Abstract
ABSTRACTExtracellular vesicles (EVs) or exosomes have been implicated in the pathophysiology of infections and cancer. The negative regulatory factor (Nef) encoded by simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) plays a critical role in the progression to AIDS and impairs endosomal trafficking. Whether HIV-1 Nef can be loaded into EVs has been the subject of controversy, and nothing is known about the connection between SIV Nef and EVs. We find that both SIV and HIV-1 Nef proteins are present in affinity-purified EVs derived from cultured cells, as well as in EVs from SIV-infected macaques. Nef-positive EVs were functional, i.e., capable of membrane fusion and depositing their content into recipient cells. The EVs were able to transfer Nef into recipient cells. This suggests that Nef readily enters the exosome biogenesis pathway, whereas HIV virions are assembled at the plasma membrane. It suggests a novel mechanism by which lentiviruses can influence uninfected and uninfectable, i.e., CD4-negative, cells.
Highlights
Extracellular vesicles (EVs) or exosomes have been implicated in the pathophysiology of infections and cancer
To test the hypothesis that negative regulatory factor (Nef) could be incorporated into EVs independently of other viral components, we transiently expressed the human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) Nef proteins in human embryonic kidney (HEK-293) cells
This phenotype was repeatable with cells transfected with hemagglutinin (HA) epitopetagged HIV Nef, demonstrating that Nef’s presence in EVs was not due to being tagged with a cytoplasmic protein such as green fluorescent protein (GFP) (Fig. S1)
Summary
Extracellular vesicles (EVs) or exosomes have been implicated in the pathophysiology of infections and cancer. We show that both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) incorporate the virusencoded Nef protein into EVs, including EVs circulating in the blood of SIV-infected macaques and that this presents a novel mechanism of Nef transfer to naive and even otherwise non-infectable cells. EVs package biologically active materials, such as enzymes, mRNAs, long non-coding RNAs, microRNAs (miRNAs), small-molecule metabolites, etc., and deliver them to recipient cells [2,3,4,5,6,7,8,9] Herpesviruses such as herpes simplex virus 1, Epstein-Barr virus, and Kaposi’s sarcoma-associated herpesvirus incorporate virus-encoded miRNA into EVs [2, 3, 10, 11]. Distinct pathophysiological changes, such as HIV-associated neurocognitive disorders, persist in latently infected individuals or in individuals on successful antiretroviral therapy (ART), implying an indirect mechanism of pathogenesis
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