Abstract

Extracellular Vesicle Isolation Methods Identify Distinct HIV-1 Particles Released from Chronically Infected T-cells

Fatah Kashanchi, Professor, George Mason University

In 2022, 1.5 million people acquired Human Immunodeficiency Virus (HIV-1), and an estimated 37.7 million individuals lived with HIV-1 (PLWH) worldwide. While combination antiretroviral therapy suppresses viral replication, it does not silence viral transcription. We have identified presence of HIV-1 products, including non-coding viral RNA and proteins, within extracellular vesicles (EVs). These EVs are not infectious and can be isolated from cell culture supernatants of HIV-1 chronically infected cell lines and biofluids. Here we expanded upon a sequential differential ultracentrifugation (DUC) method by employing higher g-force with longer spin times to recover smaller EVPs (<100 nm) and have found presence of virus in both large and very small EVPs. Furthermore, we modified a virus recovery assay which indicates that these EVPs were infectious, including the novel EVPs under 100 nm. Standard assays for EV characterizations were used for validation.  Data was further validated using filtrations and other methods of EV purification. Viral and EV markers were used to quantify each prep. Collectively, we identified unique, infectious particles smaller than the currently accepted size for HIV-1. This methodology may be employed for other viruses or infectious agents where EVPs may impact disease progression by transmitting highly replicating virulent nucleic acids.