TY - JOUR T1 - Serum extracellular vesicle depletion processes affect release and infectivity of HIV-1 in culture JF - bioRxiv DO - 10.1101/081687 SP - 081687 AU - Zhaohao Liao AU - Dillon C. Muth AU - Erez Eitan AU - Meghan Travers AU - Elin Lehrmann AU - Kenneth W. Witwer Y1 - 2016/01/01 UR - http://biorxiv.org/content/early/2016/12/08/081687.abstract N2 - Background Extracellular vesicles (EVs) are involved in intercellular communication in health and disease and affect processes including immune and antiviral responses. We and others have previously demonstrated that ultracentrifuged serum is depleted of EVs and, when used in cell culture media, is associated with declines in growth and viability of some cell types. Although EVs had been reported to enhance or interfere with HIV-1 infection, depending on the setting, the effects of EVs on HIV-1 production and infectivity of released virions remain largely unknown. In this study, we examined the effects of serum EV depletion processes on HIV-1 replication in primary cells and cell lines, including two HIV-1 latency models.Methods Cell culture media were prepared with EV-replete fetal bovine serum (FBS) or serum depleted of EVs via ultracentrifugation or a proprietary method (ThermoFisher/Gibco). T-cell and myeloid-lineage cell lines, including ACH-2 and U1 HIV-1 latency models, and primary cells were grown in 10% FBS-based culture media. Cell counts, viability, and proliferation were assessed throughout. HIV-1 production and infectivity were measured by p24 ELISA and luciferase reporter cell lines, respectively. Flow cytometry, Seahorse assays, and miRNA and mRNA expression arrays were done to assess cellular responses to EV-depleted conditions.Results Increases in HIV-1 production were observed in certain EV-depleted conditions, along with, in some cases, morphology changes and decreased cell viability. Add-back of ultracentrifuge pellets reduced HIV-1 production almost to baseline. Primary cells appeared to be less sensitive to EV depletion. ACH-2 and U1 latency models also produced more HIV-1 under EV-depleted conditions. Virus produced under processed serum conditions was also more infectious. Finally, changes in cellular metabolism and gene expression were associated with EV-depleted culture conditions.Conclusions The EV environment of HIV-1 infected cells appears to have a significant effect on virus production and infectivity. In cell lines of HIV-latency, significantly higher concentrations of p24 were observed in those cells cultured in EVD conditions. EV-dependence of cell cultures should be examined carefully prior to examining additional experimental variables. However, we would also sound a cautionary note that EVs are unlikely to be the only particles depleted by ultracentrifugation or other processes. Effects of EVs may be accompanied by or confused with the effects of closely associated or physically similar particles. ER -