Abstract
HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Several recent studies including ours have demonstrated that Nef can be transferred to neighboring cells and alters the function of these cells. However, how the intercellular Nef transfer occurs is in dispute. In the current study, we attempted to address this important issue using several complementary strategies, a panel of exosomal markers, and human CD4+ T lymphocyte cell line Jurkat and a commonly used cell line 293T. First, we showed that Nef was transferred from Nef-expressing or HIV-infected Jurkat to naïve Jurkat and other non-Jurkat cells and that the transfer required the membrane targeting function of Nef and was cell density-dependent. Then, we showed that Nef transfer was cell-cell contact-dependent, as exposure to culture supernatants or exosomes from HIV-infected Jurkat or Nef-expressing Jurkat and 293T led to little Nef detection in the target cells Jurkat. Thirdly, we demonstrated that Nef was only detected to be associated with HIV virions but not with acetylcholinesterase (AChE+) exosomes from HIV-infected Jurkat and not in the exosomes from Nef-expressing Jurkat. In comparison, when it was over-expressed in 293T, Nef was detected in detergent-insoluble AChE+/CD81low/TSG101low exosomes, but not in detergent-soluble AChE-/CD81high/TSG101high exosomes. Lastly, microscopic imaging showed no significant Nef detection in exosomal vesicle-like structures in and out 293T. Taken together, these results show that exosomes are unlikely involved in intercellular Nef transfer. In addition, this study reveals existence of two types of exosomes: AChE+/CD81low/TSG101low exosomes and AChE-/CD81high/TSG101high exosomes.
Highlights
Intercellular protein transfer has been recognized as a common phenomenon for cell-cell communication in multi-cellular organisms including plants and animals; it can occur among immune cells and nonimmune cells [1, 2]
To determine whether intercellular Nef transfer could occur between Nef-expressing CD4+ T lymphocytes and other types of cells, we took advantage of a Jurkat cell clone stably expressing HIV-1 Nef.GFP fusion protein [56] and used it as the donor cells in the co-culture assay with target cells Jurkat, human monocytic cells THP-1, or human primary astrocytes (HPA)
membrane vesicles (MV) can be either shedding vesicles, which are outward blebbing of small vesicles of 100– 1000 nm in diameter directly from cellular plasma membrane [79, 80], or exosomes, which are exocytosis of internal luminal vesicles of 30–100 nm in diameter formed in the multivesicular bodies and released from the cells as a result of fusion of multivesicular bodies with plasma doi:10.1371/journal.pone.0124436.g010
Summary
Intercellular protein transfer has been recognized as a common phenomenon for cell-cell communication in multi-cellular organisms including plants and animals; it can occur among immune cells and nonimmune cells [1, 2]. The cell-cell contact-dependent protein transfer includes tunneling nanotubes (TNT) and trogocytosis. TNT are characterized by long cytoplasmic bridges that enable long-range cell-cell communication and function to transfer large cellular. Cell-cell contact-independent protein transfer is accomplished through release of protein-bearing membrane vesicles (MV) or exosomes by one cell and uptake of protein-bearing MV or exosomes by the other cell [5, 9]. Intercellular protein transfer regulates immune response and other cellular function of neighboring cells such as cellular homoeostasis and anti-tumor activities [10,11,12,13]
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