The microRNA miR-29a is associated with human immunodeficiency virus latency

Abstract

Background: Latent reservoirs of HIV-1 provide a major challenge to its cure. There are increasing reports of interplay between HIV-1 replication and host miRNAs. Several host miRNAs, which potentially target the nef-3'LTR region of HIV-1 RNA, including miR-29a, are proposed to promote latency.

Findings: We used two established cellular models of HIV-1 latency - the U1 monocytic and J1.1 CD4+ T cell lines to show an inverse relationship between HIV-1 replication and miR-29a levels, which was mediated by the HIV-1 Nef protein. Using a miR-29a responsive luciferase reporter plasmid, an expression plasmid and an anti-miR29a LNA, we further demonstrate increased miR-29a levels during latency and reduced levels following active HIV replication. Finally, we show that miR-29a levels in the PBMCs and plasma of HIV infected persons also correlate inversely with latency and active viral replication.

Conclusions: The levels of miR-29a correlate inversely with active HIV-1 replication in cell culture models and in HIV infected persons. This links miR-29a to viral latency and suggests another approach to activate and destroy latent HIV-1 reservoirs.

Figure 1

MiR-29a levels are inversely correlated with activation in cellular models of HIV latency. (A) The two cellular models of latency – U1 cells and J1.1 cells were activated with PMA, RNA was isolated and quantified for miR-29a levels as described in Materials and Methods. (B) U1 cells were treated with vehicle (DMSO) or PMA, and western blotting of cell lysates (upper panel) and culture supernatants (lower panel) was carried out with anti-p24 antibody. (C) U937 and Jurkat cell lines were activated with PMA, RNA was isolated and quantified for miR-29a levels as described in Materials and Methods. (D) U937 cells or stable U937 cell lines expressing either the HIV-1 Nef or Vpu protein were also used for quantifying miR-29a levels. Western blotting of the indicated cell lysates was carried out using anti-GFP antibodies. All data represents at least three independent experiments; *p < 0.05.

Figure 2

Functional correlation between miR-29a levels and HIV replication. (A) U1 cells and J1.1 cells were transfected with the pMIR-Report-Nef3′UTR reporter plasmid or the control pMIR-Report plasmid together with plasmid pRLTK. After 48 hr the cells were activated with PMA and the cell lysates quantified for luciferase activity as described in Materials and Methods. The luciferase activity in PMA activated cells is shown relative to unactivated cells after two normalizations - one with the Renilla luciferase activity to control for transfection efficiency and the other with luciferase activity observed with the control plasmid. Data represents three independent experiments; *p < 0.05. (B) J1.1 cells were transfected with the miR-29a-EGFP or EGFP expression vector. After 48 hr the cells were activated with PMA (or not) and HIV-1 production in culture supernatants was measured using a quantitative p24 ELISA. Transfection efficiency was around 30-40%. Data are shown as ng/ml of p24 and represent three independent experiments; the p-values for different sets are indicated. (C) U1 cells were transfected with a miR-29a specific LNA or a control LNA. After 48 hr HIV-1 production in culture supernatants was measured using a quantitative p24 ELISA. Data are shown relative to the EGFP control and represent three independent experiments; *p < 0.05. The western blot shows intracellular levels of Gag for the same cells; Actin was used as a loading control.

Figure 3

MiR-29a levels in HIV-infected persons correlate with disease stage. A cohort of HIV-infected persons was categorized into two groups – asymptomatic and symptomatic based on their CD4 counts. (A) PBMCs and (B) plasma from this cohort as well as healthy persons were quantified for miR-29a levels as described in Materials and Methods. The normalized 1/Ct values are plotted for each individual sample. The p values are shown.