Establishment, Persistence, and Reactivation of Latent HIV-1 Infection in Renal Epithelial Cells

Abstract

HIV-1 persistence in different cell types presents the main obstacle to an HIV-1 cure. We have previously shown that the renal epithelium is a site of HIV-1 infection and that the kidney represents a separate viral compartment from blood. Whether renal cells can harbor latent virus that can be reactivated upon treatment with latency reversing agents (LRAs) is unknown. To address this question, we developed an in vitro HIV-1 latency model in renal tubule epithelial (RTE) cells using a dual color HIV-1 reporter virus, R7/E-/GFP/EF1a-mCherry (R7GEmC), and evaluated the effect of LRAs, both as single agents and in combination, on viral reactivation. Our data show that HIV-1 can establish latency in RTE cells early postinfection. While the pool of latently infected cells expanded overtime, the percentage of productively infected cells declined. Following LRA treatment only a small fraction of latently infected cells, both T cells and RTE cells, could be reactivated, and the drug combinations more effective in reactivating HIV transcription in RTE cells differed from those more active in T cells. Our study demonstrates that HIV can establish latency in RTE cells and that current LRAs are only marginally effective in inducing HIV-1 reactivation. This suggests that further study of LRA dynamics in non-T cells may be warranted to assess the suitability of LRAs as a sterilizing cure strategy. IMPORTANCE Anti-retroviral therapy (ART) has dramatically reduced HIV-related morbidity and mortality. Despite this success, a number of challenges remain, including the long-term persistence of multiple, clinically latent viral reservoirs capable of reactivation in the absence of ART. As efforts proceed toward HIV eradication or functional cure, further understanding of the dynamics of HIV-1 replication, establishment of latency and mechanisms of reactivation in reservoirs harboring the virus throughout the body is necessary. HIV-1 can infect renal epithelial cells and the expression of viral genes in those cells contributes to the development of HIV associated nephropathy (HIVAN) in untreated individuals. The significance of our work is in developing the first model of HIV-1 latency in renal epithelial cells. This model enhances our understanding of HIV-1 latency and persistence in the kidney and can be used to screen candidate latency reversing agents.

Keywords: HIV-1; LRAs; latency; renal epithelial cells; reservoir.

FIG 1

HIV-1 Latency is established early in renal cells. (a) The renal tubule epithelial Hpt1-b and the Jurkat T cell lines were infected with 1 multiplicity of infection (MOI) of the VSV-G pseudotyped R7GEmC construct and followed over time by fluorescence microscopy. (b) Time course FACS analysis of Hpt1-b cells infected with 0.1 or 0.5 MOI of R7GEmC. Expression of GFP and mCherry was analyzed at day 3, 7 and 14 postinfection. Representative of 3 independent experiments.

FIG 2

Long term persistence of HIV-1 latency in renal epithelial cells. Gating strategy for flow cytometry sort of latently infected and actively infected Hpt1-b renal epithelial cells (day 5 postinfection). Uninfected cells were used as negative control. Positive controls include Hpt1-b cells stably transduced with a lentiviral vector expressing either GFP or mCherry. (b) Flow-sorted primary renal cells (HRCEp cells) were reseeded and the expression of mCherry and GFP was analyzed at 2 weeks post-sort in both the latent (top panel) and active cells (bottom panel). (c) Flow-sorted mCherry only (latent) Hpt1-b cells were cultured for 12 weeks postinfection. mCherry expression could be detected in about half of the cells, while the other half did not express any fluorescent marker. No GFP expression (active infection) was detected at this late time point. Each panel represents a separate experiment. (d) The presence of the HIV-R7GEmC construct DNA was assessed in the four populations of flow-sorted Hpt1-b cells: GFP only, GFP/mCherry double positive (DP), latently infected (mCherry only) and double negative (DN). Serial dilutions of genomic DNA extracted from Hpt-1b cells stably transduced with a lentiviral vector expressing mCherry were used to generate a standard curve. The amount of DNA and the corresponding number of cells used to generate the standard curve are indicated. Uninfected Hpt1-b cells were included as negative control. NTC = no template control.

FIG 3

Moderate HIV-1 reactivation following LRAs treatment of latently infected Hpt1-b renal cells. Latently infected renal cells were flow sorted 2 weeks postinfection and cultured for an additional week before LRAs treatment. Cells were then treated for 24 h with the indicated LRAs or DMSO either once (a) or twice (b). FACS analyses of representative single and double treatments are shown. Histograms represent the fold change in GFP expression compared to untreated controls for the single (c) or double treatments (d). Bars represent mean+SD of the percentage of GFP+ population in three independent experiments. Asterisks indicate a significant difference (P value <0.05; nonparametric Kruskall-Wallis test) between LRA treated cells and DMSO.

FIG 4

Moderate HIV-1 reactivation following LRAs treatment of latently infected Jurkat T cells. Latently infected Jurkat T cells were flow sorted 2 weeks postinfection and cultured for an additional week before LRAs treatment. Cells were then treated for 24 h with the indicated LRAs or DMSO either once (a) or twice (b). FACS analyses of representative single and double treatments are shown. Histograms represent the fold change in GFP expression compared to untreated controls for the single (c) or double treatments (d). (e) Heatmap comparing fold change in GFP expression for both Hpt1-b and Jurkat cells. Bars represent mean+SD of the percentage of GFP+ population in three independent experiments. Asterisks indicate a significant difference (P value <0.05; nonparametric Kruskall-Wallis test) between LRA treated cells and DMSO.

FIG 5

HIV-1 reactivation following JQ1 treatment of latently infected primary HRCEp cells. Latently infected primary HRCEp cells were flow sorted 11 days postinfection and cultured for an additional 8 days before treatment with JQ1. (a) Gating strategy for flow cytometry sort of latently infected HRCEp cells (day 11 postinfection). (b) Latently infected (mCherry only) flow-sorted HRCEp cells were reseeded and the expression of mCherry and GFP was analyzed at 8 days post-sort. Cells were then treated twice with JQ1 for 24 h and imaged 24 h after the first treatment, and 24 and 48 h after the second treatment. (b) Shown are images of mCherry and GFP positive HRCEp cells from eight separate fields taken at the indicated time points from untreated and JQ1 treated cells. Number of GFP+ cells present in each field are indicated in the top left corner of each image. (c) Fold change in number of GFP+ cells in untreated and JQ1 treated cells at the indicated time points.

FIG 6

Moderate HIV-1 reactivation following SMACm treatment of latently infected Hpt1-b renal cells and Jurkat T cells. Latently infected Hpt-1b renal cells or Jurkat T cells were flow sorted 2 weeks postinfection and cultured for an additional week before SMACm treatment. Cells were then treated for 24 h with SMACm, JQ1 or DMSO either once (a–c) or twice (b–d). Bars represent mean+SD of the percentage of GFP+ population in three independent experiments. Asterisks indicate a significant difference (P value <0.05; nonparametric Kruskall-Wallis test) between LRA treated cells and DMSO.