The Trojan Exosome Hypothesis
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The Trojan exosome hypothesis Stephen J. Gould*†, Amy M. Booth*, and James E. K. Hildreth‡ Departments of *Biological Chemistry and ‡Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 We propose that retroviruses exploit a cell-encoded pathway of intercellular vesicle traffic, exosome exchange, for both the biogene- sis of retroviral particles and a low-efficiency but mechanistically important mode of infection. This Trojan exosome hypothesis rec- onciles current paradigms of retrovirus-directed transmission with the unique lipid composition of retroviral particles, the host cell proteins present in retroviral particles, the complex cell biology of retroviral release, and the ability of retroviruses to infect cells in- dependently of Envelope protein–receptor interactions. An exosomal origin also predicts that retroviruses pose an unsolvable para- dox for adaptive immune responses, that retroviral antigen vaccines are unlikely to provide prophylactic protection, and that alloim- munity is a central component of antiretroviral immunity. Finally, the Trojan exosome hypothesis has important implications for the fight against HIV and AIDS, including how to develop new antiretroviral therapies, assess the risk of retroviral infection, and gener- ate effective antiretroviral vaccines. etroviruses are enveloped posi- of ILVs and their constituents can vary. plasma membrane (9, 17–19). Retrovi- tive-strand RNA viruses that rep- For example, the ILV biogenesis pathway ruses and exosomes also share many pro- licate through a DNA intermedi- can be reversed, as when proteins that are tein components that are enriched relative ate inserted in the host cell targeted into discrete ILVs return to the to the PM (tetraspannins, GPI proteins Rgenome (1). Current models of retroviral endosomal limiting membrane (14). ILVs and Lamps; refs. 3–5 and 20–23), mem- biology adhere to basic principles of virol- can also be degraded in lysosomes if the brane proteins that are present at high ogy (2), explain most empirical data on endosomes that carry them fuse with, or levels on exosomes as well as the cell sur- retroviruses, and assume a complete reli- mature into, lysosomes (13). Alternatively, face (integrins, MHC proteins, etc.; refs. ance on retroviral Env proteins for the MVBs can fuse with the plasma mem- 3–5, 22, and 24–26), and numerous cyto- binding and fusion of retroviral particles brane (PM), releasing ILVs into the extra- plasmic proteins (actin, cyclophilin, tsg101, with host cells (1, 2). However, these cellular milieu as exosomes (delayed exo- heat shock proteins, etc.; refs. 5, 22, and models do not provide a mechanistic ex- some biogenesis). MVB-PM fusion also 27–30). Moreover, side-by-side analyses planation for many important properties generates a patch of endosomal mem- show identical host cell protein profiles of retroviruses, including the array of host brane at the cell surface that can shed for retroviral particles and exosomal prep- cell molecules in retroviral particles (3–5), exosomes directly into the extracellular arations (31, 32). It should be noted that the observation of receptor-independent fluid (immediate exosome biogenesis). the host cell proteins present in retroviral and Env-independent retroviral infections Once released, exosomes can fuse with particles and exosomes are not merely the (6–8), and the ability of retroviruses to membranes of neighboring cells, delivering abundant components of the PM (22, 23). thrive in the presence of otherwise healthy membrane and cytoplasmic proteins from Also, the host cell proteins in retroviruses adaptive immune systems (1, 2). one cell to another. Exosome exchange are not just trace components, as some In an effort to reconcile these observa- plays important roles in numerous physio- (MHC class II) can exceed the abundance tions with the main body of data on retro- logical events, including prostate-induced of Env proteins (33, 34). Much of this viral biology, we propose the Trojan exo- sperm motility, wingless-mediated pattern data are from the HIV field, but similar some hypothesis. Many eukaryotic cells formation, lymphocyte activation, and in- results have been reported for other retro- synthesize and release small extracellular duction of immunological tolerance (10– viruses (35–43). vesicles called exosomes, which can fuse 12, 15, 16). Exosome uptake appears to with membranes of neighboring cells to involve clathrin-mediated endocytosis fol- Similarities in Protein Targeting and Vesicle complete an intercellular vesicle traffick- lowed by backfusion of exosomes with the Biogenesis. Expression of Gag alone is ing pathway (9–12). The Trojan exosome limiting membrane of the endosome. The sufficient to drive the formation of retro- hypothesis states that retroviruses use the net effect of the reaction is the transfer of virus-like particles (44, 45). The Trojan preexisting, nonviral exosome biogenesis membranes and cytosol from one cell to exosome hypothesis predicts that Gag pathway for the formation of infectious another in the proper topology. should therefore be targeted to ILVs. Pro- particles, and the preexisting, nonviral tein targeting into ILVs appears to be pathway of exosome uptake for a recep- Implications for Retroviral Biogenesis mediated by several mechanisms, includ- tor-independent, Env-independent mode and Transmission ing binding to ILV components, fatty acy- of infection. The following presents a por- The Trojan exosome hypothesis predicts lation, aggregation, and monoubiquityla- tion, and Gag proteins from several tion of the empirical support for this hy- that retroviral particles and exosomes will retroviruses possess these properties (13, pothesis and its major implications for the contain a similar array of host cell lipids 44). For instance, HIV Gag binds ILV fight against HIV and AIDS. and proteins, use the same protein target- components and ILV-biogenesis factors ing and vesicle biogenesis pathway, and (cyclophilin, tsg101), is N-terminally myr- Exosome Biogenesis and Uptake move between cells in the absence of a istoylated, forms large aggregates, and is Exosomes are small (50–200 nm) mem- retroviral Env protein. A review of the monoubiquitylated (44). In addition, Gag brane-bound vesicles that are released into empirical data finds support for each of mutants that interfere with these targeting the extracellular milieu (10–12). The early these predictions. mechanisms, such as the late domain mu- stages in exosome synthesis follow that of tants, can block the formation of retrovi- intralumenal vesicles (ILVs), which form Similarities in Host Cell Lipids and Proteins. rus-like particles (44). Additional support by inward budding of the endosome mem- Retroviruses and exosomes have a shared brane (13); endosomes enriched in ILVs lipid composition that includes signifi- are also referred to as multivesicular bod- cantly higher levels of cholesterol and gly- ‡To whom correspondence should be addressed. E-mail: ies (MVBs) (Fig. 1). The immediate fate cosphingolipids as compared with the sgould@jhmi.edu. 10592–10597 ͉ PNAS ͉ September 16, 2003 ͉ vol. 100 ͉ no. 19 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1831413100 Downloaded by guest on September 26, 2021 PERSPECTIVE distinct properties of different retroviruses or the unique features of different retrovi- rus-induced pathologies. However, an exo- somal origin provides retroviruses with complex physical and functional properties that have important implications for routes of pathogenesis. In the following section we will discuss how these proper- ties allow retroviruses to thrive in the face of adaptive immune responses while ren- dering them susceptible to destruction by histocompatibility, or alloimmune, reac- tions. The Failure of Adaptive Immunity It is well established that humans and other mammals combat many enveloped Fig. 1. The formation (Left) and fates (Right) of ILVs. virusesby(i) the selective proliferation of B cells that secrete neutralizing antibodies (primarily IgGs) able to block Env-medi- for the Trojan exosome hypothesis comes their Env proteins and independently of ated entry and (ii) the selective prolifera- from the observation that retroviral bio- the retroviral receptors. Perhaps the clear- tion of virus-reactive T cell clones that genesis requires ILV biogenesis factors est demonstration of Env-independent detect and kill infected cells, amplify the such as tsg101 and VPS4 (44, 46). infection is that of the gypsy retrovirus, antiviral immune response, and provide which is transmitted in vivo as efficiently immunological memory (2, 64). In healthy Similarities in Cell Biology of Release. Exo- in the absence of its Env gene as in the individuals, these mechanisms are usually somes can form at the limiting membrane presence of its Env gene (7). Another ex- sufficient to either clear viruses from the of discrete endosomes (delayed exosome ample of Env-independent infection body or drive viruses into latent states biogenesis) or at endosomal patches of comes from in vitro studies of mdg3,afly where replication occurs only infrequently. the cell surface (immediate exosome bio- retroelement that lacks an Env-like gene Furthermore, most viruses that cause genesis). The Trojan exosome hypothesis even in the WT state (56). acute pathogenesis (influenza, poliomyeli- predicts that retroviruses should also form Env-independent infection has also tis, smallpox, etc.) are easily controlled in at both endosomal membranes and at been documented