Ana Plemenitaš

HIV Nef is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells.

The HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed viral proteins. It is also found in the serum of infected individuals (Caby MP, Lankar D, Vincendeau‐Scherrer C, Raposo G, Bonnerot C. Exosomal‐like vesicles are present in human blood plasma. Int Immunol 2005;17:879–887). Extracellular Nef protein has deleterious effects on CD4+ T cells (James CO, Huang MB, Khan M, Garcia‐Barrio M, Powell MD, Bond VC. Extracellular Nef protein targets CD4+ T cells for apoptosis by interacting with CXCR4 surface receptors. J Virol 2004;78:3099–3109), the primary targets of HIV, and can suppress immunoglobulin class switching in bystander B cells (Qiao X, He B, Chiu A, Knowles DM, Chadburn A, Cerutti A. Human immunodeficiency virus 1 Nef suppresses CD40‐dependent immunoglobulin class switching in bystander B cells. Nat Immunol 2006;7:302–310). Nevertheless, the mode of exit of Nef from infected cells remains a conundrum. We found that Nef stimulates its own export via the release of exosomes from all cells examined. Depending on its intracellular location, these Nef exosomes form at the plasma membrane, late endosomes or both compartments in Jurkat, SupT1 and primary T cells, respectively. Nef release through exosomes is conserved also during HIV‐1 infection of peripheral blood lymphocytes (PBLs). Released Nef exosomes cause activation‐induced cell death of resting PBLs in vitro. Thus, HIV‐infected cells export Nef in bioactive vesicles, which facilitate the depletion of CD4+ T cells that is a hallmark of acquired immunodeficiency syndrome (AIDS).

Halotolerant and halophilic fungi.

Extreme environments have for long been considered to be populated almost exclusively by prokaryotic organisms and therefore monopolized by bacteriologists. Solar salterns are natural hypersaline environments characterized by extreme concentrations of NaCl, often high concentrations of other ions, high uv irradiation and in some cases extremes in pH. In 2000 fungi were first reported to be active inhabitants of solar salterns. Since then many new species and species previously known only as food contaminants have been discovered in hypersaline environments around the globe. The eukaryotic microorganism most studied for its salt tolerance is Saccharomyces cerevisiae. However, S. cerevisiae is rather salt sensitive and not able to adapt to hypersaline conditions. In contrast, some species like Debaryomyces hansenii, Hortaea werneckii, and Wallemia ichthyophaga have been isolated globally from natural hypersaline environments. We believe that all three are more suitable model organisms to study halotolerance in eukaryotes than S. cerevisiae. Furthermore, they belong to different and distant taxonomic groups and have developed different strategies to cope with the same problems of ion toxicity and loss of water.

Nef increases the synthesis of and transports cholesterol to lipid rafts and HIV-1 progeny virions

HIV buds from lipid rafts and requires cholesterol for its egress from and entry into cells. Viral accessory protein Nef plays a major role in this process. In this study, it not only increased the biosynthesis of lipid rafts and viral particles with newly synthesized cholesterol, but also enriched them. Furthermore, via the consensus cholesterol recognition motif at its C terminus, Nef bound cholesterol. When this sequence was mutated, Nef became unable to transport newly synthesized cholesterol into lipid rafts and viral particles. Interestingly, although its levels in lipid rafts were not affected, this mutant Nef protein was poorly incorporated into viral particles, and viral infectivity decreased dramatically. Thus, Nef also transports newly synthesized cholesterol to the site of viral budding. As such, it provides essential building blocks for the formation of viruses that replicate optimally in the host.

Nef increases infectivity of HIV via lipid rafts

Lipid rafts, also known as detergent-resistant membranes (DRM), are microdomains in the plasma membrane enriched in sphingolipids and cholesterol (reviewed in [1, 2]). Human immunodeficiency virus 1 (HIV) buds via lipid rafts [3, 4]. However, the targeting of viral structural components to DRM and its consequences for viral replication are not understood. Moreover, the negative factor Nef from HIV increases viral infectivity (reviewed in [5, 6]). With no apparent differences in structural components and morphology between wild-type and DeltaNef virons, the latter viruses display less efficient reverse transcription in target cells. As Nef is expressed abundantly early in the viral replicative cycle [7], we hypothesized that Nef could affect viral morphogenesis and budding to render viruses more infectious. In this report, we demonstrated first that Nef increases viral budding from lipid rafts. Second, in the presence of Nef, viral envelopes contain more ganglioside (GM1), which is a major component of lipid rafts. This finding correlated directly with the increased infectivity of HIV. Finally, the depletion of exogenous and endogenous cholesterol biochemically and genetically, which disrupted lipid rafts, decreased viral infectivity only in the presence of Nef. Importantly, HIV lacking the nef gene remained unaffected by these manipulations. We conclude that lipids in virions are essential for viral infectivity. Thus, HIV becomes more infectious when it buds from lipid rafts, and Nef plays a major role in this process.

CDC42 and Rac1 are implicated in the activation of the Nef-associated kinase and replication of HIV-1.

BACKGROUND The negative factor (Nef) of human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV) is required for high levels of viremia and progression to AIDS. Additionally, Nef leads to cellular activation, increased viral infectivity and decreased expression of CD4 on the cell surface. Previously, we and others demonstrated that Nef associates with a cellular serine kinase (NAK) activity. Recently, it was demonstrated that NAK bears structural and functional similarity to p21-activated kinases (PAKs). RESULTS In this study, we demonstrate that Nef not only binds to but also activates NAK via the small GTPases CDC42 and Rac1. First, the dominant-negative PAK (PAKR), via its GTPase-binding domain, and dominant-negative GTPases (CDC42Hs-N17 and Rac1-N17) block the ability of Nef to associate with and activate NAK. Second, constitutively active small GTPases (CDC42Hs-V12 and Rac1-V12) potentiate the effects of Nef. Third, interactions between Nef and NAK result in several cellular effector functions, such as activation of the serum-response pathway. And finally, PAKR, CDC42Hs-N17 and Rac1-N17 decrease levels of HIV-1 production to those of virus from which the nef gene is deleted. CONCLUSIONS By activating NAK via small GTPases and their downstream effectors, Nef interacts with regulatory pathways required for cell growth, cytoskeletal rearrangement and endocytosis. Thus, NAK could participate in the budding of new virions, the modification of viral proteins and the increased endocytosis of surface molecules such as CD4. Moreover, blocking the activity of these GTPases could lead to new therapeutic interventions against AIDS.

Salt-induced changes in lipid composition and membrane fluidity of halophilic yeast-like melanized fungi.

The halophilic melanized yeast-like fungi Hortaea werneckii, Phaeotheca triangularis, and the halotolerant Aureobasidium pullulans, isolated from salterns as their natural environment, were grown at different NaCl concentrations and their membrane lipid composition and fluidity were examined. Among sterols, besides ergosterol, which was the predominant one, 23 additional sterols were identified. Their total content did not change consistently or significantly in response to raised NaCl concentrations in studied melanized fungi. The major phospholipid classes were phosphatidylcholine and phosphatidylethanolamine, followed by anionic phospholipids. The most abundant fatty acids in phospholipids contained C16 and C18 chain lengths with a high percentage of C18:2Δ9,12. Salt stress caused an increase in the fatty acid unsaturation in the halophilic H. werneckii and halotolerant A. pullulans but a slight decrease in halophilic P. triangularis. All the halophilic fungi maintained their sterol-to-phospholipid ratio at a significantly lower level than did the salt-sensitive Saccharomyces cerevisiae and halotolerant A. pullulans. Electron paramagnetic resonance (EPR) spectroscopy measurements showed that the membranes of all halophilic fungi were more fluid than those of the halotolerant A. pullulans and salt-sensitive S. cerevisiae, which is in good agreement with the lipid composition observed in this study.

The Halophilic Fungus Hortaea werneckii and the Halotolerant Fungus Aureobasidium pullulans Maintain Low Intracellular Cation Concentrations in Hypersaline Environments

ABSTRACT Hortaea werneckii and Aureobasidium pullulans, black yeast-like fungi isolated from hypersaline waters of salterns as their natural ecological niche, have been previously defined as halophilic and halotolerant microorganisms, respectively. In the present study we assessed their growth and determined the intracellular cation concentrations of salt-adapted and non-salt-adapted cells of both species at a wide range of salinities (0 to 25% NaCl and 0 to 20% NaCl, respectively). Although 5% NaCl improved the growth of H. werneckii, even the minimal addition of NaCl to the growth medium slowed down the growth rate of A. pullulans, confirming their halophilic and halotolerant nature. Salt-adapted cells of H. werneckii and A. pullulans kept very low amounts of internal Na+ even when grown at high NaCl concentrations and can be thus considered Na+ excluders, suggesting the existence of efficient mechanisms for the regulation of ion fluxes. Based on our results, we can conclude that these organisms do not use K+ or Na+ for osmoregulation. Comparison of cation fluctuations after a hyperosmotic shock, to which nonadapted cells of both species were exposed, demonstrated better ionic homeostasis regulation of H. werneckii compared to A. pullulans. We observed small fluctuations of cation concentrations after a hyperosmotic shock in nonadapted A. pullulans similar to those in salt-adapted H.werneckii, which additionally confirmed better regulation of ionic homeostasis in the latter. These features can be expected from organisms adapted to survival within a wide range of salinities and to occasional exposure to extremely high NaCl concentrations, both characteristic for their natural environment.

Melanized halophilic fungi are eukaryotic members of microbial communities in hypersaline waters of solar salterns

Abstract Until recently, it was believed that microbial communities at high salinities are dominated exclusively by Archaea and Bacteria and one eukaryotic species, the alga Dunaliella salina. Recently, it became evident that melanized fungi, so far described only in the crystallization pond of Adriatic salterns within the season of salt production, can be considered as a new group of eukaryotic halophiles. They were represented by black, yeast-like hyphomycetes: Hortaea werneckii, Phaeotheca triangularis, Trimmatostroma salinum, Aureobasidium pullulans, together with phylogenetically closely related Cladosporium species. In the present study, the distribution of the melanized fungal population in five different evaporating ponds in the Adriatic salterns [covering the entire salinity range (3–30% NaCl)] was followed throughout the year. It appeared in three peaks, at 5–8%, 10–20% and 18–25% NaCl, which correlated primarily with high nitrogen values. At the highest environmental salinities, melanized fungi represented 85–100% of the total isolated mycobiota, but with lowering salinities they were partially replaced by non-melanized fungi and, at the end of the season, with NaCl concentrations below 5%, they were detected only occasionally. Melanized fungi have been isolated from hypersaline waters on three continents, indicating that they are present globally in hypersaline waters of man-made salterns.

Interactions between Nef and AIP1 proliferate multivesicular bodies and facilitate egress of HIV-1

BackgroundNef is an accessory protein of primate lentiviruses, HIV-1, HIV-2 and SIV. Besides removing CD4 and MHC class I from the surface and activating cellular signaling cascades, Nef also binds GagPol during late stages of the viral replicative cycle. In this report, we investigated further the ability of Nef to facilitate the replication of HIV-1.ResultsTo this end, first the release of new viral particles was much lower in the absence of Nef in a T cell line. Since the same results were obtained in the absence of the viral envelope using pseudo-typed viruses, this phenomenon was independent of CD4 and enhanced infectivity. Next, we found that Nef not only possesses a consensus motif for but also binds AIP1 in vitro and in vivo. AIP1 is the critical intermediate in the formation of multivesicular bodies (MVBs), which play an important role in the budding and release of viruses from infected cells. Indeed, Nef proliferated MVBs in cells, but only when its AIP1-binding site was intact. Finally, these functions of Nef were reproduced in primary macrophages, where the wild type but not mutant Nef proteins led to increased release of new viral particles from infected cells.ConclusionWe conclude that by binding GagPol and AIP1, Nef not only proliferates MVBs but also contributes to the egress of viral particles from infected cells.

Ostreolysin, a pore-forming protein from the oyster mushroom, interacts specifically with membrane cholesterol-rich lipid domains.

Ostreolysin, a 15 kDa pore‐forming protein from the edible oyster mushroom (Pleurotus ostreatus), is lytic to membranes containing both cholesterol and sphingomyelin. Its cytotoxicity to Chinese hamster ovary cells correlates with their cholesterol contents and with the occurrence of ostreolysin in the cells detergent resistant membranes. Moreover, ostreolysin binds to supported monolayers and efficiently permeabilizes sonicated lipid vesicles, only if cholesterol is combined with either sphingomyelin or dipalmitoylphosphatidylcholine. Addition of mono‐ or di‐unsaturated phosphatidylcholine to the cholesterol/sphingomyelin vesicles dramatically reduces the ostreolysins activity. It appears that the protein recognizes specifically a cholesterol‐rich lipid phase, probably the liquid‐ordered phase.