Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages

HIV type 1 (HIV-1) infects CD4⁺ T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as “Trojan horses” carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages.HIV type 1 (HIV-1) infects CD4⁺ T lymphocytes, myeloid dendritic cells, and monocyte-macrophages, cell types sharing the expression of the primary viral receptor (R) CD4 on their surface together with one or more chemokine R, usually CCR5 and/or CXCR4 (1, 2). The discovery of combination antiretroviral therapy (cART) in the mid 1990s has significantly impacted the natural history of the infection by virtually suppressing, in optimal conditions, the capacity of the virus to infect target cells, thereby resulting in a prolonged, near-normal life expectation of infected individuals. Therapy suspension almost inevitably results in the resumption of virus replication and disease progression in most individuals, owing to the existence of viral reservoirs of likely heterogeneous cellular origin that are unaffected by prolonged cART (3, 4). Consequently, complete eradication of HIV-1 from infected individuals is not currently achievable without targeting these cART-insensitive sources of infection. Latently infected CD4⁺ T cells with both “central memory” and “transitional effector memory” phenotypes have been well characterized as reservoirs of replication-competent HIV-1 (4, 5); in contrast, the contribution of non–T-cell subsets to the overall viral reservoir in cART-treated individuals has remained more elusive (6–8).Concerning mononuclear phagocytes, whether bone marrow precursor cells and circulating monocytes are truly infected in vivo is a matter of debate, whereas a greater consensus exists on the role played by resident tissue macrophages of different organs. In particular, brain-associated macrophages or microglia, together with astrocytes, have been reported as the main targets and source of virus in the central nervous system (4, 5, 8, 9). In addition, infected mononuclear phagocytes are considered responsible for the slower second-phase decay of plasma viremia observed in patients starting cART (9).Unlike CD4⁺ T cells, mononuclear phagocytes are depleted neither in vivo (at least in terms of circulating monocytes) nor in vitro on HIV-1 infection. In addition, macrophage infection, both in vitro and in vivo, has been described as characterized by budding and accumulation of HIV-1 particles in virus-containing compartments (VCC) of debated origin (10–13). These peculiar features of macrophage infection led to the “Trojan horse” hypothesis of a pathogenic role of mononuclear phagocytes as capable of accumulating virions in subcellular compartments invisible to immune recognition (14, 15) and relatively insensitive to antiretrovirals (15, 16). It is hypothesized that VCC-associated virions are released either as a consequence of cell death or by functional stimulation of the infected cells. In this regard, we have previously observed that IFN-γ stimulation of the chronically infected promonocytic U1 cell line differentiated to macrophage-like cells by phorbol-12, myristate, 13-acetate (PMA) caused a profound redirection of the major site of virion production from the plasma membrane to VCC (17). This observation was later extended to other stimulants of PMA-differentiated U1 cells, including urokinase-type plasminogen activator (uPA) and CD11b/CD18 (Mac-1) integrin ligands (18, 19). Of interest, a similar phenotype has been reported in infected primary human monocyte-derived macrophages (MDM) by interference with endogenously released CCL2/monocyte chemotactic protein-1 (MCP-1) (20).Regarding the nature of VCC, Gould et al. (14) proposed that retroviruses exploit the exosome biogenesis pathway for both the formation and release of infectious particles as well as for the uptake of a R-independent, gp120 envelope (Env)-dependent infection. More recently, VCC are believed to represent intracellular sequestration of plasma membrane areas rich in a subset of tetraspanins (12). Likely because of their origin, VCC can be transiently (11) connected to the extracellular medium through microchannels or conduits (21, 22). Of interest, VCC-like compartments accessible by the extracellular medium preexist in uninfected macrophages and express similar markers, including CD9, CD81, CD63 (23), and, identified more recently, CD36 (24). Recently, it was demonstrated that on HIV-1 infection, Gag is recruited to these preexisting compartments in which virion assembly occurs, leading to their conversion into VCC (24, 25). Whether the release of virions is a regulated process, and the nature of the signal(s) that may induce virion discharge from VCC, remain largely unknown, however.In the present study, we investigated the hypothesis that signals from an inflamed microenvironment may affect HIV-1 accumulation and release from macrophage-associated VCC. We focused on extracellular ATP (eATP), a molecule passively released during necrotic cell death (26), but also actively released on cell stimulation via innate immunity R, as reviewed previously (27). We indeed observed that eATP induced a rapid release of HIV-1 virions accumulated in VCC of both primary human MDM acutely infected with HIV-1 and chronically infected U1 cells differentiated to macrophage-like cells (D-U1 cells) (28). eATP-induced release of HIV-1 virions was not associated with gross cytopathic effects and was blocked by imipramine, an antidepressant agent known to inhibit the membrane-associated acid sphingomyelinase (aSMase), an enzyme involved in the formation of membrane microvesicles (29, 30). Finally, we found that eATP-dependent discharge of virions from VCC was attributed mainly to engagement of the P2X7R on the surface of infected macrophages.     

Dopamine- and cAMP-regulated phosphoprotein DARPP-32: phosphorylation of Ser-137 by casein kinase I inhibits dephosphorylation of Thr-34 by calcineurin.

Although protein phosphatases appear to be highly controlled in intact cells, relatively little is known about the physiological regulation of their activity. DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of apparent M(r) 32,000, is phosphorylated in vitro by casein kinase I, casein kinase II, and cAMP-dependent protein kinase on sites phosphorylated in vivo. DARPP-32 phosphorylated on Thr-34 by cAMP-dependent protein kinase is a potent inhibitor of protein phosphatase 1 and an excellent substrate for calcineurin, a Ca2+/calmodulin-dependent protein phosphatase. Here we provide evidence, using both purified proteins and brain slices, that phosphorylation of DARPP-32 on Ser-137 by casein kinase I inhibits the dephosphorylation of Thr-34 by calcineurin. This inhibition occurs only when phospho-Ser-137 and phospho-Thr-34 are located on the same DARPP-32 molecule and is not dependent on the mode of activation of calcineurin. The results demonstrate that the inhibition is due to a modification in the properties of the substrate which alters its dephosphorylation rate. Thus, casein kinase I may play a physiological role in striatonigral neurons as a modulator of the regulation of protein phosphatase 1 via DARPP-32.     

CD36-specific antibodies block release of HIV-1 from infected primary macrophages and its transmission to T cells

HIV-1–infected macrophages likely represent viral reservoirs, as they accumulate newly formed virions in internal virus-containing compartments (VCCs). However, the nature and biogenesis of VCCs remain poorly defined. We show that upon HIV-1 infection of primary human macrophages, Gag is recruited to preexisting compartments containing the scavenger receptor CD36, which then become VCCs. Silencing of CD36 in HIV-1–infected macrophages decreases the amount of virions released. Strikingly, soluble anti-CD36 antibodies, but not the natural ligands of CD36, inhibit release of virions from HIV-1–infected macrophages and the transmission of virus to CD4⁺ T cells. The effect of the antibodies is potent, rapid, and induces the retention of virions within VCCs. Ectopic expression of CD36 in HeLa cells renders them susceptible to the inhibitory effect of the anti-CD36 mAb upon HIV-1 infection. We show that the anti-CD36 mAb inhibits HIV-1 release by clustering newly formed virions at their site of budding, and that signaling via CD36 is not required. Thus, HIV-1 reservoirs in macrophages may be tackled therapeutically using anti-CD36 antibodies to prevent viral dissemination.Early after its discovery, it has been established that HIV-1 infects not only CD4⁺ T lymphocytes but also macrophages, like other lentiviruses. The presence of HIV-1–infected macrophages in vivo has been documented in various tissues (Gyorkey et al., 1985; Koenig et al., 1986; Pomerantz et al., 1988; Jarry et al., 1990). The precise contribution of macrophages to the infection and pathogenesis of HIV-1 still remains to be established. Nevertheless, macrophages are considered as viral reservoirs because they are long-lived cells resistant to the cytopathic effects of HIV-1. Indeed, HIV-1–infected macrophages can survive for months (Salahuddin et al., 1986; Orenstein et al., 1988) and store infectious virions for extended periods of time (Sharova et al., 2005). Supporting the idea of a viral reservoir in macrophages, newly formed virions are assembled and stored in unusual intracellular compartments, often referred to as virus-containing compartments (VCCs; Tan and Sattentau, 2013), which may protect virions from the immune response and antiviral drug treatments.The VCC appears to be a macrophage-specific compartment, clearly distinct from the endocytic pathway as it possesses a neutral pH (Jouve et al., 2007) and expresses a subset of endocytic markers such as CD81 and CD9 but not Lamp1 nor Lamp2 (Pelchen-Matthews et al., 2003; Marsh et al., 2009). Moreover, its limiting membrane is often decorated by a thick molecular coat, which contains β2 integrins (Pelchen-Matthews et al., 2012) and members of the ESCRT (endosomal sorting complexes required for transport) machinery (Benaroch et al., 2010). Although the exact origin and nature of the VCC remains obscure so far, indirect evidence suggests that VCCs represent specialized domains of the plasma membrane that have been sequestered intracellularly (Deneka et al., 2007; Welsch et al., 2007). Supporting this plasma membrane origin, VCCs can remain accessible to the external medium through conduits or narrow microchannels (Deneka et al., 2007; Welsch et al., 2007; Bennett et al., 2009). One third of the VCCs are accessible to the external medium overtime, but this access can be transient and therefore suggests that such connections are dynamic (Gaudin et al., 2013). VCCs evolve with time post infection (p.i.) as the density of viral particles present in their lumen increases (Gaudin et al., 2013). Such compartments are absent from T lymphocytes where viral assembly takes place at the plasma membrane. In macrophages, HIV-1 assembly occurs at the limiting membrane of the VCCs through mechanisms that remain to be deciphered (Tan and Sattentau, 2013).To approach these mechanisms, we examined the role of proteins specific for macrophages as compared with T lymphocytes. More precisely, we looked for macrophage proteins that could potentially be involved in the functioning of the VCC and may represent targets to treat the intracellular stocks of virus present in the infected macrophages. Macrophages are equipped with a collection of phagocytic receptors (including lectins, integrins, GPI-anchored proteins, and scavenger receptors) that allow the internalization of many self-, nonself-, or modified self-components such as modified low density lipoproteins (LDLs; Taylor et al., 2005). We focused on the scavenger receptor family, which is highly expressed in monocytes/macrophages as compared with T lymphocytes (Areschoug and Gordon, 2009). CD36 belongs to the class B scavenger receptor family and is expressed by endothelial cells, smooth muscle cells, adipocytes, platelets, and macrophages but not by T lymphocytes (Talle et al., 1983; Swerlick et al., 1992; Matsumoto et al., 2000; Kuniyasu et al., 2003). In macrophages, it binds to multivalent ligands such as oxidized LDL, components of the bacterial surface, and apoptotic cells (Savill et al., 1992; Endemann et al., 1993; Hoebe et al., 2005; Stuart et al., 2005).Here, we show that HIV-1 hijacks preexisting CD36⁺ compartments for its own assembly in macrophages. Exposure to CD36-specific antibodies inhibits virus release due to retention into VCCs. This effect is rapid, potent, long lasting, and does not require signaling through the known pathway of CD36 signal transduction. Our results further suggest that exposure to CD36 antibodies induces a tethering of virions within VCCs thereby inhibiting their release and transmission to CD4⁺ T lymphocytes.     

Modulation of the voltage-gated sodium current in rat striatal neurons by DARPP-32, an inhibitor of protein phosphatase

DARPP-32 is a cyclic adenosine monophosphate-regulated inhibitor of protein phosphatase 1, highly enriched in striatonigral neurons. Stimulation of dopamine D₁ receptors increases phosphorylation of DARPP-32, whereas glutamate acting on N-methyl-d -aspartate receptors induces its dephosphorylation. Yet, to date, there is little direct evidence for the function of DARPP-32 in striatal neurons. Using a whole cell patch-clamp technique, we have studied the role of DARPP-32 in the regulation of voltage-gated sodium channels in rat striatal neurons maintained in primary culture. Injection of phospho-DARPP-32, but not of the unphosphorylated form, reduced the sodium current amplitude. This effect was similar to those induced by okadaic acid, with which there was no additivity and by tautomycin. Our results indicate that, in striatal neurons, sodium channels are under dynamic control by phosphorylation/dephosphorylation, and that phospho-DARPP-32 reduces sodium current by stabilizing a phosphorylated state of the channel or an associated regulatory protein. We propose that the DARPP-32-mediated modulation of sodium channels, via inhibition of phosphatase 1, contributes to the regulation of these channels by D₁ receptors and other neurotransmitters which influence the state of phosphorylation of DARPP-32.     

Use of Human Intestinal Enteroids to Evaluate Persistence of Infectious Human Norovirus in Seawater

Little data on the persistence of human norovirus infectivity are available to predict its transmissibility. Using human intestinal enteroids, we demonstrate that 2 human norovirus strains can remain infectious for several weeks in seawater. Such experiments can improve understanding of factors associated with norovirus survival in coastal waters and shellfish.     

Transcytosis of HTLV-1 across a tight human epithelial barrier and infection of subepithelial dendritic cells

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to blood transfusion and sexual transmission, HTLV-1 is transmitted mainly through prolonged breastfeeding, and such infection represents a major risk for the development of adult T-cell leukemia/lymphoma. Although HTLV-1-infected lymphocytes can be retrieved from maternal milk, the mechanisms of HTLV-1 transmission through the digestive tract remain unknown. In the present study, we assessed HTLV-1 transport across the epithelial barrier using an in vitro model. Our results show that the integrity of the epithelial barrier was maintained during coculture with HTLV-1-infected lymphocytes, because neither morphological nor functional alterations of the cell monolayer were observed. Enterocytes were not susceptible to HTLV-1 infection, but free infectious HTLV-1 virions could cross the epithelial barrier via a transcytosis mechanism. Such virions were able to infect productively human dendritic cells located beneath the epithelial barrier. Our data indicate that HTLV-1 crosses the tight epithelial barrier without disruption or infection of the epithelium to further infect target cells such as dendritic cells. The present study provides the first data pertaining to the mode of HTLV-1 transport across a tight epithelial barrier, as can occur during mother-to-child HTLV-1 transmission during breastfeeding.