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

Background: HIV-1 proteins and RNA are packaged into extracellular vesicles (EVs) through interaction with the multivesicular endosomes of the EV biogenesis machinery. This interaction also allows functional or abortive viruses to exit the cells as exosomes, microvesicles or apoptotic vesicles. HIV-1 viral particles and EVs share significant similarities in size, composition, and molecular cargo, making their separation challenging. Current HIV-1 purification methods neglect the effects of EVs on virus infectivity, which could influence experiment outcomes. Here, we co-characterized HIV-1 particles co-purified with exosomes, microvesicles and apoptotic vesicles to determine their impact on HIV-1 infection. Methods: 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 protein and HIV-1 RNA associated with proteins outside the EVs. The supernatants were fractionated into three pellets by differential centrifugation. The 3K pellet contained the largest EVs, such as apoptotic vesicles. The 17K and 100K pellets were respectively associated with microvesicles and exosomes. 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. Results: Proteinase K treatment notably lowered HIV-1 RNA concentration in the EV pellet and did not affect viral p24 capsid protein 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 (measured by p24) from each pellet. Conclusion: Productively infected cells released functional viruses in the three EV subtypes, each exerting a distinct effect on virus infectivity. For future experiments, the presence of EVs in viral preparations should be taken into account, as they influence the progression of HIV-1 infection.