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. 2016 Feb;32(2):187-93.
doi: 10.1089/aid.2015.0106. Epub 2015 Jul 14.

Modeling HIV-1 Latency in Primary T Cells Using a Replication-Competent Virus

Laura J Martins  1 , Pawel Bonczkowski  2 , Adam M Spivak  3 , Ward De Spiegelaere  2 , Camille L Novis  1 , Ana Beatriz DePaula-Silva  1 , Eva Malatinkova  2 , Wim Trypsteen  2 , Alberto Bosque  1 , Linos Vanderkerckhove  2 , Vicente Planelles  1
Affiliations

Modeling HIV-1 Latency in Primary T Cells Using a Replication-Competent Virus

Laura J Martins et al. AIDS Res Hum Retroviruses. 2016 Feb.

Abstract

HIV-1 latently infected cells in vivo can be found in extremely low frequencies. Therefore, in vitro cell culture models have been used extensively for the study of HIV-1 latency. Often, these in vitro systems utilize defective viruses. Defective viruses allow for synchronized infections and circumvent the use of antiretrovirals. In addition, replication-defective viruses cause minimal cytopathicity because they fail to spread and usually do not encode env or accessory genes. On the other hand, replication-competent viruses encode all or most viral genes and better recapitulate the nuances of the viral replication cycle. The study of latency with replication-competent viruses requires the use of antiretroviral drugs in culture, and this mirrors the use of antiretroviral treatment (ART) in vivo. We describe a model that utilizes cultured central memory CD4(+) T cells and replication-competent HIV-1. This method generates latently infected cells that can be reactivated using latency reversing agents in the presence of antiretroviral drugs. We also describe a method for the removal of productively infected cells prior to viral reactivation, which takes advantage of the downregulation of CD4 by HIV-1, and the use of a GFP-encoding virus for increased throughput.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Generation of cultured TCM cells latently infected with HIV-1NL4-3. (A) Protocol used for the generation of latently infected cultured central memory T cell subset (TCM) cells using HIV-1NL4-3. From days 0 to 3, naive CD4 T cells were activated with αCD3/αCD28 beads in the presence of αIL-4, αIL-12, and tumor growth factor (TGF)-β1. From days 3 to 7, cultured TCM cells proliferated rapidly and were maintained from 1–3×106 cells/ml. On day 7, cells were infected with HIV-1NL4-3 and from days 7–10, cells were cultured in standard tissue culture flasks. From days 10 to 13, a crowded condition was imposed by culturing in U-bottom 96-well plates. From days 13 to 17, cells were cultured in standard tissue culture flasks in the presence of antiretroviral treatment (ART). (B) Cells from a single blood donor (Donor 5) were cultured and infected with HIV-1NL4-3 following Protocol B. On day 17, CD4+ cells were isolated using positive magnetic selection. Cells before isolation are denoted UPL and purified cells are denoted UL. Cells were stained with a cell viability dye followed by cell-surface staining with a CD4-APC antibody then stained intracellularly with a p24-FITC antibody. Dot plots of the viable fraction are shown. (C) Cultured TCM cells latently infected with HIV-1NL4-3 were generated as indicated in (A). On day 17 HIV-1-infected cells containing uninfected, productively infected, and latently infected cells (UPL) were subjected to magnetic isolation based on cell-surface CD4 expression. CD4+ cells contain uninfected and latently infected cells (UL) and CD4− cells contain productively infected cells (P). The UL fraction was treated with either interleukin (IL)-2 alone or IL-2+αCD3/αCD28 for 48 h.
<b>FIG. 2.</b>
FIG. 2.
Reactivation of HIV-1 from latently infected cultured TCM cells. (A) Fourteen CD4+ purified samples were treated with IL-2 alone or IL-2+αCD3/αCD28 for 48 h. ICp24 was analyzed by flow cytometry. Significance was calculated using a two-tailed paired t-test analysis (p values provided). Asterisk indicates data corresponding to dot plot figures in (B). (B) Representative dot plots of IL-2 and IL-2+αCD3/αCD28-stimulated UL fractions. (C) Four CD4+ purified samples were treated with IL-2 alone or IL-2+αCD3/αCD28 for 48 h. CA HIV-1 RNA copies were measured by quantitative polymerase chain reaction (qPCR) in triplicate samples. Normalization of cell-associated HIV-1 RNA to a cellular RNA would not be feasible for the comparison of HIV-1 transcripts produced from quiescent cells to those generated from cells treated with a strong cell activation stimulus. We, therefore, report HIV-1 RNA values normalized to input cell number. Mean values are plotted and error bars denote standard deviations. (D) UL fractions were stimulated with 330 nM SAHA, 10 μg/ml PAM3CSK4, 100 nM bryostatin-1, 100 nM ingenol 3,20-dibenzoate and ICp24 and viability from Donor 5-7 (E) were measured using flow cytometry. Significance was calculated using a two-tailed paired t-test analysis (p values provided).
<b>FIG. 3.</b>
FIG. 3.
Generation of cultured TCM cells latently infected with HIV-1 NLENG1-IRES and reactivation of latent HIV-1. (A) Plasmid used for the generation of latently infected cells with an EGFP reporter. (B) Protocol for the generation of cultured TCM cells latently infected with HIV-1 NLENG1-IRES. (C) Cells were cultured and infected with HIV-1 NLENG1-IRES. On day 14, cultures were treated with IL-2 alone or IL-2+αCD3/αCD28 or IL-2+PHA. EGFP expression was measured using flow cytometry on day 16. Representative dot plots are shown for IL-2 and IL-2+αCD3/αCD28-treated cultures.
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