Rho GTPases Modulate Entry of Ebola Virus and Vesicular Stomatitis Virus Pseudotyped Vectors

To explore mechanisms of entry for Ebola virus (EBOV) glycoprotein (GP) pseudotyped virions, we used comparative gene analysis to identify genes whose expression correlated with viral transduction. Candidate genes were identified by using EBOV GP pseudotyped virions to transduce human tumor cell lines that had previously been characterized by cDNA microarray. Transduction profiles for each of these cell lines were generated, and a significant positive correlation was observed between RhoC expression and permissivity for EBOV vector transduction. This correlation was not specific for EBOV vector alone as RhoC also correlated highly with transduction of vesicular stomatitis virus GP (VSVG) pseudotyped vector. Levels of RhoC protein in EBOV and VSV permissive and nonpermissive cells were consistent with the cDNA gene array findings. Additionally, vector transduction was elevated in cells that expressed high levels of endogenous RhoC but not RhoA. RhoB and RhoC overexpression significantly increased EBOV GP and VSVG pseudotyped vector transduction but had minimal effect on human immunodeficiency virus (HIV) GP pseudotyped HIV or adeno-associated virus 2 vector entry, indicating that not all virus uptake was enhanced by expression of these molecules. RhoB and RhoC overexpression also significantly enhanced VSV infection. Similarly, overexpression of RhoC led to a significant increase in fusion of EBOV virus-like particles. Finally, ectopic expression of RhoC resulted in increased nonspecific endocytosis of fluorescent dextran and in formation of increased actin stress fibers compared to RhoA-transfected cells, suggesting that RhoC is enhancing macropinocytosis. In total, our studies implicate RhoB and RhoC in enhanced productive entry of some pseudovirions and suggest the involvement of actin-mediated macropinocytosis as a mechanism of uptake of EBOV GP and VSVG pseudotyped viral particles.Enveloped viruses enter cells by a variety of different pathways. Productive internalization of enveloped viruses with targeted cells is mediated through interactions of the viral glycoprotein(s) (GPs) with moieties on the surface of the cell. In general, enveloped viral entry occurs through viral adherence to the cell surface, interaction with a specific plasma membrane-associated receptor that results in a series of GP conformational changes leading to fusion of viral and cellular membranes, and delivery of the viral core particle into the cytoplasm. Fusion of the two membranes can occur at the plasma membrane or by uptake of the intact virions into endosomes with subsequent membrane fusion between the viral membrane and the lipid bilayer of the endocytic vesicle. Human immunodeficiency virus (HIV) is an example of a virus that fuses directly to the plasma membrane (5), whereas influenza virus must be internalized into acidified vesicles where the appropriate GP conformational changes can occur, mediating membrane fusion (21). Most enveloped viruses that enter through vesicles utilize a low-pH environment to mediate the necessary conformational changes in GP that induce membrane fusion (37).Ebola virus (EBOV) and vesicular stomatitis virus (VSV) are enveloped, single-stranded, negative-sense RNA viruses belonging to the families Filoviridae and Rhabdoviridae, respectively. Though they share similarity in genome organization and a broad tropism for a variety of cell types, they differ greatly in their pathogenicities (29, 39). EBOV causes severe hemorrhagic fever that is frequently fatal, whereas VSV infects mainly livestock, generating fluid-filled vesicles on mucosal surfaces.Interestingly, the receptor(s) that mediate entry of these two viruses have yet to be definitively identified. C-type lectins such as DC-SIGN and DC-SIGNR are thought to serve as adherence factors for EBOV (26). Other plasma membrane-associated proteins have been implicated in EBOV uptake including folate receptor alpha and the tyrosine kinase receptor Axl (6, 35, 36, 38), but the physical interaction of EBOV GP and these proteins has not been demonstrated, and cells that do not express these proteins are permissive for EBOV GP-mediated virion uptake. VSV was shown to bind ubiquitously to cells via phosphatidylserine (PS) (31). However, a more recent study reports that PS is not a receptor for VSV as no correlation was found between cell surface PS levels and VSV infection, and annexin V, which binds specifically to PS, did not inhibit infection of VSV (9).Both viruses enter cells through a low-pH-dependent, endocytosis-mediated process. A large body of evidence indicates that VSV is internalized via clathrin-coated pits, with a reduction in pH mediating reversible alterations in the GP leading to membrane fusion (40). EBOV may also enter cells by clathrin-mediated endocytosis (30), but lipid raft-associated, caveolin-mediated endocytosis has also been proposed as a mechanism of EBOV uptake (11). Low-pH events lead to cathepsin-dependent cleavage of EBOV GP that is required for productive uptake of the virus (8, 19, 33). Other low-pH-dependent events have been postulated to be required as well (33).To identify genes whose expression correlated with EBOV GP-dependent transduction, we compared the relative transduction efficiency of EBOV GP pseudotyped virions on a panel of human tumor cell lines with gene expression data from cDNA microarrays developed for the same panel of cell lines (20). The gene array data are available from the Developmental Therapeutics Program at the National Cancer Institute (NCI) website (http://dtp.nci.nih.gov/). A significant correlation was observed between expression of RhoC, a member of the small GTP-binding Rho GTPase family, and permissivity for EBOV transduction. Surprisingly, a significant correlation was also observed between VSV glycoprotein (VSVG)-mediated transduction and RhoC expression. In this study, we report that modulation of RhoC expression by transfection of expression plasmids or treatment with an inhibitor alters transduction by virions pseudotyped with either EBOV GP or VSVG and fusion of EBOV virus-like particles (VLPs). RhoC expression also significantly enhanced wild-type VSV infection. We also examine the differential effect each Rho GTPase has on nonspecific endocytotic uptake of exogenous material and on organization of the actin filament. Our findings suggest that RhoC enhances entry of EBOV GP and VSVG pseudovirions through modulation of fluid-phase endocytosis.     

Influence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence

Background

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family) from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models.

Methodology/Principal Findings

We followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes) obtained during sequential sub-cultivation in mammalian cells. ATPase/ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin), or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites.

Conclusions/Significance

Our results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target candidates in chemotherapy of Chagas disease.     

Tracking and quantification of P-labeled phosphopeptides in liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry

Phosphoamino acid modifications on substrate proteins are critical components of protein kinase signaling pathways. Thus, diverse methodologies have been developed and applied to identify the sites of phosphorylated amino acids within proteins. Despite significant progress in the field, even the determination of phosphorylated residues in a given highly purified protein is not a matter of routine and can be difficult and time-consuming. Here we present a practicable approach that integrates into a liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry (LC–MALDI MS) workflow and allows localization and quantification of phosphorylated peptides on the MALDI target plate prior to MS analysis. Tryptic digests of radiolabeled proteins are fractionated by reversed-phase LC directly onto disposable MALDI target plates, followed by autoradiographic imaging. Visualization of the radiolabel enables focused analysis of selected spots, thereby accelerating the process of phosphorylation site mapping by decreasing the number of spectra to be acquired. Moreover, absolute quantification of the phosphorylated peptides is permitted by the use of appropriate standards. Finally, the manual sample handling is minimal, and consequently the risk of adsorptive sample loss is very low. Application of the procedure allowed the targeted identification of six novel autophosphorylation sites of AMP-activated protein kinase (AMPK) and displayed additional unknown phosphorylated peptide species not amenable to detection by MS. Furthermore, autoradiography revealed topologically inhomogeneous distribution of phosphorylated peptides within individual spots. However, accurate analysis of defined areas within single spots suggests that, rather than such quantitative differences, mainly the manner of matrix crystallization significantly affects ionization of phosphopeptides.     

Deletion of BCY1 from the Saccharomyces cerevisiae genome is semidominant and induces autolytic phenotypes suitable for improvement of sparkling wines

Autolysis of Saccharomyces cerevisiae is the main source of molecules that contribute to the quality of sparkling wines made by the traditional method. In this work the possibility of accelerating this slow process in order to improve the quality of sparkling wines by using genetically engineered wine yeast strains was explored. The effect of partial or total deletion of BCY1 (which encodes a regulatory subunit of cAMP-dependent protein kinase A) in haploid and diploid (heterozygous and homozygous) yeast strains was studied. We proved that heterozygous strains having partial or complete BCY1 deletions have a semidominant phenotype for several of the properties studied, including autolysis under simulated second-fermentation conditions, in contrast to previously published reports describing mutations in BCY1 as recessive. Considering the degree of autolysis, ethanol tolerance, and technical feasibility, we propose that deletion of the 3' end of the open reading frame of a single copy of BCY1 is a way to improve the quality of sparkling wines.     

A multiple PCR-primer approach to access the microeukaryotic diversity in environmental samples

The Cariaco Basin off the Venezuelan coast in the Caribbean Sea is the world's largest truly marine body of anoxic water. The first rRNA survey of microbial eukaryotes in this environment revealed a number of novel lineages, but sampled only a fraction of the entire diversity. The goal of this study was to significantly improve recovery of protistan rRNA from the Basin. This was achieved by a systematic application of multiple PCR primer sets and substantially larger sequencing efforts. We focused on the most diverse habitat in the basin, anoxic waters approximately 100m below the oxic-anoxic interface, and detected novel lineages that escaped the single PCR primer approach. All clones obtained proved unique. A 99% sequence similarity cut-off value combined these clones into operational taxonomic units (OTUs), over 75% of which proved novel. Some of these OTUs form deep branches within established protistan groups. Others signify discovery of novel protistan lineages that appear unrelated to any known microeukaryote. Surprisingly, even this large-scale multi-primer rRNA approach still missed a substantial part of the samples' rRNA diversity. The overlap between the species lists obtained with different primers is low, with only 4% of OTUs shared by all three libraries, and the number of species detected only once is large (55%). This strongly indicates that, at least in anoxic environments, protistan diversity may be much larger than is commonly thought. A single sample appears to contain thousands of largely novel protistan species. Multiple PCR primer combinations may be needed to capture these species.     

Influence of head models on neuromagnetic fields and inverse source localizations

Background  

The magnetoencephalograms (MEGs) are mainly due to the source currents. However, there is a significant contribution to MEGs from the volume currents. The structure of the anatomical surfaces, e.g., gray and white matter, could severely influence the flow of volume currents in a head model. This, in turn, will also influence the MEGs and the inverse source localizations. This was examined in detail with three different human head models.     

Time-related improvement of survival in resectable gastric cancer: the role of Japanese-style gastrectomy with D2 lymphadenectomy and adjuvant chemotherapy

Background

Late presentation of patients at advanced stages when little or no benefit can be derived from any form of therapy is the hallmark of breast cancer in Nigerian women. Recent global cancer statistics indicate rising global incidence of breast cancer and the increase is occurring at a faster rate in populations of the developing countries that hitherto enjoyed low incidence of the disease. Worried by this prevailing situation and with recent data suggesting that health behavior may be influenced by level of awareness about breast cancer, a cross-sectional study was designed to assess the knowledge, attitude and practices of community-dwelling women in Nigeria towards breast cancer.

Methods

One thousand community-dwelling women from a semi-urban neighborhood in Nigeria were recruited for the study in January and February 2000 using interviewer-administered questionnaires designed to elicit sociodemographic information and knowledge, attitude and practices of these women towards breast cancer. Data analysis was carried out using Statistical Analysis Software (SAS) version 8.2.

Results

Study participants had poor knowledge of breast cancer. Mean knowledge score was 42.3% and only 214 participants (21.4%) knew that breast cancer presents commonly as a painless breast lump. Practice of breast self examination (BSE) was low; only 432 participants (43.2%) admitted to carrying out the procedure in the past year. Only 91 study participants (9.1%) had clinical breast examination (CBE) in the past year. Women with higher level of education (X² = 80.66, p < 0.0001) and those employed in professional jobs (X² = 47.11, p < 0.0001) were significantly more knowledgeable about breast cancer. Participants with higher level of education were 3.6 times more likely to practice BSE (Odds ratio [OR] = 3.56, 95% Confidence interval [CI] 2.58–4.92).

Conclusion

The results of this study suggest that community-dwelling women in Nigeria have poor knowledge of breast cancer and minority practice BSE and CBE. In addition, education appears to be the major determinant of level of knowledge and health behavior among the study participants. We recommend the establishment and sustenance of institutional framework and policy guidelines that will enhance adequate and urgent dissemination of information about breast cancer to all women in Nigeria.