Characterization of HIV‐1 Particles Co‐Purified With Three Extracellular Vesicle Subtypes From the Raji CD4 DCIR Cell Line, a Hybrid Model of CD4 T Cells and Dendritic Cells

HIV-1 proteins and RNA are incorporated into extracellular vesicles (EVs) via the EV biogenesis machinery. Due to their similar size and content, EVs and HIV-1 particles are hard to separate, and current purification methods often overlook EVs' effects on infectivity. This study co-characterized HIV-1 particles and three EV subtypes to assess their impact on infection. The HIV-infected Raji CD4 DCIR cells' supernatants were harvested 2 and 8 days after infection. The 2-day supernatant was treated with proteinase K to discard viral components outside the EVs. The supernatants were fractionated into three pellets by differential centrifugation: 3K, 17K and 100K. EVs and viral particles were co-characterized for their host and viral contents and the pellets obtained after 8 days post-infection were tested for infectivity. Proteinase K reduced HIV-1 RNA in EVs without affecting p24 concentration. The p24 protein was mostly found in the 17K pellet and HIV-1 RNA was the most abundant in the 100K pellet for both 2- and 8-day productions. Nevertheless, the 3K pellet had the highest infectivity when cells were infected with an equal quantity of virus. Each EV subtype were co-purified with functional virus and uniquely influenced HIV-1 infectivity, underscoring the importance of considering EVs in viral preparations.