Molecular Biology

, Volume 52, Issue 2, pp 251–261 | Cite as

Protection of Lymphocytes Against HIV using Lentivirus Vector Carrying a Combination of TRIM5α-HRH Genes and microRNA Against CCR5

  • D. O. Omelchenko
  • D. V. Glazkova
  • E. V. Bogoslovskaya
  • F. A. Urusov
  • Y. A. Zhogina
  • G. M. Tsyganova
  • G. A. Shipulin
Molecular Cell Biology


Gene therapy is considered a promising approach to treating infections caused by human immunodeficiency virus (HIV). One strategy is to introduce antiviral genes into cells in order to impart resistance to HIV. In this work, the antiviral activity of new anti-HIV lentiviral vector pT has been studied. The vector carries a combination that consists of two identical artificial miRNA mic13lg and the TRIM5α-HRH gene. Two mic13lg microRNAs suppress the expression of the CCR5 gene, which encodes the HIV coreceptor and, thus, prevents the penetration of R5-tropic HIV strains into the cell. It has been shown that pT effectively inhibits the expression of CCR5 in both the HT1080 CCR5-EGFP model cell line and in human primary lymphocytes. The second line of protection against R5- and X4-tropic HIV is provided by the TRIM5α-HRH protein, which binds virus capsids after the virus enters the cell. Indeed, when infecting cells of the SupT1 line, which contains four copies of the vector per cell, with the X-4 tropic HIV, more than 1000-fold suppression of viral replication has been observed. The process of generation of the pT vector and conditions of transduction of CD4+ lymphocytes were optimized for testing the antiviral activity of the vector on primary human lymphocytes. As a result, the transduction efficiency for the pT vector was 28%. After infection with the R5-tropic strain of the virus, the survival of cells in the culture of lymphocytes with the vector was significantly higher than in the control. However, the complete suppression of HIV replication was not achieved, presumably due to the inadequate fraction of cells that carry the vector in culture. In the future, it is planned to find the best way to enrich the lymphocyte culture with modified cells to increase resistance to HIV.


microRNA CCR5 TRIM5α-HRH HIV lentiviral transduction SupT1 HT1080 lymphocytes CD4+ 


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  1. 1.
    Pokrovsky V.V. 2016. Infection caused by human immunodeficiency virus in Russia. Ter. Arkh. 88, 4–11.CrossRefPubMedGoogle Scholar
  2. 2.
    Bhatti A.B., Usman M., Kandi V. 2016. Current scenario of HIV/AIDS, treatment options, and major challenges with compliance to antiretroviral therapy. Cureus. 8, e515.PubMedPubMedCentralGoogle Scholar
  3. 3.
    Zhogina Y.A., Glazkova D.V., Vetchinova A.S., et al. 2014. Comparison of activity of different gene constructions aimed at inhibiting HIV 1 replication. Biopharm. J. 6, 11–18.Google Scholar
  4. 4.
    Deng H., Liu R., Ellmeier W., et al. 1996. Identification of a major co-receptor for primary isolates of HIV-1. Nature. 381, 661–666.CrossRefPubMedGoogle Scholar
  5. 5.
    Dragic T., Litwin V., Allaway G.P., et al. 1996. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature. 381, 667–673.CrossRefPubMedGoogle Scholar
  6. 6.
    Novembre J., Galvani A.P., Slatkin M. 2005. The geographic spread of the CCR5Δ32 HIV-resistance allele. PLoS Biol. 3, e339.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Liu R., Paxton W.A., Choe S., et al. 1996. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell. 86, 367–377.CrossRefPubMedGoogle Scholar
  8. 8.
    Ghorban K., Dadmanesh M., Hassanshahi G., et al. 2013. Is the CCR5Δ32 mutation associated with immune system-related diseases? Inflammation. 36, 633–642.CrossRefPubMedGoogle Scholar
  9. 9.
    Swamy M.N., Wu H., Shankar P. 2016. Recent advances in RNAi-based strategies for therapy and prevention of HIV-1/AIDS. Adv. Drug Delivery Rev. 103, 174–186.CrossRefGoogle Scholar
  10. 10.
    Glazkova D.V., VetchinovaA.S., Bogoslovskaya E.V., et al. 2013. Downregulation of human CCR5 gene expression with artificial microRNAs. Mol. Biol. (Moscow). 47 (3), 419–428.CrossRefGoogle Scholar
  11. 11.
    Stremlau M., Owens C.M., Perron M.J., et al. 2004. The cytoplasmic body component TRIM5α restricts HIV-1 infection in Old World monkeys. Nature. 427, 848–853.CrossRefPubMedGoogle Scholar
  12. 12.
    Anderson J., Akkina R. 2008. Human immunodeficiency virus type 1 restriction by human–rhesus chimeric tripartite motif 5α (TRIM 5α) in CD34+ cellderived macrophages in vitro and in T cells in vivo in severe combined immunodeficient (SCID-hu) mice transplanted with human fetal tissue. Hum. Gene Ther. 19, 217–228.CrossRefPubMedGoogle Scholar
  13. 13.
    Lu X., Humeau L., Slepushkin V., et al. 2004. Safe twoplasmid production for the first clinical lentivirus vector that achieves >99% transduction in primary cells using a one-step protocol. J. Gene Med. 6, 963–973.CrossRefPubMedGoogle Scholar
  14. 14.
    Kuroda H., Kutner R.H., Bazan N.G., Reiser J. 2009. Simplified lentivirus vector production in protein-free media using polyethylenimine-mediated transfection. J. Virol. Methods. 157, 113–121.CrossRefPubMedGoogle Scholar
  15. 15.
    Hesselgesser J., Liang M., Hoxie J., et al. 1998. Identification and characterization of the CXCR4 chemokine receptor in human T cell lines: Ligand binding, biological activity, and HIV-1 infectivity. J. Immunol. 160, 877–883.PubMedGoogle Scholar
  16. 16.
    Segura M.M., Mangion M., Gaillet B., Garnier A. 2013. New developments in lentiviral vector design, production and purification. Expert Opin. Biol. Ther. 13, 987–1011.CrossRefPubMedGoogle Scholar
  17. 17.
    Yang S., Shi H., Chu X., et al. 2016. A rapid and efficient polyethylenimine-based transfection method to prepare lentiviral or retroviral vectors: Useful for making iPS cells and transduction of primary cells. Biotechnol. Lett. 38, 1631–1641.CrossRefPubMedGoogle Scholar
  18. 18.
    Merten O.-W., Hebben M., Bovolenta C. 2016. Production of lentiviral vectors. Mol. Ther. Methods Clin. Dev. 3, 16017.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Canté-Barrett K., Mendes R.D., Smits W.K., et al. 2016. Lentiviral gene transfer into human and murine hematopoietic stem cells: Size matters. BMC Res. Notes. 9,312.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Pazare A.R. 2015. Evolution of anti-retroviral therapy: Multiple pills to fixed drug combinations. J. Assoc. Physicians India. 63, 11–13.PubMedGoogle Scholar
  21. 21.
    Voit R.A., McMahon M.A., Sawyer S.L., Porteus M.H. 2013. Generation of an HIV resistant T-cell line by targeted ‘stacking’ of restriction factors. Mol. Ther. 21, 786–795.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Anderson J.S., Javien J., Nolta J.A., Bauer G. 2009. Preintegration HIV-1 inhibition by a combination lentiviral vector containing a chimeric TRIM5αprotein, a CCR5 shRNA, and a TAR decoy. Mol. Ther. 17, 2103–2114.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Wolstein O., Boyd M., Millington M., et al. 2014. Preclinical safety and efficacy of an anti–HIV-1 lentiviral vector containing a short hairpin RNA to CCR5 and the C46 fusion inhibitor. Mol. Ther.–Methods Clin. Dev. 1,11.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Myburgh R., Cherpin O., Schlaepfer E., et al. 2014. Optimization of critical hairpin features allows miRNA-based gene knockdown upon single-copy transduction. Mol. Ther.–Nucleic Acids. 3, e207.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Walker J.E., Chen R.X., McGee J., et al. 2012. Generation of an HIV-1-resistant immune system with CD34+ hematopoietic stem cells transduced with a triple-combination anti-HIV lentiviral vector. J. Virol. 86, 5719–5729.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Kustikova O.S., Wahlers A., Kühlcke K., et al. 2003. Dose finding with retroviral vectors: Correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population. Blood. 102, 3934–3937.CrossRefPubMedGoogle Scholar
  27. 27.
    Sakuma R., Noser J.A., Ohmine S., Ikeda Y. 2007. Rhesus monkey TRIM5α restricts HIV-1 production through rapid degradation of viral Gag polyproteins. Nat. Med. 13, 631–635.CrossRefPubMedGoogle Scholar
  28. 28.
    Cribbs A.P., Kennedy A., Gregory B., Brennan F.M. 2013. Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells. BMC Biotechnol. 13,98.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Bilal M.Y., Vacaflores A., Houtman J.C.D. 2015. Optimization of methods for the genetic modification of human T cells. Immunol. Cell Biol. 93, 896–908.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Richardson M.W., Carroll R.G., Stremlau M., et al. 2008. Mode of transmission affects the sensitivity of human immunodeficiency virus type 1 to restriction by rhesus TRIM5α. J. Virol. 82, 11117–11128.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Pleiades Publishing, Inc. 2018

Authors and Affiliations

  • D. O. Omelchenko
    • 1
  • D. V. Glazkova
    • 1
  • E. V. Bogoslovskaya
    • 1
  • F. A. Urusov
    • 1
  • Y. A. Zhogina
    • 1
  • G. M. Tsyganova
    • 1
  • G. A. Shipulin
    • 1
  1. 1.Central Research Institute of EpidemiologyMoscowRussia

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