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Isolation, Transfection, and Culture of Primary Human Monocytes
Published on: December 16, 2019
The HIV Nef protein modulates cellular and exosomal miRNA profiles in human monocytic cells
Madeeha Aqil1, Afsar Raza Naqvi1, Saurav Mallik2
1Virology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India;
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.
Insights
The HIV Nef protein alters microRNA (miRNA) secretion in exosomes, impacting viral pathogenesis. Nef dysregulates cellular miRNAs, promoting their exosomal release to influence host pathways and viral replication.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- The human immunodeficiency virus (HIV) Nef protein is a key virulence factor influencing cellular processes and is found in exosomes.
- While exosome proteomic profiles are known, the miRNA cargo of Nef-containing exosomes remains uncharacterized.
- Nef's role in suppressing RNA interference suggests a potential impact on miRNA export.
Purpose of the Study:
- To investigate the effect of HIV Nef expression on miRNA profiles within cells and secreted exosomes.
- To identify specific miRNAs dysregulated by Nef and their potential targets.
- To understand how Nef-mediated miRNA export contributes to HIV pathogenesis.
Main Methods:
- Purification of exosomes from U937 monocytic cells stably expressing HIV-1 Nef.
- Comprehensive miRNA profiling of cells and exosomes using Taqman Low Density Array (667 miRNAs).
- Validation of selected miRNAs and mRNA targets via quantitative RT-PCR and bioinformatics analysis.
Main Results:
- Nef expression significantly altered cellular miRNA populations.
- 47 miRNAs were selectively secreted into exosomes, while 2 were retained in Nef-expressing cells.
- Predicted exosomal miRNA targets included genes in inflammatory cytokine pathways and the HIV genome.
Conclusions:
- This study presents the first miRnome analysis of HIV Nef-expressing monocytes and their exosomes.
- HIV Nef induces large-scale miRNA dysregulation, including altered exosomal secretion.
- This represents a novel viral strategy to modulate pathogenesis and evade RNA interference for viral persistence.
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