Nef Secretion into Extracellular Vesicles or Exosomes Is Conserved across Human and Simian Immunodeficiency Viruses

Extracellular 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. IMPORTANCE Extracellular vesicles (EVs) transfer biologically active materials from one cell to another, either within the adjacent microenvironment or further removed. EVs also package viral RNAs, microRNAs, and proteins, which contributes to the pathophysiology of infection. In this report, we show that both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) incorporate the virus-encoded 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. Nef is dispensable for viral replication but essential for AIDS progression in vivo. Demonstrating that Nef incorporation into EVs is conserved across species implicates EVs as novel mediators of the pathophysiology of HIV. It could help explain the biological effects that HIV has on CD4-negative cells and EVs could become biomarkers of disease progression.