Exosomes in the pathogenesis of HIV-1

Cells secrete various membrane-enclosed microvesicles from their cell surface (shedding microvesicles) and from internal, endosome-derived membranes (exosomes). Intriguingly, these vesicles have many characteristics in common with viruses, including biophysical properties, biogenesis, and uptake by cells. Recent discoveries describing the microvesicle-mediated intercellular transfer of functional cellular proteins, RNAs, and mRNAs have revealed additional similarities between viruses and cellular microvesicles. Apparent differences include the complexity of viral entry, temporally regulated viral expression, and self-replication proceeding to infection of new cells. Interestingly, many virally infected cells secrete microvesicles that differ in content from their virion counterparts but may contain various viral proteins and RNAs. Accumulative findings have demonstrated that exosomes highly resembled HIV-1 particles in many aspects, from their physical properties to composition. The similarity of the composition (i.e., lipids, proteins, carbohydrates, and RNAs) between viral particles and exosomes suggests that exosomes may play an indispensable role in HIV-1 pathogenesis. Infact, HIV-1 infected cells release exosomes associating viral proteins, such as Nef and Gag, and RNA. Their potential functions during HIV-1 infection are just beginning to emerge. The main aim of this PhD thesis is the analysis of possible influence of exosomes released from HIV-1 infected cells on different stages of viral infection: in the primary infection, in HIV-1 infection during the cART (combination anti-retroviral therapy) and in HIV-1 latency, the major hurdle toward HIV-1 eradication. As CD4+ T lymphocytes are the major target of HIV-1 infection and replicate HIV-1 when activated, but resist HIV-1 replication when they are in a quiescent/resting state, the first aim of the PhD project has been the analysis in primary infection and under cART of the effects of exosomes from HIV-1 infected cells on activation state of quiescent CD4+ T lymphocytes and their susceptibility to HIV-1 infection. However, it has been reported that exosomes from cells expressing Nef upload activated ADAM17, a disintegrin and metalloprotease, which converts the pro-TNF-? in its mature form, and induce release of TNF-? from unstimulated peripheral blood mononuclear cells (PBMCs). We investigated whether a similar mechanism occurs in resting CD4+ T lymphocytes challenged with exosomes from HIV-1 infected cells and the possible consequences in terms of HIV-1 replication. First, we confirmed that cells infected with either replication-competent or defective HIV-1 release exosomes, uploading activated ADAM17. Next, we demonstrated that in HIV-1 primary infection and during cART these "armedࢠnanovesicles induce cell activation and render resting human primary CD4+ T lymphocytes permissive to HIV-1 replication. We found that the expression of HIV-1 Nef in exosome-producing cells is both necessary and sufficient for cell activation as well as HIV-1 replication in target CD4+ T lymphocytes. We also identified a Nef protein domain important for the effects we observed, i.e., the 62EEEE65 acidic cluster domain. The data presented here are consistent with a model where Nef induces intercellular communication through exosomes to activate bystander quiescent CD4+ T lymphocytes, thus stimulating viral spread. Overall, our findings support the idea that HIV-1 in primary infection and under cART infection evolved to usurp the exosome-based intercellular communication network to favor its spread in infected hosts. In the second part of this thesis we analyzed the effects of exosomes release from HIV-1 infected cells on viral latency. HIV-1 latency is the major hurdle toward HIV-1 eradication. Infact, HIV-1 infection is efficiently counteracted by cART which, despite preventing disease progression, does not eradicate virus infection which persists in a latent form. The latent reservoir is generated by virus entry in activated CD4+ T lymphocytes committed to return to a resting state, even though also resting CD4+ T lymphocytes can be latently infected. Thus, we may assume that HIV-1 reservoir is prevalently composed by memory CD4+ T lymphocytes. We asked whether exosomes from cells infected with either replication-competent or defective HIV-1 can activate latent HIV-1 in CD4+ T lymphocytes. Our results demonstrated that latent HIV-1 can be activated by TNF? released by cells upon ingestion of exosomes released by infected cells, and that this effect depends on the activity of exosome-associated ADAM17. These pieces of evidence shed new light on the mechanism of HIV-1 reactivation in latent reservoirs, and might also be relevant to design new therapeutic interventions focused on HIV-1 eradication.