Trans-dissemination of exosomes from HIV-1-infected cells fosters both HIV-1 trans-infection in resting CD4+T lymphocytes and reactivation of the HIV-1 reservoir

Abstract Intact HIV-1 and exosomes can be internalized by dendritic cells (DCs) through a common pathway leading to their transmission to CD4? T lymphocytes by means of mechanisms defined as trans-infection and trans- dissemination, respectively. We previously reported that exosomes from HIV-1-infected cells activate both unin- fected quiescent CD4? T lymphocytes, which become permissive to HIV-1, and latently infected cells, with release of HIV-1 particles. However, nothing is known about the effects of trans-dissemination of exosomes pro- duced by HIV-1-infected cells on uninfected or latently HIV-1-infected CD4? T lymphocytes. Here, we report that trans-dissemination of exosomes from HIV-1-infected cells induces cell activation in resting CD4? T lympho- cytes, which appears stronger with mature than immature DCs. Using purified preparations of both HIV-1 and exo- somes, we observed that mDC-mediated trans-dissemina- tion of exosomes from HIV-1-infected cells to resting CD4? T lymphocytes induces efficient trans-infection and HIV-1 expression in target cells. Most relevant, when both mDCs and CD4? T lymphocytes were isolated from combination anti-retroviral therapy (ART)-treated HIV-1- infected patients, trans-dissemination of exosomes from HIV-1-infected cells led to HIV-1 reactivation from the viral reservoir. In sum, our data suggest a role of exosome trans-dissemination in both HIV-1 spread in the infected host and reactivation of the HIV-1 reservoir. & Maurizio Federico maurizio.federico@iss.it 1 Introduction HIV-1 can be delivered to uninfected target cells by den- dritic cells (DCs) through a process referred to as trans- infection [1, 2], which is mediated by the formation of virological synapses containing HIV-1-loaded DCs and target CD4? T lymphocytes [3, 4]. DCs support HIV-1 replication poorly, which is mostly a consequence of the intracellular expression of the Sam domain and HD domain-containing protein (SAMHD)-1 cell restriction factor, which acts by a twofold mechanism, i.e., by limiting the availability of triphosphate-dNTP needed for reverse transcription [5] and by degrading incoming viral RNA [6]. On the other hand, DCs can deliver intact HIV-1 viral particles to target cells in the absence of viral infection. Exactly how virions are transported by DCs is still a matter of debate. Fluorescence microscopy studies have indicated that viral particles are stored in non-acidic tetraspanin-rich (i.e., CD9?, CD63?, CD81?) endosomal compartments before their retrograde delivery to target cells [7]. It has also been hypothesized that viral particles traffic to invaginated pockets [8] that are partially accessible to proteases but exclude neutralizing antibodies [9]. Trans- infection efficiency differs between immature (i) and mature (m)DCs [10]. Most of the HIV-1 particles captured by iDCs are delivered to lysosomes for degradation. Con- versely, the interaction between CD169, which is highly expressed by mDCs [11, 12], and both monosialotetra- hexosylganglioside (GM)-1 [13] and GM-3 [14] embedded in the viral membrane favors the trafficking of virions to non-acidic vesicular compartments of mDCs that are con- tiguous with the extracellular milieu. Exosomes are vesicles of 50-100 nanometers that are released by all cell types [for a recent review, see reference 15]. They are generated by invagination of endosome National Center for Global Health, Istituto Superiore di Sanita`, Viale Regina Elena, 299, 00161 Rome, Italy 2 Department of Public Health and Infectious Diseases, ‘Sapienza’ University of Rome, Rome, Italy 123 membranes, leading to the formation of intraluminal vesi- cles which then become part of multivesicular bodies. They can traffic either to lysosomes for degradation or to the plasma membrane, thereby releasing their vesicular con- tents upon membrane fusion. Exosomes are part of the intercellular communication network [16]. They incorpo- rate messenger RNAs, microRNAs, and proteins, which can be active in target cells [17]. As a consequence, exo- somes play a key role in both physiological and patho- logical processes. An increasing body of literature supports the idea that exosomes are involved in the mechanisms of HIV-1-related pathogenesis [18–20]. On this subject, we previously demonstrated that the expression of HIV-1 Nef induces the release of exosomes incorporating active ADAM (a disin- tegrin and metalloprotease) 17 [21], i.e., a multi-domain, transmembrane, Zn2?-dependent proteinase whose most studied function is processing pro-tumor necrosis factor (TNF)a to its active form [22]. Resting CD4? T lympho- cytes targeted by exosome-associated ADAM17 are induced to release TNFa. This initiates events leading to activation of quiescent human primary CD4? T lympho- cytes, which thereby become competent for HIV-1 expression and replication [23–25]. Similar to HIV-1, exosomes also can be captured by DCs and delivered to bystander cells through a mechanism referred to as trans-dissemination [26]. It has been reported that both trans-infection and trans-dissemination recognize a similar intracellular pathway, since HIV-1 and exosomes are both retained in the same CD81? intracellular com- partments. Although it is known that exosomes and HIV-1 particles compete for the same pathway for entry into DCs [26], little is known about the virological consequences of trans-dissemination. We sought to fill this gap by investi- gating the effects on trans-infection of the DC-mediated delivery of exosomes from HIV-1-infected cells. Here, we provide evidence that mDC-mediated delivery of exosomes from HIV-1-infected cells induces cell activation and HIV- 1 permissiveness in resting primary CD4? T lymphocytes, as well as HIV-1 reactivation in the viral reservoir