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.