21 suppresses target mRNA translation. Mol. Cell 47, 648–655 (2012).

  38. 38.

    Gong, C. & Maquat, L. E. lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3′ UTRs via Alu elements. Nature 470, 284–288 (2011).

  39. 39.

    Kretz, M. et al. Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature 493, 231–235 (2013).

  40. 40.

    Xiao, Z. D. et al. Energy stress-induced lncRNA FILNC1 represses c-Myc-mediated energy metabolism and inhibits renal tumor development. Nat. Commun. 8, 783 (2017).

  41. 41.

    Rehfuss, R. P., Walton, K. M., Loriaux, M. M. & Goodman, R. H. The cAMP-regulated enhancer-binding protein ATF-1 activates transcription in response to cAMP-dependent protein kinase A. J. Biol. Chem. 266, 18431–18434 (1991).

  42. 42.

    Thio, C. L. et al. Genetic protection against hepatitis B virus conferred by CCR5Delta32: evidence that CCR5 contributes to viral persistence. J. Virol. 81, 441–445 (2007).

  43. 43.

    Glass, W. G. et al. CCR5 deficiency increases risk of symptomatic West Nile virus infection. J. Exp. Med. 203, 35–40 (2006).

  44. 44.

    Kakimi, K., Guidotti, L. G., Koezuka, Y. & Chisari, F. V. Natural killer T cell activation inhibits hepatitis B virus replication in vivo. J. Exp. Med. 192, 921–930 (2000).

  45. 45.

    Moreno, C. et al. CCR5 deficiency exacerbates T-cell-mediated hepatitis in mice. Hepatology 42, 854–862 (2005).

  46. 46.

    Sun, Q., Hao, Q. & Prasanth, K. V. Nuclear long noncoding RNAs: key regulators of gene expression. Trends Genet. 34, 142–157 (2018).

  47. 47.

    Noh, J. H., Kim, K. M., McClusky, W. G., Abdelmohsen, K. & Gorospe, M. Cytoplasmic functions of long noncoding RNAs. Wiley Interdiscip. Rev. RNA 9, e1471 (2018).

  48. 48.

    Maurano, M. T. et al. Systematic localization of common disease-associated variation in regulatory DNA. Science 337, 1190–1195 (2012).

  49. 49.

    Degner, J. F. et al. DNase I sensitivity QTLs are a major determinant of human expression variation. Nature 482, 390–394 (2012).

  50. 50.

    ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

  51. 51.

    Vlahov, D. et al. The ALIVE study, a longitudinal study of HIV-1 infection in intravenous drug users: description of methods and characteristics of participants. NIDA Res. Monogr. 109, 75–100 (1991).

  52. 52.

    Emu, B. et al. Phenotypic, functional, and kinetic parameters associated with apparent T-cell control of human immunodeficiency virus replication in individuals with and without antiretroviral treatment. J. Virol. 79, 14169–14178 (2005).

  53. 53.

    Naruto, T. et al. HLA class I-mediated control of HIV-1 in the Japanese population, in which the protective HLA-B*57 and HLA-B*27 alleles are absent. J. Virol. 86, 10870–10872 (2012).

  54. 54.

    Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408 (2001).

  55. 55.

    Engreitz, J. M. et al. RNA-RNA interactions enable specific targeting of noncoding RNAs to nascent Pre-mRNAs and chromatin sites. Cell 159, 188–199 (2014).

  56. 56.

    Josic, D. et al. Proteomic characterization of inter-alpha inhibitor proteins from human plasma. Proteomics 6, 2874–2885 (2006).

  57. 57.

    Lahaye, X. et al. The capsids of HIV-1 and HIV-2 determine immune detection of the viral cDNA by the innate sensor cGAS in dendritic cells. Immunity 39, 1132–1142 (2013).

  58. 58.

    Martin-Gayo, E. et al. Potent cell-intrinsic immune responses in dendritic cells facilitate HIV-1-specific T cell immunity in HIV-1 elite controllers. PLoS Pathog. 11, e1004930 (2015).

  59. 59.

    Unutmaz, D., KewalRamani, V. N., Marmon, S. & Littman, D. R. Cytokine signals are sufficient for HIV-1 infection of resting human T lymphocytes. J. Exp. Med. 189, 1735–1746 (1999).

  60. 60.

    Chen, H. et al. CD4+T cells from elite controllers resist HIV-1 infection by selective upregulation of p21. J. Clin. Invest. 121, 1549–1560 (2011).

  61. 61.

    Li, H., Wright, P. W., McCullen, M. & Anderson, S. K. Characterization of KIR intermediate promoters reveals four promoter types associated with distinct expression patterns of KIR subtypes. Genes Immun. 17, 66–74 (2016).

Download references

Acknowledgements

The project was supported by NIAID (grant nos AI120900 and AI140956 to S.K.), HU-CFAR (to S.K.), Cowles fellowship (to S.S.), institutional funds from the Texas Biomedical Research Institute and the Ragon Institute of MGH, MIT and Harvard. This project has been funded in whole or in part with federal funds from the Frederick National Laboratory (contract no. HHSN261200800001E). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. This research was supported in part by the Intramural Research Program of the NIH, Frederick National Lab and Center for Cancer Research. See extended acknowledgements in Supplementary Information for full details.

Author information

S.K., S.L.G., X.G.Y., S.K.A. and M.C. designed the study. S.K., M.P.M., V.W.-S., A.L., V.K., H.N., S.S., R.E., M.R. and F.Z.C. designed and performed experiments, and analyzed and interpreted the data. M.V. and V.N. analyzed data in African American and Hispanic cohorts infected with HIV and cohorts with HBV. C.Z., Z.L., H.G., S.O. and M.T. analyzed data in Japanese patients. M.C. directed the study and wrote the manuscript with S.K., M.P.M. and M.V. The clinical samples and data were contributed by P.J.M., C.L.T., J.M., D.W.H., G.D.K., J.J.G., W.K.H., S.G.D., D.W.H., N.M. and B.W. Intellectual input was provided by all authors.

Correspondence to Smita Kulkarni or Mary Carrington.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 The rs1015164 SNP marking expression of CCR5AS associates independently with HIV viral load in Japanese subjects.

a, Sanger sequencing was carried out to determine rs1015164 genotypes of 504 HIV infected Japanese subjects (GG=51%, AG=44% and AA=5%). b, Patients with AA genotype showed significantly higher HIV viral load as compared to the patients with GG or AG/GG genotypes.

Supplementary Figure 2 Gating strategy for CD4+ T cell subsets and characterization of CCR5AS transcript expression.

a, Flow cytometry gating strategy used to characterize CD4+ T cell subsets. b, rs1015164A marks high expression of CCR5AS (ENSG00000223552.1) in blood, Colon, Adipose-Visceral, Heart left ventricle, Lung, Adipose-subcutaneous, Brain (https://www.gtexportal.org/). c, Amplification of the 3’ and 5’ ends of the CCR5AS transcript uncovered the presence of two distinct isoforms: a long isoform of 699bp and a 431bp short form that is differentially spliced and lacks exon 2. d, Total RNA was extracted from the cytoplasmic and nuclear fractions of primary CD4+ T cells and Hut-78 cells. Relative expression levels of CCR5AS in the cytoplasm (black) vs. the nucleus (white) were assessed using qPCR. Unspliced β-actin mRNA and spliced GAPDH mRNA, which are enriched in the nuclear and cytoplasmic fractions, respectively, were used as controls to assess purity of the extracts.

Supplementary Figure 3 CCR5AS enhances CCR5 mRNA and cell surface expression.

a, Peripheral blood CD4+ T cells were transfected with 300 nm siRNA targeting CCR5AS (siLnc1) or a control siRNA (siCon1). CCR5AS mRNA was measured 24 hours post-transfection. Cells transfected with siLnc1 showed 70–80% downregulation of CCR5AS mRNA as compared to the siCon1 treated cells. b, Cells transfected with siLnc2, which targets a region of CCR5AS distinct from that of siLinc1, showed 60–70% downregulation of CCR5AS mRNA as compared to siCon2 treated cells. c, Cells transfected with siLnc2 showed lower CCR5 mRNA expression as compared to the siCon2 transfected cells. d, Cell surface expression of CCR5 was measured 24 hours after siRNA transfection. The cells transfected with siLnc2 (red curve) showed lower CCR5 cell surface expression as compared to the siCon2 (blue curve) transfected cells. The open black curve depicts isotype control. Fold change in expression levels was calculated as the ratio of mean fluorescence intensity (MFI) of CCR5 vs isotype control. A histogram of one of 6 comparable experiments performed is shown for each silencing. The mean ± SE (n = 6) are depicted as horizontal and vertical bars for each group, respectively. Paired t test was used for statistical comparisons and two tailed p value is indicated. e, CD4+ T cells were transfected with the in vitro transcribed long form of CCR5AS [CCR5AS(L)] or scrambled RNA controls (ConRNA). CCR5 mRNA expression was measured 24 hours post-transfection. Cells transfected with CCR5AS(L) showed higher CCR5 mRNA levels as compared to the scrambled control. f, The cell surface expression of CCR5 in cells transfected with CCR5AS(L) (red curve) was higher as compared to the scrambled control (blue curve) transfected cells. The grey curve depicts isotype control. Fold change in expression levels was calculated as the ratio of mean fluorescence intensity (MFI) of CCR5 vs isotype control. A histogram of one of 6 comparable experiments performed is shown. Statistics are as described in (d).

Supplementary Figure 4 CCR5AS silencing inhibits CCR5 expression in Hut-78 cells by reducing CCR5 mRNA stability.

a, Hut-78 cells were transfected with 300 nm siRNA targeting CCR5AS (siLnc1) or control siRNA (siCon1). CCR5 mRNA was measured 24 hours post-transfection. Cells transfected with siLnc1 showed lower expression as compared to the controls. b, Cell surface expression of CCR5 on Hut-78 cells was measured 48 hours post-transfection. The cells transfected with siLnc1 (red curve) showed lower cell surface expression as compared to siCon1 transfected cells (blue curve). The gray curve depicts isotype controls. Fold change in expression levels was calculated as the ratio of mean fluorescence intensity (MFI) of CCR5 vs isotype control. A histogram of one of three comparable experiments performed is shown. The mean ± SE (n = 3) are depicted as horizontal and vertical bars for each group, respectively. Paired t test was used for statistical comparisons and two tailed p value is indicated. c, CCR5 mRNA RNA decay was determined by 5-Ethylene uridine (EU) pulse-labeling of RNA using the Click-iT Nascent RNA Capture Kit. Hut-78 cells were transfected with siCon1 or siLnc1 and pulsed with EU. Eighteen hours after EU-pulsing, the cells were washed, supplemented with fresh growth medium and harvested at one hour intervals for 4 hours. Total RNA was isolated from cells and quantitated. The EU-labeled RNA was biotinylated, precipitated, and captured using streptavidin coated magnetic beads as per the manufacturer’s protocol. The RNA captured on beads was used for cDNA synthesis and qPCR analysis. Data are represented as relative expression levels of CCR5 mRNA in the siCon1 or siLnc1 transfected cells. The mRNA half-life (50% mRNA remaining, t1/2) for the CCR5 mRNA in siCon1 (3h) was higher as compared to the siLnc1 treated cells (2.2h). Data represent mean ± SEM. One of two comparable experiments performed is shown. Paired t test was used for statistical comparisons and one tailed p value is indicated.

Supplementary Figure 5 CCR5AS mediated regulation of CCR5 expression is independent of shared miRNA binding sites.

Hut-78 cells were transfected with in vitro transcribed CCR5AS or scrambled RNA controls and cell surface expression of CCR5 was measured 48h post-transfection. a, b, Cells transfected with the wild type long (L) form (a) or the short (S) form (b) of CCR5AS transcript showed higher CCR5 cell surface expression. c,d,e, Cells transfected with CCR5AS mutant transcripts with disruption in binding sites of miR-1224 (c), miR-197 (d), or both (e) showed higher CCR5 cell surface expression as compared to the respective controls. Surface expression of cells transfected with CCR5AS are shown as various colored curves whereas the scrambled RNA controls are represented by blue curves. The open black curves depict isotype control. A histogram of one of three comparable experiments performed is shown for each overexpression. f, Fold change in CCR5 expression levels was calculated as the ratio of mean fluorescence intensity (MFI) when staining with CCR5-specific antibody vs isotype control. g, Fold changes in CCR5 mRNA upon transfection with the various forms of the CCR5AS mRNA are shown. CCR5 mRNA levels were normalized to GAPDH mRNA and the mean ±SE (n=3) are depicted as horizontal and vertical bars for each group, respectively. CCR5AS RNA transfections were compared to those with Control RNA and an unpaired t test was used for statistical comparisons with two tailed p value indicated.

Supplementary Figure 6 Raly binds CCR5AS and the 3’UTR of CCR5 mRNA.

a, Western blot analysis was performed to confirm the presence of Raly protein in the (AS) pulldown as compared to the SC pulldown. b, In silico prediction of the interaction between the long (left) and short (right) forms of the CCR5AS transcript and Raly protein was done using a freely available algorithm (http://service.tartaglialab.com/newsubmission/globalscore), which integrates properties of protein and RNA structures into overall binding propensity. Global score predicts global and local interactions between RBP and lncRNA. A high interaction score (≈1) predicts a strong interaction between Raly protein and CCR5AS transcript. c, A Myc-tagged Raly protein sequence was transfected in Hut-78 cells. The c-Myc tagged protein was immunoprecipitated using anti-Myc antibody coated magnetic beads, but not with nonspecific IgG, as confirmed by Western blot. d, The Hut-78 cell line was transfected with either control siRNA (siCon) or Raly siRNA (siRaly). Cells were harvested at 48h and 72h post-transfection. Western blot analysis showed downregulation of Raly after 72h. Alpha tubulin was used as the housekeeping control. e CCR5 3’UTR was cloned downstream of Renilla luciferase into a dual luciferase reporter psicheck2 vector. The Raly binding site in the CCR5 3’UTR sequence is indicated by a red bar. f, Hut-78 cells were transfected with a vector encoding Myc-tagged Raly protein (Raly-Myc) along with either Control siRNA (siCon1) or CCR5AS siRNA (siLnc1). Cells were lysed and RNA immunoprecipitation was carried out using anti-Myc antibody (Raly-IP) or control IgG (IgG-IP). Precipitation of Raly protein by anti-Myc antibody, but not control IgG, was confirmed by Western blot. Input represents straight lysate (that is no immunoprecipitation). g, Fold enrichment of CCR5AS in the pulldown was determined using qPCR. CCR5AS was enriched in the Raly pulldown (Raly-IP) as compared to IgG in the siCon1 treated cells. No enrichment of CCR5AS was observed in the Raly pulldown of cells transfected with siLnc1. h, CCR5 mRNA decay was determined by 5-Ethylene uridine (EU) pulse-labeling of RNA using the Click-iT Nascent RNA Capture Kit. Hut-78 cells were transfected with siRaly or siCon and pulsed with EU after 60 hours. Eighteen hours after EU-pulsing, the cells were washed, supplemented with fresh growth medium and harvested at one hour intervals for 4 hours. Total RNA was isolated from cells and quantitated. The EU-labeled RNA was biotinylated, precipitated, and captured using streptavidin coated magnetic beads as per the manufacturer’s protocol. The RNA captured on beads was used for cDNA synthesis and qPCR analysis. Data are represented as relative expression levels of CCR5 mRNA in the siCon or siRaly transfected cells. The mRNA half-life (50% mRNA remaining, t1/2) for the CCR5 mRNA in siRaly (4h) was higher as compared to the siCon treated cells (2.9h).

Supplementary Figure 7 In vitro Infection of CD4+T cells with HIV-1.

a, Schematic presentation of experimental design. Peripheral blood CD4+ T cells were transfected with siRNA. Twenty-four hours post transfection, the cells were infected with VSV-G pseudotyped HIV virus encoding GFP or CCR5-tropic HIV virus encoding GFP. The proportions of VSV-G/GFP+ or CCR5-tropic/GFP+ cells were determined by flow cytometry. b, c, d Flow cytometry gating strategy is represented here uninfected (b), and VSV-G/GFP (c) or CCR5-tropic/GFP (d) virus infected cells of a single experiment. Debris was excluded based on light scattering characteristics. Proportions of VSV-G/GFP+ or CCR5-tropic/GFP+ cells were analyzed for the cells in P1 gate. The uninfected cells did not show GFP+(R1 gate). e, f Peripheral blood CD4+ T cells from 8 unrelated donors were transfected with control siRNA (siCon1) or CCR5AS siRNA (siLnc1). Twenty-four hours post transfection, the cells were infected with VSV-G/GFP (e) or CCR5-tropic/GFP virus (f). The proportions of VSV-G/GFP+ cells in the siLnc1 treated cells were comparable to that of the siCon1 treated cells, but silencing of CCR5AS diminished infection of CD4+ T cells with the CCR5-tropic/GFP virus.

Supplementary Figure 8 Schematic representation of the effect of rs1015164A/G variant on HIV infection.

The rs1015164A/G variant associates with CCR5AS expression, where rs1015164A marks high CCR5AS expression. High CCR5AS expression enhances CCR5 expression post-transcriptionally by sequestering the RNA binding protein Raly, inhibiting the binding of Raly to the CCR5 3’UTR, and protecting CCR5 mRNA from Raly-mediated decay. Higher expression levels of CCR5AS result in increased cell surface expression of the HIV co-receptor CCR5 and higher levels of HIV infection, providing the functional basis for the association between rs1015164A and poor HIV control.

Supplementary information

Supplementary Information

Supplementary Figures 1–8, Supplementary Notes, and Supplementary Table 1

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Further reading

Fig. 1: rs1015164A/G variation associates with HIV-1 viral load and CD4+ T cell counts across distinct populations.
Fig. 2: rs1015164A/G variation marks differential expression of CCR5AS and CCR5.
Fig. 3: CCR5AS enhances CCR5 mRNA and cell surface expression.
Fig. 4: CCR5AS binds Raly.
Fig. 5: Raly regulates CCR5 expression.
Fig. 6: CCR5AS inhibits binding of Raly to the CCR5 3′ UTR.
Fig. 7: CCR5AS expression levels associate with susceptibility to CCR5-dependent HIV infection.
Fig. 8: ATF1 binds rs2027820G more strongly than rs2027820A.
Supplementary Figure 1: The rs1015164 SNP marking expression of CCR5AS associates independently with HIV viral load in Japanese subjects.
Supplementary Figure 2: Gating strategy for CD4+ T cell subsets and characterization of CCR5AS transcript expression.
Supplementary Figure 3: CCR5AS enhances CCR5 mRNA and cell surface expression.
Supplementary Figure 4: CCR5AS silencing inhibits CCR5 expression in Hut-78 cells by reducing CCR5 mRNA stability.
Supplementary Figure 5: CCR5AS mediated regulation of CCR5 expression is independent of shared miRNA binding sites.
Supplementary Figure 6: Raly binds CCR5AS and the 3’UTR of CCR5 mRNA.
Supplementary Figure 7: In vitro Infection of CD4+T cells with HIV-1.
Supplementary Figure 8: Schematic representation of the effect of rs1015164A/G variant on HIV infection.