EXTRACELLULAR VESICLES AND MICRORNA IN BIOFLUIDS, CELL CULTURE AND PRIMARY CELLS IN HEALTH AND HIV-1 AND SIV INFECTIONS

Extracellular vesicle (EV) research is a rapidly growing field, with renewed interest due in part to the discovery of the association of EVs with small extracellular RNAs (exRNA), in particular microRNAs. Roles of EVs and their RNA cargo in facilitating or fighting infection with viruses such as HIV-1 are likely but remain incompletely understood. To investigate one aspect through which EVs might play a role in infection, we first evaluated the EV and exRNA population of cervical-vaginal fluid in Asian macaques, initially in endometriosis and subsequently in animals with naturally suppressed SIV infections. Evaluating the miRNA cargo of EVs from infected animals relative to uninfected animals, we found that miR-186 was more abundant in the infected animals, suggesting it could be a potential restriction factor of SIV. We then used in vitro culture to establish the importance of EVs in HIV proliferation and maturation. We found that when EVs are removed from culture media, cell lines, either acutely or latently infected with HIV-1, experienced increased viral production. Virus produced by these cells was also more infectious than baseline. Restoration of cell-derived EVs to EV-depleted cell culture rescued virus production to normal levels. Finally, we investigated miRNA expression in different blood T-cell subsets of HIV-1 elite suppressors (ES), for comparison with those of healthy (i.e. uninfected) controls (HC) and HIV-1 chronic progressors (CP). EVs and cell-free exRNAs have also been collected from all donors. We found that several miRNA species were differentially regulated in T-cell types in ES compared with HC or CP, consistent with previous studies examining PBMCs. Interestingly, we also found that the miRNA profile of different classes of cells, i.e., Naïve, Central Memory or Effector Memory, clustered together irrespective of CD4+ or CD8+ phenotype in HC. These data taken together demonstrate the importance of EVs and their cargo in the context of retroviral infection and set the stage for future studies to further elucidate the complex relationship between HIV-1, miRNA and EVs