Prediction of arenavirus fusion peptides on the basis of computer analysis of envelope protein sequences

AbstractTheoretical search and selection criteria for putative fusion peptides of enveloped viruses are proposed. Arena virus fusion peptides are predicted on the basis of computer-assisted analysis of amino acid sequences of arenavirus envelope proteins and elements of their secondary and tertiary structure. Accordingly, two regions of GP2 surface protein from 5 viruses of Arenaviridae family have been detected with properties typical of fusion peptides of other enveloped viruses. One region, named peptide IV, located at the N-terminus of the GP2 protein, is followed by the other region or peptide V, more likely candidate for the arenavirus fusion peptide

Yeasts producing zeatin

The present paper describes the first screening study of the ability of natural yeast strains to synthesize in culture the plant-related cytokine hormone zeatin, which was carried out using HPLC-MS/MS. A collection of 76 wild strains of 36 yeast species (23 genera) isolated from a variety of natural substrates was tested for the production of zeatin using HPLC-MS/MS. Zeatin was detected in more than a half (55%) of studied strains and was more frequently observed among basidiomycetous than ascomycetous species. The amount of zeatin accumulated during the experiment varied among species and strains. Highest zeatin values were recorded for basidiomycete Sporobolomyces roseus and ascomycete Taphrina sp. that produced up to 8,850.0 ng and 5,166.4 ng of zeatin per g of dry biomass, respectively. On average, the ability to produce zeatin was more pronounced among species isolated from the arctic-alpine zone than among strains from tropical and temperate climates. Our study also demonstrated that epiphytic strains and pigmented yeast species, typically for phyllosphere, are able to more often produce a plant hormone zeatin than other yeasts

Merozoite release from Plasmodium falciparum-infected erythrocytes involves the transfer of DiIC16 from infected cell membrane to Maurer’s clefts

Merozoite release from infected erythrocytes is a complex process, which is still not fully understood. Such process was characterised at ultra-structural level in this work by labelling erythrocyte membrane with a fluorescent lipid probe and subsequent photo-conversion into an electron-dense precipitate. A lipophilic DiIC16 probe was inserted into the infected erythrocyte surface and the transport of this phospholipid analogue through the erythrocyte membrane was followed up during 48 h of the asexual erythrocyte cycle. The lipid probe was transferred from infected erythrocyte membranes to Maurer’s clefts during merozoite release, thereby indicating that these membranes remained inside host cells after parasite release. Fluorescent structures were never observed inside infected erythrocytes preceding merozoite exit and merozoites released from infected erythrocyte were not fluorescent. However, specific precipitated material was localised bordering the parasitophorous vacuole membrane and tubovesicular membranes when labelled non-infected erythrocytes were invaded by merozoites. It was revealed that lipids were interchangeable from one membrane to another, passing from infected erythrocyte membrane to Maurer’s clefts inside the erythrocyte ghost, even after merozoite release. Maurer’s clefts became photo-converted following merozoite release, suggesting that these structures were in close contact with infected erythrocyte membrane during merozoite exit and possibly played some role in malarial parasite exit from the host cell

Biophysics of Malarial Parasite Exit from Infected Erythrocytes

Upon infection and development within human erythrocytes, P. falciparum induces alterations to the infected RBC morphology and bio-mechanical properties to eventually rupture the host cells through parasitic and host derived proteases of cysteine and serine families. We used previously reported broad-spectrum inhibitors (E64d, EGTA-AM and chymostatin) to inhibit these proteases and impede rupture to analyze mechanical signatures associated with parasite escape. Treatment of late-stage iRBCs with E64d and EGTA-AM prevented rupture, resulted in no major RBC cytoskeletal reconfiguration but altered schizont morphology followed by dramatic re-distribution of three-dimensional refractive index (3D-RI) within the iRBC. These phenotypes demonstrated several-fold increased iRBC membrane flickering. In contrast, chymostatin treatment showed no 3D-RI changes and caused elevated fluctuations solely within the parasitophorous vacuole. We show that E64d and EGTA-AM supported PV breakdown and the resulting elevated fluctuations followed non-Gaussian pattern that resulted from direct merozoite impingement against the iRBC membrane. Optical trapping experiments highlighted reduced deformability of the iRBC membranes upon rupture-arrest, more specifically in the treatments that facilitated PV breakdown. Taken together, our experiments provide novel mechanistic interpretations on the role of parasitophorous vacuole in maintaining the spherical schizont morphology, the impact of PV breakdown on iRBC membrane fluctuations leading to eventual parasite escape and the evolution of membrane stiffness properties of host cells in which merozoites were irreversibly trapped, recourse to protease inhibitors. These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.Singapore. Agency for Science, Technology and ResearchSingapore-MIT AllianceGlobal Enterprise for Micro-Mechanics and Molecular MedicineNational University of SingaporeNational Institutes of Health (U.S.) (Grant R01 HL094270-01A1)National Institutes of Health (U.S.) (Grant 1-R01-GM076689-01)National Institutes of Health (U.S.) (P41-RR02594-18-24

Replication of Epstein-Barr Virus Primary Infection in Human Tonsil Tissue Explants

Epstein-Barr virus (EBV) may cause a variety of virus-associated diseases, but no antiviral agents have yet been developed against this virus. Animal models are thus indispensable for the pathological analysis of EBV-related infections and the elucidation of therapeutic methods. To establish a model system for the study of EBV infection, we tested the ability of B95–8 virus and recombinant EBV expressing enhanced green fluorescent protein (EGFP) to replicate in human lymphoid tissue. Human tonsil tissues that had been surgically removed during routine tonsillectomy were sectioned into small blocks and placed on top of collagen sponge gels in culture medium at the air-interface, then a cell-free viral suspension was directly applied to the top of each tissue block. Increasing levels of EBV DNA in culture medium were observed after 12–15 days through 24 days post-infection in tissue models infected with B95–8 and EGFP-EBV. Expression levels of eight EBV-associated genes in cells collected from culture medium were increased during culture. EBV-encoded small RNA-positive cells were detected in the interfollicular areas in paraffin-embedded sections. Flow cytometric analyses revealed that most EGFP+ cells were CD3− CD56− CD19+ HLA-DR+, and represented both naïve (immunoglobulin D+) and memory (CD27+) B cells. Moreover, EBV replication in this model was suppressed by acyclovir treatment in a dose-dependent manner. These data suggest that this model has potential for use in the pathological analysis of local tissues at the time of primary infection, as well as for screening novel antiviral agents

A protease cascade regulates release of the human malaria parasite Plasmodium falciparum from host red blood cells

Malaria parasites replicate within a parasitophorous vacuole in red blood cells (RBCs). Progeny merozoites egress upon rupture of first the parasitophorous vacuole membrane (PVM), then poration and rupture of the RBC membrane (RBCM). Egress is protease-dependent1, but none of the effector molecules that mediate membrane rupture have been identified and it is unknown how sequential rupture of the two membranes is controlled. Minutes before egress, the parasite serine protease SUB1 is discharged into the parasitophorous vacuole2,3,4,5,6 where it cleaves multiple substrates2,5,7,8,9 including SERA6, a putative cysteine protease10,11,12. Here, we show that Plasmodium falciparum parasites lacking SUB1 undergo none of the morphological transformations that precede egress and fail to rupture the PVM. In contrast, PVM rupture and RBCM poration occur normally in SERA6-null parasites but RBCM rupture does not occur. Complementation studies show that SERA6 is an enzyme that requires processing by SUB1 to function. RBCM rupture is associated with SERA6-dependent proteolytic cleavage within the actin-binding domain of the major RBC cytoskeletal protein β-spectrin. We conclude that SUB1 and SERA6 play distinct, essential roles in a coordinated proteolytic cascade that enables sequential rupture of the two bounding membranes and culminates in RBCM disruption through rapid, precise, SERA6-mediated disassembly of the RBC cytoskeleton

The M18 aspartyl aminopeptidase of Plasmodium falciparum binds to human erythrocyte spectrin in vitro

<p>Abstract</p> <p>Background</p> <p>During erythrocytic schizogony, <it>Plasmodium falciparum </it>interacts with the human erythrocyte membrane when it enters into, grows within and escapes from the erythrocyte. An interaction between the <it>P. falciparum </it>M18 aspartyl aminopeptidase (<it>Pf</it>M18AAP) and the human erythrocyte membrane protein spectrin was recently identified using phage display technology. In this study, recombinant (r) <it>Pf</it>M18AAP was characterized and the interaction between the enzyme and spectrin, as well as other erythrocyte membrane proteins, analyzed.</p> <p>Methods</p> <p>r<it>Pf</it>M18AAP was produced as a hexahistidine-fusion protein in <it>Escherichia coli </it>and purified using magnetic bead technology. The pI of the enzyme was determined by two-dimensional gel electrophoresis and the number of subunits in the native enzyme was estimated from Ferguson plots. The enzymatic activity over a pH and temperature range was tested by a coupled enzyme assay. Blot overlays were performed to validate the spectrin-<it>Pf</it>M18AAP interaction, as well as identify additional interactions between the enzyme and other erythrocyte membrane proteins. Sequence analysis identified conserved amino acids that are expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization.</p> <p>Results</p> <p>r<it>Pf</it>M18AAP has a molecular weight of ~67 kDa and the enzyme separated as three entities with pI 6.6, 6.7 and 6.9. Non-denaturing gel electrophoresis indicated that r<it>Pf</it>M18AAP aggregated into oligomers. An <it>in vitro </it>coupled enzyme assay showed that r<it>Pf</it>M18AAP cleaved an N-terminal aspartate from a tripeptide substrate with maximum enzymatic activity at pH 7.5 and 37°C. The spectrin-binding region of <it>Pf</it>M18AAP is not found in <it>Homo sapiens, Saccharomyces cerevisiae </it>and other<it>Plasmodium </it>species homologues. Amino acids expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization, are conserved. Blot overlays with r<it>Pf</it>M18AAP against spectrin and erythrocyte membrane proteins indicated that r<it>Pf</it>M18AAP binds to spectrin, as well as to protein 4.1, protein 4.2, actin and glyceraldehyde 3-phosphate dehydrogenase.</p> <p>Conclusion</p> <p>Studies characterizing r<it>Pf</it>M18AAP showed that this enzyme interacts with erythrocyte spectrin and other membrane proteins. This suggests that, in addition to its proposed role in hemoglobin digestion, <it>Pf</it>M18AAP performs other functions in the erythrocyte host and can utilize several substrates, which highlights the multifunctional role of malaria enzymes.</p

Efficient Transduction of Vascular Endothelial Cells with Recombinant Adeno-Associated Virus Serotype 1 and 5 Vectors

Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC). rAAV vectors with AAV2 inverted terminal repeats containing the human α1-antitrypsin (hAAT) gene were transcapsidated using helper plasmids to provide viral capsids for the AAV1 through 5 serotypes. True type rAAV2 and 5 vectors encoding β-galactosidase or green fluorescence protein were also studied. Infection with rAAV1 resulted in the most efficient transduction in both HAEC and RAEC compared to other serotypes (p < 0.001) at 7 days posttransduction. Interestingly, expression was increased in cells transduced with rAAV5 to levels surpassing rAAV1 by day 14 and 21. Transduction with rAAV1 was completely inhibited by removal of sialic acid with sialidase, while heparin had no effect. These studies are the first demonstration that sialic acid residues are required for rAAV1 transduction in endothelial cells. Transduction of rat aortic segments ex vivo and in vivo demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors, in comparison to rAAV2. These results suggest the unique potential of rAAV1 and rAAV5-based vectors for vascular-targeted gene-based therapeutic strategies