Abstract
One of the main methods to generate the HIV reservoir is during the transition of infected activated effector CD4 T cells to a memory phenotype. The QUECEL (Quiescent Effector Cell Latency) protocol mimics this process efficiently and allows for production of large numbers of latently infected CD4+ T cells. After polarization and expansion, CD4+ T cells are infected with a single round reporter virus which expressed GFP/CD8a. The infected cells are purified and coerced into quiescence using a defined cocktail of cytokines including TGF-β, IL-10, and IL-8, producing a homogeneous population of latently infected cells. Since homogeneous populations of latently infected cells can be recovered, the QUECEL model has an excellent signal-to-noise ratio, and has been extremely consistent and reproducible in numerous experiments performed during the last 5 years. The ease, efficiency, and accurate mimicking of physiological conditions make the QUECEL model a robust and reproducible tool to study the molecular mechanisms underlying HIV latency.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Finzi D, Blankson J, Siliciano JD, Margolick JB, Chadwick K, Pierson T, Smith K, Lisziewicz J, Lori F, Flexner C et al (1999) Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med 5:512–517
Yukl SA, Gianella S, Sinclair E, Epling L, Li Q, Duan L, Choi AL, Girling V, Ho T, Li P et al (2010) Differences in HIV burden and immune activation within the gut of HIV-positive patients receiving suppressive antiretroviral therapy. J Infect Dis 202:1553–1561
Pearson R, Kim YK, Hokello J, Lassen K, Friedman J, Tyagi M, Karn J (2008) Epigenetic silencing of human immunodeficiency virus (HIV) transcription by formation of restrictive chromatin structures at the viral long terminal repeat drives the progressive entry of HIV into latency. J Virol 82:12291–12303
Jadlowsky JK, Wong JY, Graham AC, Dobrowolski C, Devor RL, Adams MD, Fujinaga K, Karn J (2014) The negative elongation factor (NELF) is required for the maintenance of proviral latency but does not induce promoter proximal pausing of RNAP II on the HIV LTR. Mol Cell Biol 34:1911–1928
Jordan A, Bisgrove D, Verdin E (2003) HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. EMBO J 22:1868–1877
Williams SA, Chen LF, Kwon H, Ruiz-Jarabo CM, Verdin E, Greene WC (2006) NF-kappaB p50 promotes HIV latency through HDAC recruitment and repression of transcriptional initiation. EMBO J 25:139–149
Spina CA, Anderson J, Archin NM, Bosque A, Chan J, Famiglietti M, Greene WC, Kashuba A, Lewin SR, Margolis DM et al (2013) An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog 9:e1003834
Nguyen K, Das B, Dobrowolski C, Karn J (2017) Multiple histone lysine methyltransferases are required for the establishment and maintenance of HIV-1 latency. MBio 8:e00133-17
Kim YK, Mbonye U, Hokello J, Karn J (2011) T-cell receptor signaling enhances transcriptional elongation from latent HIV proviruses by activating P-TEFb through an ERK-dependent pathway. J Mol Biol 410:896–916
Kulpa DA, Talla A, Brehm JH, Ribeiro SP, Yuan S, Bebin-Blackwell A-G, Miller M, Barnard R, Deeks SG, Hazuda D et al (2019) Differentiation into an effector memory phenotype potentiates HIV-1 latency reversal in CD4+ T cells. J Virol 93:e00969–e00919
Shan L, Deng K, Gao H, Xing S, Capoferri AA, Durand CM, Rabi SA, Laird GM, Kim M, Hosmane NN et al (2017) Transcriptional reprogramming during effector-to-memory transition renders CD4(+) T cells permissive for latent HIV-1 infection. Immunity 47:766–775.e763
Cameron PU, Saleh S, Sallmann G, Solomon A, Wightman F, Evans VA, Boucher G, Haddad EK, Sekaly RP, Harman AN et al (2010) Establishment of HIV-1 latency in resting CD4+ T cells depends on chemokine-induced changes in the actin cytoskeleton. Proc Natl Acad Sci U S A 107:16934–16939
Hakre S, Chavez L, Shirakawa K, Verdin E (2012) HIV latency: experimental systems and molecular models. FEMS Microbiol Rev 36:706–716
Bosque A, Planelles V (2008) Induction of HIV-1 latency and reactivation in primary memory CD4+ T cells. Blood 113:58–65
Bosque A, Planelles V (2011) Studies of HIV-1 latency in an ex vivo model that uses primary central memory T cells. Methods 53:54–61
Tyagi M, Pearson RJ, Karn J (2010) Establishment of HIV latency in primary CD4+ cells is due to epigenetic transcriptional silencing and P-TEFb restriction. J Virol 84:6425–6437
Pace MJ, Graf EH, Agosto LM, Mexas AM, Male F, Brady T, Bushman FD, O’Doherty U (2012) Directly infected resting CD4+T cells can produce HIV gag without spreading infection in a model of HIV latency. PLoS Pathog 8:e1002818
Agosto LM, Herring MB, Mothes W, Henderson AJ (2018) HIV-1-infected CD4+ T cells facilitate latent infection of resting CD4+ T cells through cell-cell contact. Cell Rep 24:2088–2100
Dobrowolski C, Valadkhan S, Graham AC, Shukla M, Ciuffi A, Telenti A, Karn J (2019) Entry of polarized effector cells into quiescence forces HIV latency. MBio 10:e00337-19
Das B, Dobrowolski C, Luttge B, Valadkhan S, Chomont N, Johnston R, Bacchetti P, Hoh R, Gandhi M, Deeks SG et al (2018) Estrogen receptor-1 is a key regulator of HIV-1 latency that imparts gender-specific restrictions on the latent reservoir. Proc Natl Acad Sci U S A 115:E7795–E7804
Das B, Dobrowolski C, Shahir AM, Feng Z, Yu X, Sha J, Bissada NF, Weinberg A, Karn J, Ye F (2015) Short chain fatty acids potently induce latent HIV-1 in T-cells by activating P-TEFb and multiple histone modifications. Virology 474:65–81
Bosque A, Famiglietti M, Weyrich AS, Goulston C, Planelles V (2011) Homeostatic proliferation fails to efficiently reactivate HIV-1 latently infected central memory CD4+ T cells. PLoS Pathog 7:e1002288
Ivanov II, RdL F, Manel N, Yoshinaga K, Rifkin DB, Sartor RB, Finlay BB, Littman DR (2008) Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. Cell Host Microbe 4:337–349
Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ, Cua DJ, Littman DR (2006) The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126:1121–1133
Sun H, Kim D, Li X, Kiselinova M, Ouyang Z, Vandekerckhove L, Shang H, Rosenberg ES, Yu XG, Lichterfeld M (2015) Th1/17 polarization of CD4 T cells supports HIV-1 persistence during antiretroviral therapy. J Virol 89:11284–11293
Friedman J, Cho WK, Chu CK, Keedy KS, Archin NM, Margolis DM, Karn J (2011) Epigenetic silencing of HIV-1 by the histone H3 lysine 27 methyltransferase enhancer of Zeste 2 (EZH2). J Virol 85:9078–9089
Mbonye UR, Gokulrangan G, Datt M, Dobrowolski C, Cooper M, Chance MR, Karn J (2013) Phosphorylation of CDK9 at Ser175 enhances HIV transcription and is a marker of activated P-TEFb in CD4(+) T lymphocytes. PLoS Pathog 9:e1003338
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Shukla, M., Kizito, F., Mbonye, U., Nguyen, K., Dobrowolski, C., Karn, J. (2022). A Reliable Primary Cell Model for HIV Latency: The QUECEL (Quiescent Effector Cell Latency) Method. In: Poli, G., Vicenzi, E., Romerio, F. (eds) HIV Reservoirs. Methods in Molecular Biology, vol 2407. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1871-4_5
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1871-4_5
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1870-7
Online ISBN: 978-1-0716-1871-4
eBook Packages: Springer Protocols