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. 2014 Mar 25:3.
doi: 10.3402/jev.v3.23129. eCollection 2014.

The HIV Nef protein modulates cellular and exosomal miRNA profiles in human monocytic cells

Madeeha Aqil  1 , Afsar Raza Naqvi  1 , Saurav Mallik  2 , Sanghamitra Bandyopadhyay  2 , Ujjwal Maulik  3 , Shahid Jameel  1
Affiliations

The HIV Nef protein modulates cellular and exosomal miRNA profiles in human monocytic cells

Madeeha Aqil et al. J Extracell Vesicles. .

Abstract

Introduction: The HIV Nef protein is a multifunctional virulence factor that perturbs intracellular membranes and signalling and is secreted into exosomes. While Nef-containing exosomes have a distinct proteomic profile, no comprehensive analysis of their miRNA cargo has been carried out. Since Nef functions as a viral suppressor of RNA interference and disturbs the distribution of RNA-induced silencing complex proteins between cells and exosomes, we hypothesized that it might also affect the export of miRNAs into exosomes.

Method: Exosomes were purified from human monocytic U937 cells that stably expressed HIV-1 Nef. The RNA from cells and exosomes was profiled for 667 miRNAs using a Taqman Low Density Array. Selected miRNAs and their mRNA targets were validated by quantitative RT-PCR. Bioinformatics analyses were used to identify targets and predict pathways.

Results: Nef expression affected a significant fraction of miRNAs in U937 cells. Our analysis showed 47 miRNAs to be selectively secreted into Nef exosomes and 2 miRNAs to be selectively retained in Nef-expressing cells. The exosomal miRNAs were predicted to target several cellular genes in inflammatory cytokine and other pathways important for HIV pathogenesis, and an overwhelming majority had targets within the HIV genome.

Conclusions: This is the first study to report miRnome analysis of HIV Nef expressing monocytes and exosomes. Our results demonstrate that Nef causes large-scale dysregulation of cellular miRNAs, including their secretion through exosomes. We suggest this to be a novel viral strategy to affect pathogenesis and to limit the effects of RNA interference on viral replication and persistence.

Keywords: HIV; Nef; exosomes; inflammatory cytokine; miRNA.

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Figures

Fig. 1
Fig. 1
Characterization of exosomes from U937/Nef-EYFP and U937/EYFP cells. (A) Electron microscopy of exosomes from U937/Nef-EYFP cells; bar 100 nm. (B) Flow cytometry of exosomes for the marker protein CD81. (C) Exosomes were labelled with lipid dye PKH26; these were (a, b) observed directly at 100X magnification, or (c, d) first bound to 4 μm latex beads and then observed at 100X magnification. The insets on the right show enlarged views of the images. (D) Amount of exosomes secreted by U937/EYFP and U937/Nef-EYFP cell lines presented as protein concentration from 1×106 cells. Data is presented as mean±SD from 3 independent experiments. The p-value calculated using Students t-test is shown.
Fig. 2
Fig. 2
Nef expression modulates cellular and exosomal miRNAs profiles. The miRNAs in (A) cells and (B) exosomes were profiled and relative fold-changes calculated as described in Methods. (C) The Venn diagrams show the intersection of detected miRNAs as follows: (i) Upregulated miRNAs in both U937/Nef-EYFP cells and exosomes; (ii) Downregulated miRNAs in U937/Nef-EYFP cells and upregulated miRNAs in U937/Nef-EYFP exosomes; (iii) Upregulated miRNAs in U937/Nef-EYFP cells and downregulated miRNAs in U937/Nef-EYFP exosomes.
Fig. 3
Fig. 3
Validation of differentially expressed miRNAs in cells and exosomes. Total RNA isolated from (A) cells and (B) exosomes was used to quantify expression levels of selected miRNAs as described in Methods. The data represents relative fold-increase±SD from 3 independent experiments; *p<0.05.
Fig. 4
Fig. 4
Nef induces the expression of proinflammatory cytokines in U937 cells. (A) RNA isolated from U937/Nef-EYFP and U937/EYFP cells were tested for the expression of selected cytokine genes by qRT-PCR; relative fold-changes were calculated from raw Ct values using the Data Assist Software (ABI). Error bars represent standard errors from 3 experiments, each with triplicate measurements; *p<0.05. (B) Western blots of lysates from U937/Nef-EYFP and U937/EYFP cells for IL1β and IL6 levels. Actin was used as a loading control.
Fig. 5
Fig. 5
HIV targeting host miRNAs are preferentially secreted into U937/Nef-EYFP exosomes. (A) Diagram showing the correlation between HIV targeting miRNAs and Nef exosomal miRNAs. All the detected miRNAs were screened for potential binding sites on 2 different HIV-1 genomes (NL4-3 and IndieC1) using the MicroInspector algorithm (http://bioinfo.uni-plovdiv.bg/microinspector/); the identified miRNAs were verified using the RNAHybrid algorithm (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/). (B) miRNAs present in exosomes that can potentially target the HIV-1 genome are separated based on whether their levels are higher or lower in Nef compared to control exosomes.
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