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

Abstract

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.

Large‐scale, cross‐flow based isolation of highly pure and endocytosis‐competent extracellular vesicles

“…Isolation of EV from human plasma can present several issues that must be addressed such as: contamination with virus particles, contamination with high levels of lipoprotein-associated with LDLs and HDLs, and contamination with coagulation factors. To separate EV from viral particles an large lipoproteins, subsequent steps such as affinity purification using antibody coated beads should be employed [7,11]. …”

The Ambiguous Roles of Extracellular Vesicles in HIV Replication and Pathogenesis

“…On the other hand, the non-structural Nef protein, a major determinant of HIV-1 pathogenicity that manipulates protein trafficking in host cells (reviewed by Pereira and daSilva, 2016 ), is the most well-known HIV-1 factor released within EVs ( Campbell et al, 2008 ; Muratori et al, 2009 ; Lenassi et al, 2010 ; De Carvalho et al, 2014 ; McNamara et al, 2018 ). HEK293 cells expressing Nef-GFP secrete this fusion protein within EVs, which can be taken up by neighboring cells.…”

Extracellular Vesicles in Herpes Viral Spread and Immune Evasion

“…On the other hand, EVs may also transfer virus-encoded proteins and nucleic acids independently of the viral particles. For instance, both simian immunodeficiency virus (SIV) ( McNamara et al, 2018 ) and HIV-1 ( Lenassi et al, 2010 ; Pereira and daSilva, 2016 ; Puzar Dominkus et al, 2017 ) induce the release of Nef protein in exosomes; EBV LMP1 is also secreted in EVs ( Meckes et al, 2010 ; Nkosi et al, 2018 ) and functional exosomes containing miRNAs have been detected in human clinical samples and mouse models of KSHV-associated malignancies ( Chugh et al, 2013 ).…”