Robert Lodge

A tubular EHD1‐containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane

The Eps15 homology (EH) domain‐containing protein, EHD1, has recently been ascribed a role in the recycling of receptors internalized by clathrin‐mediated endocytosis. A subset of plasma membrane proteins can undergo internalization by a clathrin‐independent pathway regulated by the small GTP‐binding protein ADP‐ribosylation factor 6 (Arf6). Here, we report that endogenous EHD proteins, as well as transgenic tagged EHD1, are associated with long, membrane‐bound tubules containing Arf6. EHD1 appears to induce tubule formation, which requires nucleotide cycling on Arf6 and intact microtubules. Mutations in the N‐terminal P‐loop domain or deletion of the C‐terminal EH domain of EHD1 prevent association of EHD1 with tubules or induction of tubule formation. The EHD1 tubules contain internalized major histocompatibility complex class I (MHC‐I) molecules that normally traffic through the Arf6 pathway. Recycling assays show that overexpression of EHD1 enhances MHC‐I recycling. These observations suggest an additional function of EHD1 as a tubule‐inducing factor in the Arf6 pathway for recycling of plasma membrane proteins internalized by clathrin‐independent endocytosis.

The membrane‐proximal intracytoplasmic tyrosine residue of HIV‐1 envelope glycoprotein is critical for basolateral targeting of viral budding in MDCK cells

Budding of retroviruses from polarized epithelial Madin–Darby canine kidney cells (MDCK) takes place specifically at the basolateral membrane surface. This sorting event is suspected to require a specific signal harbored by the viral envelope glycoprotein and it was previously shown that, as for most basolateral proteins, the intracytoplasmic domain plays a crucial role in this targeting phenomenon. It is well known that tyrosine‐based motifs are a central element in basolateral targeting signals. In the present study, site‐directed mutagenesis was used to generate conservative or non‐conservative substitutions of each four intracytoplasmic tyrosines of the human immunodeficiency virus (HIV‐1) envelope glycoprotein. This approach revealed that the membrane‐proximal tyrosine is essential to ensure both the basolateral localization of envelope glycoprotein and the basolateral targeting of HIV‐1 virions. Substitutions of the membrane‐proximal tyrosine did not appear to affect incorporation of envelope glycoprotein into the virions, as assayed by virion infectivity and protein content, nor its capability to ensure its role in viral infection, as determined by viral multiplication kinetics. Altogether, these results indicate that the intracytoplasmic domain of the HIV‐1 envelope glycoprotein harbors a unique, tyrosine‐based, basolateral targeting signal. Such a tyrosine‐based targeting signal may play a fundamental role in HIV transmission and pathogenesis.

Divalent interaction of the GGAs with the Rabaptin-5–Rabex-5 complex

Cargo transfer from trans‐Golgi network (TGN)‐derived transport carriers to endosomes involves a still undefined set of tethering/fusion events. Here we analyze a molecular interaction that may play a role in this process. We demonstrate that the GGAs, a family of Arf‐dependent clathrin adaptors involved in selection of TGN cargo, interact with the Rabaptin‐5–Rabex‐5 complex, a Rab4/Rab5 effector regulating endosome fusion. These interactions are bipartite: GGA‐GAE domains recognize an FGPLV sequence (residues 439–443) in a predicted random coil of Rabaptin‐5 (a sequence also recognized by the γ1‐ and γ2‐adaptin ears), while GGA‐GAT domains bind to the C‐terminal coiled‐coils of Rabaptin‐5. The GGA–Rabaptin‐5 interaction decreases binding of clathrin to the GGA‐hinge domain, and expression of green fluorescent protein (GFP)–Rabaptin‐5 shifts the localization of endogenous GGA1 and associated cargo to enlarged early endosomes. These observations thus identify a binding sequence for GAE/γ‐adaptin ear domains and reveal a functional link between proteins regulating TGN cargo export and endosomal tethering/fusion events.

Efficient magnetic bead-based separation of HIV-1-infected cells using an improved reporter virus system reveals that p53 up-regulation occurs exclusively in the virus-expressing cell population

HIV-1 infection in cell lines is very efficient, since the target population is clonal and highly dividing. However, infection of primary cells such as CD4 T lymphocytes and monocyte-derived macrophages is much more difficult, resulting in a very small percentage of infected cells. In order to study events occurring in productively infected primary cells, we determined that a way to isolate this population from bystander cells was needed. We engineered a novel HIV-1-based reporter virus called NL4-3-IRES-HSA that allows for the magnetic separation of cells infected with fully competent virions. This X4-using virus encodes for the heat-stable antigen (HSA/murine CD24) without the deletion of any viral genes by introducing an IRES sequence between HSA and the auxiliary gene Nef. Using commercial magnetic beads, we achieved efficient purification of HIV-1-infected cells (i.e. purity >85% and recovery >90%) from diverse primary cell types at early time points following infection. We used this system to accurately quantify p53 protein levels in both virus-infected and uninfected bystander primary CD4(+) T cells. We show that p53 up-regulation occurs exclusively in the infected population. We devised a strategy that allows for an efficient separation of HIV-1 infected cells from bystanders. We believe that this new reporter virus system will be of great help to study in depth how HIV-1 interacts with its host in a primary cells context.

Nelfinavir, an HIV-1 Protease Inhibitor, Induces Oxidative Stress–Mediated, Caspase-Independent Apoptosis in Leishmania Amastigotes

Background Visceral leishmaniasis has now emerged as an important opportunistic disease in patients coinfected with human immunodeficiency virus type-1 (HIV-1). Although the effectiveness of HIV-1 protease inhibitors, such as nelfinavir, in antiretroviral therapies is well documented, little is known of the impact of these drugs on Leishmania in coinfected individuals. Methodology and Principal Findings Here, we show that nelfinavir generates oxidative stress in the parasite, leading to altered physiological parameters such as an increase in the sub-G1 DNA content, nuclear DNA fragmentation and loss of mitochondrial potential, which are all characteristics of apoptosis. Pretreatment of axenic amastigotes with the caspase inhibitor z-VAD-fmk did not inhibit the increase in sub-G1 DNA content in nelfinavir-treated parasites, suggesting therefore that this antiviral agent does not kill Leishmania amastigotes in a caspase-dependent manner. Furthermore, we observed that the mitochondrial resident protein endonuclease G is involved. We also demonstrate that parasites overexpressing GSH1 (the rate limiting enzyme of glutathione biosynthesis) were more resistant to nelfinavir when compared to untransfected controls. Conclusions and Significance These data suggest that nelfinavir induces oxidative stress in Leishmania amastigotes, culminating in caspase-independent apoptosis, in which DNA is degraded by endonuclease G. This study provides a rationale for future, long-term design of new therapeutic strategies to test nelfinavir as a potential antileishmanial agent as well as for possible future use in Leishmania/HIV-1 coinfections.

C5a- and ASP-mediated C5L2 activation, endocytosis and recycling are lost in S323I-C5L2 mutation.

UNLABELLED C5L2, a G-protein-coupled receptor (GPCR), has been identified as an ASP (C3adesArg) and C5a receptor. Controversy exists regarding both ligand binding and functionality. ASP activation of C5L2 is proposed to regulate fat storage. C5L2 is also proposed as a decoy receptor for C5a, an inflammatory mediator, based on absence of Ca(2+) or chemotaxis changes. AIMS (i) to evaluate C5L2 receptor activation and recycling using recombinant ASP (rASP) and rC5a and (ii) assess receptor trafficking of S323I-C5L2 mutation previously identified in a family and demonstrated to have altered functionality. RESULTS stably transfected C5L2-HEK cells were sorted using fluorescent-ASP (Fluos-ASP) binding. Following 2-h serum-free pretreatment, C5L2 was typically localized to the cell-surface. beta-Arrestin-2-GFP transiently transfected C5L2-HEK cells demonstrated rASP and rC5a-dependent beta-arrestin-2-GFP translocation, which showed time-dependent intracellular colocalization with C5L2. Without ligand or C5L2 transfection, no translocation was identified at any time point. Ligand-dependent (rASP and rC5a) C5L2 endocytosis was time-dependent with a 1-h nadir, and was clathrin- and cholesterol-dependent. Transiently transfected Rab-GFP proteins (Rabs 5, 7 and 11) demonstrated time-dependent colocalization of Rab5, Rab7, and Rab11 with C5L2. In contrast to C5L2, a large proportion of stably transfected S323I-C5L2 did not localize to the cell-surface. While S323I-C5L2 was competent for Fluos-ASP and (125)I-ASP binding, although at a reduced level, there was no ligand-mediated receptor phosphorylation. Further, there was no ligand-mediated activation of beta-arrestin-2-GFP translocation, and no downstream functional activation of glucose transport or triglyceride synthesis. CONCLUSION C5L2 is a functional metabolic receptor, and serine 323 is important for ASP induced functionality.

Intense pseudotransport of a cationic drug mediated by vacuolar ATPase: procainamide-induced autophagic cell vacuolization.

Cationic drugs frequently exhibit large apparent volumes of distribution, consistent with various forms of cellular sequestration. The contributions of organelles and metabolic processes that may mimic drug transport were defined in human vascular smooth muscle cells. We hypothesized that procainamide-induced vacuolar cytopathology is driven by intense pseudotransport mediated by the vacuolar (V)-ATPase and pursued the characterization of vesicular trafficking alterations in this model. Large amounts of procainamide were taken up by intact cells (maximal in 2 h, reversible upon washout, apparent KM 4.69 mM; fluorometric determination of cell-associated drug). Procainamide uptake was extensively prevented or reversed by pharmacological inhibition of the V-ATPase with bafilomycin A1 or FR 167356, decreased at low extracellular pH and preceded vacuolar cell morphology. However, the uptake of procainamide was unaffected by mitochondrial poisons that reduced the uptake of rhodamine 6G. Large vacuoles induced by millimolar procainamide were labeled with the late endosome/lysosome markers Rab7 and CD63 and the autophagy effector LC3; their osmotic formation (but not procainamide uptake) was reduced by extracellular mannitol and parallel to LC3 II formation. Procainamide-induced vacuolization is associated with defective endocytosis of fluorophore-labeled bovine serum albumin, but not with induction of the unfolded protein response. The contents of a vacuole subset slowly (> or =24 h) become positive for Nile red staining (phospholipidosis-like response). V-ATPase-driven ion trapping is a form of intense cation pseudotransport that concerns the uncharged form of the drugs, and is associated with a vacuolar, autophagic and evolutive cytopathology and profound effects on vesicular trafficking.

Macropinocytosis-Like HIV-1 Internalization in Macrophages Is CCR5 Dependent and Leads to Efficient but Delayed Degradation in Endosomal Compartments

ABSTRACT HIV-1 endocytosis by a macropinocytosis-like mechanism has been shown to lead to productive infection in macrophages. However, little is known of this pathway. In this study, we examined HIV-1 endocytosis using biochemical approaches and imaging techniques in order to better understand the mechanisms that allow for productive infection of these cells via the endosomal pathway. We show here that this macropinocytosis-like mechanism is not the sole pathway involved in HIV-1 endocytosis in macrophages. However, this pathway specifically requires CCR5 engagement at the cell surface, which in turn suggests that the virus and its coreceptor are present in the endosomal environment simultaneously. Furthermore, although we observed efficient viral degradation following endocytosis, analyses of HIV-1 transport through the endolysosomal pathway revealed that viral degradation is delayed following endosomal internalization, possibly allowing the virus to complete its fusion.

Mechanisms of interaction between protozoan parasites and HIV.

Purpose of reviewThis review summarizes the current knowledge on human immunodeficiency virus type 1 (hereinafter called HIV)/protozoan co-infections in the case of three important, although neglected, tropical diseases: malaria, trypanosomiasis (Chagas disease) and leishmaniasis. The HIV pandemic has modified the immunopathogenic, epidemiological and therapeutic aspects of these human diseases. Recent findingsIn-vitro data suggests that HIV favors Leishmania infection, whereas different parasites have contrasting effects on HIV. However, many of the previous models are a limited representation of the complex interactions within the host; this situation is particularly the case when microbial products are used in place of live parasites. SummaryIn the host, protozoan parasites generally enhance HIV replication and accelerate AIDS progression. HIV alters parasite pathogenesis, often worsening disease outcome. These aspects bring significant complications for the treatment of co-infected individuals.

Leishmania infantum amastigotes enhance HIV-1 production in cocultures of human dendritic cells and CD4+ T cells by inducing secretion of IL-6 and TNF-α.

Background Visceral leishmaniasis has emerged as an important opportunistic disease among patients infected with HIV-1. Both HIV-1 and the protozoan parasite Leishmania can productively infect cells of the macrophage-dendritic cell lineage. Methodology/Principal Findings Here we demonstrate that Leishmania infantum amastigotes increase HIV-1 production when human primary dendritic cells (DCs) are cocultured together with autologous CD4+ T cells. Interestingly, the promastigote form of the parasite does not modulate virus replication. Moreover, we report that amastigotes promote virus replication in both cell types. Our results indicate that this process is due to secretion of parasite-induced soluble factors by DCs. Luminex micro-beads array system analyses indicate that Leishmania infantum amastigotes induce a higher secretion of several cytokines (i.e. IL-1α, IL-2, IL-6, IL-10 and TNF-α) and chemokines (i.e. MIP-1α, MIP-1β and RANTES) in these cells. Studies conducted with pentoxifylline and neutralizing antibodies revealed that the Leishmania-dependent augmentation in HIV-1 replication is due to a higher secretion of IL-6 and TNF-α. Conclusions/Significance Altogether these findings suggest that the presence of Leishmania within DC/T-cell conjugates leads to an enhancement of virus production and demonstrate that HIV-1 and Leishmania can establish complex interactions in such a cellular microenvironment.