Fatah Kashanchi,
Professor and Director of Research, Lab of Molecular Virology,
George Mason University
For the past eighteen years Kashanchi lab has been interested in understanding the mechanism of viral gene expression in human viruses and how the virus and the host control the dynamics of fundamental machineries needed for viral replication and/or host survival. They also have ample experience with biochemical pathways that leads to transcription and chromatin remolding using in vitro reconstituted machineries. These complexes with epigenetic modifications utilize host signaling events and therapeutic targets that control viral replication. In recent years, they have also started focusing on Extracellular vesicles (i.e., exosomes) mainly from latent virally infected cells. These cells remain in the body for a long period of time can be extended to the life of a person (i.e., CNS cells). These latent cells produce exosomes that carry markers of the infection including RNA and protein sequences specific to a given virus. The lab for the first time showed that viral release and exosome release have overlapping biogenesis in the ESCRT pathway. For instance, HIV-1 latent cells utilize ESCRT-I for viral release, and ESCRT-II for exosomal release. Using in vitro and in vivo (both patient samples and animal models), the lab has found that exosomes from HIV-1 infected cells carry short non-coding RNAs (i.e., TAR) which regulate TLR3 and other pathways in the recipient cells. Similar results were also observed from other neuro-tropic RNA viral infections including HTLV-1, Ebola, RVFV, SARS, and Zika infection.
Extracellular Vesicle Isolation Methods Identify Distinct HIV-1 Particles Released from Chronically Infected T-cells
Thursday, 4 April 2024 at 17:00
Add to Calendar ▼2024-04-04 17:00:002024-04-04 18:00:00Europe/LondonExtracellular Vesicle Isolation Methods Identify Distinct HIV-1 Particles Released from Chronically Infected T-cellsExtracellular Vesicles (EVs) and Nanoparticles 2024: Diagnostics, Delivery, Therapeutics in Miami, FloridaMiami, FloridaSELECTBIOenquiries@selectbiosciences.com
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.
