Abstract
Extracellular vesicles (EVs) and their contents (proteins, lipids, messenger RNA, microRNA, and DNA) are viewed as intercellular signals, cell-transforming agents, and shelters for viruses that allow both diagnostic and therapeutic interventions. EVs circulating in the blood of individuals infected with human immunodeficiency virus (HIV-1) may provide insights into pathogenesis, inflammation, and disease progression. However, distinguishing plasma membrane EVs from exosomes, exomeres, apoptotic bodies, virions, and contaminating proteins remains challenging. We aimed at comparing sucrose and iodixanol density and velocity gradients along with commercial kits as a means of separating EVs from HIV particles and contaminating protein like calprotectin; and thereby evaluating the suitability of current plasma EVs analysis techniques for identifying new biomarkers of HIV-1 immune activation. Multiple analysis have been performed on HIV-1 infected cell lines, plasma from HIV-1 patients, or plasma from HIV-negative individuals spiked with HIV-1. Commercial kits, the differential centrifugation and density or velocity gradients to precipitate and separate HIV, EVs, and proteins such as calprotectin, have been used. EVs, virions, and contaminating proteins were characterized using Western blot, ELISA, RT-PCR, hydrodynamic size measurement, and enzymatic assay. Conversely to iodixanol density or velocity gradient, protein and virions co-sedimented in the same fractions of the sucrose density gradient than AChE-positive EVs. Iodixanol velocity gradient provided the optimal separation of EVs from viruses and free proteins in culture supernatants and plasma samples from a person living with HIV (PLWH) or a control and revealed a new population of large EVs enriched in microRNA miR-155 and mitochondrial DNA. Although EVs and their contents provide helpful information about several key events in HIV-1 pathogenesis, their purification and extensive characterization by velocity gradient must be investigated thoroughly before further use as biomarkers. By revealing a new population of EVs enriched in miR-155 and mitochondrial DNA, this study paves a way to increase our understanding of HIV-1 pathogenesis.
Highlights
Extracellular vesicles (EVs) are heterogeneous groups of vesicles found in most biological fluids
We have reported previously that plasma EVs in ART-naïve HIV-1 patients were rich in miR-92, miR-155, and miR-223 and that the abundance of miR-155 and miR-223 was strongly correlated with EVs size and AChE activity [5]
This study showed that the sucrose density gradient or commercial kits are inadequate for this purpose and concentrate materials since virus and EVs are present in the same fractions
Summary
Extracellular vesicles (EVs) are heterogeneous groups of vesicles found in most biological fluids. These components include microRNAs [7], signaling, and apoptotic proteins from the cells of origin and sometimes viral proteins and genetic material [5,8,9,10,11] Based on their biogenesis, release pathways, size, content, and functions, EVs are classified into different subsets; namely exosomes, microvesicles (MVs), and apoptotic bodies (ApoBDs). Founded in 2012, the International Society for Extracellular Vesicles (ISEV) has contributed significantly to establishing guidelines in this new research field and has helped with classification but many challenges remain to address when it comes to their distinction from viral particles [13] We address these issues in the analysis of EVs from plasma in the context of HIV-1 infection
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
