Characterization and Identification of Large Extracellular Vesicles Released by HIV-1 Infected Myeloid and T-cells



Dehbandi, Fatemeh

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The mounting evidences suggest that extracellular vesicles (EVs) contribute to HIV-1 pathogenesis, which is likely attributed to their ability to carry viral proteins and RNA. Previously we were able to investigate the role of EVs in viral infections [1] [2] [3] [4] [5]. Since EVs are heterogenous in their sizes and intracellular origin [6], we have attempted to investigate the role of large EVs in HIV-1 infection. HIV-1 infected monocytes and T cells were synchronized in G0 phase by serum starvation for 72 hours. Afterwards, cells were released in serum-rich media containing inducers to initiate viral transcription and resume normal cell cycle. The supernatant samples were collected at prior to release (-72 hours), and then, 6 (+6 hours) and 24 (+24 hours) post release. To enrich the samples for large EVs, the supernatants were centrifuged at 2000g (2K) speed for 45 minutes and separated through size exclusion chromatography and tested for viral proteins and RNAs. As a result, 2K fractions from both infected myeloid and T-cells showed the presence of HIV-1 capsid and envelope proteins, as well as HIV-1 short non-coding RNA (TAR). Next, the isolated large EVs from both infected myeloid and T-cells were subjected to virus rescue assay on uninfected naïve myeloid and T-cells. The cells were lysed and tested on the presence of viral proteins. As a result, previously uninfected myeloid and T-cells showed the production of viral proteins after the treatment by 2K EVs collected from -72 hours samples and +24 hrs. The cell treated by +6 hours, on the other hand, did not produce viral proteins. Taken together, 2K fraction from HIV-1 infected myeloid and T-cells, collected at 24 hours post release and from serum-deprived cells, are infectious and contain fully fledged virions. However, large EVs collected at 6 hours post-release failed to initiate viral infection. Since, 2K samples, including non-infectious ones, contain short non-coding HIV-1 RNA (TAR), they might play a major role in bystander effect on uninfected neighboring cells, that was reported previously [1].



HIV-1 infected Extracellular vesicles, Amphisome, Izon Column, Large EVs characterization, Size exclusion chromatography