Rescue of Infectious Sindbis Virus by Yeast Spheroplast-Mammalian Cell Fusion.

Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites. SINV reverse genetics have many implications, not only in understanding alphavirus transmission, replication cycle, and virus-host interactions, but also in biotechnology and biomedical applications. The rescue of SINV infectious particles is usually achieved by transfecting susceptible cells (BHK-21) with SINV-infectious mRNA genomes generated from cDNA constructed via in vitro translation (IVT). That procedure is time consuming, costly, and relies heavily on reagent quality. Here, we constructed a novel infectious SINV cDNA construct that expresses its genomic RNA in yeast cells controlled by galactose induction. Using spheroplasts made from this yeast, we established a robust polyethylene glycol-mediated yeast: BHK-21 fusion protocol to rescue infectious SINV particles. Our approach is timesaving and utilizes common lab reagents for SINV rescue. It could be a useful tool for the rescue of large single strand RNA viruses, such as SARS-CoV-2.

Identification and Molecular Characterization of Novel Mycoviruses in Saccharomyces and Non-Saccharomyces Yeasts of Oenological Interest.

Wine yeasts can be natural hosts for dsRNA, ssRNA viruses and retrotransposon elements. In this study, high-throughput RNA sequencing combined with bioinformatic analyses unveiled the virome associated to 16 Saccharomyces cerevisiae and 8 non-Saccharomyces strains of oenological interest. Results showed the presence of six viruses and two satellite dsRNAs from four different families, two of which-Partitiviridae and Mitoviridae-were not reported before in yeasts, as well as two ORFan contigs of viral origin. According to phylogenetic analysis, four new putative mycoviruses distributed in Totivirus, Cryspovirus, and Mitovirus genera were identified. The majority of commercial S. cerevisiae strains were confirmed to be the host for helper L-A type totiviruses and satellite M dsRNAs associated with the killer phenotype, both in single and mixed infections with L-BC totiviruses, and two viral sequences belonging to a new cryspovirus putative species discovered here for the first time. Moreover, single infection by a narnavirus 20S-related sequence was also found in one S. cerevisiae strain. Considering the non-Saccharomyces yeasts, Starmerella bacillaris hosted four RNAs of viral origin-two clustering in Totivirus and Mitovirus genera, and two ORFans with putative satellite behavior. This study confirmed the infection of wine yeasts by viruses associated with useful technological characteristics and demonstrated the presence of complex mixed infections with unpredictable biological effects.

Prevalence, antifungal susceptibility and virulence determinants of oral yeast species isolated from immunodeficient patients in Northeastern Brazil / Prevalência, suscetibilidade a antifúngicos e determinantes de virulência de leveduras orais isoladas de pacientes imunodeficientes do Nordeste do Brasil / Prevalencia, susceptibilidad antifúngica y determinantes de virulencia de levaduras orales aisladas de pacientes inmunodeficientes del Noreste de Brasil

Justificativa e Objetivos: A candidíase oral tem uma ocorrência comum em pacientes imunocomprometidos. No entanto, outras infecções emergentes tornaram-se cada vez mais habituais. O objetivo deste estudo foi investigar a prevalência, os determinantes de virulência e a suscetibilidade a antifúngicos de leveduras que colonizam a mucosa de pacientes imunocomprometidos na região Nordeste do Brasil. Métodos: A amostra foi composta por 60 pacientes HIV positivos atendidos no Serviço de Atendimento Especializado/Hospital Dia do Hospital Universitário Prof. Alberto Antunes, vinculado à Universidade Federal de Alagoas. As amostras foram coletadas em regiões subgengivais e semeadas em CHROMagar para confirmação presuntiva de Candida spp., seguido por PCR e sequenciamento. Além disso, testamos os determinantes de virulência fosfolipase e protease e avaliamos in vitro a concentração inibitória mínima dos antifúngicos anfotericina B e fluconazol. Este projeto foi aprovado pelo Comitê de ética em pesquisa do Centro de Estudos Superiores de Maceió. Resultados: Aproximadamente 63% dos pacientes foram colonizados por leveduras. A espécie C. albicans foi predominante, enquanto as espécies de Candida não-albicans representaram 49% dos isolados, sendo C. dubliniensis e C. parapsilosis as mais comuns. Entretanto, C. intermedia, Bullera penniseticola e Naganishia liquefaciens também foram encontrados. Os determinantes da virulência protease e/ou fosfolipase também foram produzidos por Candida spp. e alguns isolados oportunistas incomuns como Kodamaea ohmeri, N. liquefaciens e Saitozyma podzolica. Além disso, a maioria dos isolados de Candida spp. e algumas espécies oportunistas incomuns apresentaram altos valores de concentração inibitória mínima. Conclusão: Os resultados obtidos indicam que C. albicans continua a ser a espécie predominante na cavidade oral de pacientes imunodeficientes e, juntamente com outras espécies incomuns, pode apresentar alta resistência aos antifúngicos testados.(AU)

Background and Objectives: Oral candidiasis has a common occurrence in immunocompromised patients. However, other emergent infections have become increasingly common. The aim of this study was to investigate the prevalence, virulence determinants and the antifungal susceptibility of yeast colonizing the mucosa of immunocompromised patients in Northeastern Brazil. Methods: Samples from sixty HIV-positive patients seen at the Specialized Service / Hospital Dia - Hospital Universitário Prof. Alberto Antunes from the Federal University of Alagoas were collected from subgingival sites and seeded on CHROMagar for presumptive confirmation of Candida spp. followed by PCR and sequencing. In addition, we tested virulence determinants, phospholipase and protease and evaluated in vitro the Minimum Inhibitory Concentration of antifungals amphotericin B and fluconazole. This project was approved by the Research Ethics Committee of the Center for Higher Studies in Maceió. Results: Approximately 63% of the patients were colonized by yeasts, with C. albicans as the predominant species, while non-Candida albicans species accounted for 49% of the isolates, with C. dubliniensis and C. parapsilosis being the commonest, but C. intermedia, Bullera penniseticola and Naganishia liquefaciens were also found. The virulence determinants protease and/or phospholipase were also produced by Candida spp. and some uncommon opportunistic isolates such as Kodamaea ohmeri, N. liquefaciens and Saitozyma podzolica. Furthermore, most of Candida spp. strains and some uncommon opportunistic species showed high values of minimal inhibitory concentration. Conclusion: Results obtained indicate that C. albicans continues to be the predominant species in oral cavity of immunodeficient patients and along with other unusual species may present high resistance to the antifungals tested.(AU)

Justificación y Objetivos: La candidiasis oral acomete con frecuencia a pacientes inmunocomprometidos. Sin embargo, otras infecciones emergentes se han vuelto cada vez más comunes. El objetivo de este estudio fue investigar la prevalencia, la producción de determinantes de virulencia y la susceptibilidad a antifúngicos de levaduras que colonizan la mucosa de pacientes inmunocomprometidos en la región Nordeste de Brasil. Métodos: Se colectaron muestras de sesenta pacientes VIH positivos atendidos en el Servicio de Atención Especializado/Hospital Día del Hospital Universitario Prof. Alberto Antunes, vinculado a la Universidad Federal de Alagoas. Se colectaron las muestras en las regiones subgingivales y las sembraron en CHROMagar para la presunta confirmación de Candida spp. seguido de PCR y secuenciación. Además, analizamos los determinantes de virulencia fosfolipasa y proteasa y evaluamos in vitro la concentración mínima inhibitoria de los antifúngicos anfotericina B y fluconazol. Este proyecto fue aprobado por el Comité de Ética en Investigación del Centro de Estudios Superiores de Maceió. Resultados: Aproximadamente el 63% de los pacientes fueron colonizados por levaduras, y la C. albicans fue la especie predominante, mientras que las especies de Candida no-albicans representaron el 49% de los aislamientos, de las cuales la C. dubliniensis y la C. parapsilosis fueron las más comunes. Sin embargo, también se encontraron C. intermedia, Bullera penniseticola y Naganishia liquefaciens. Los determinantes de virulencia de proteasa y/o fosfolipasa también fueron producidos por Candida spp. y algunos aislados oportunistas inusuales como Kodamaea ohmeri, N. liquefaciens y Saitozyma podzolica. Además, la mayoría de los asilados de Candida spp. y algunas especies oportunistas inusuales mostraron valores altos de concentración mínima inhibitoria. Conclusión: Los resultados obtenidos indican que C. albicans continúa siendo la especie predominante en la cavidad oral de pacientes inmunodeprimidos y, junto con otras especies poco comunes, puede presentar una alta resistencia a los antifúngicos evaluados.(AU)

Fatores de virulência e susceptibilidade a antifúngicos de Cryptococcus Spp / Virulence factors and susceptibility to Cryptococcus Spp. antifungals

Criptococose é uma doença grave que afeta tanto imunocomprometidos quanto imunocompetentes, com isso analisar a virulência é fundamental para novas terapêuticas. Objetivo: Analisar a capacidade de virulência e susceptibilidade aos antifúngicos de Cryptococcus spp. isolados de líquor de pacientes de hospital do norte do Paraná. Métodos: A partir de dois isolados clínicos C. neoformans e C. gattii, realizou-se a confirmação da identificação. Para a virulência, avaliou-se o tamanho da cápsula, capacidade de sobrevivência após exposição a neutrófilos, produção de melanina e urease. No antifungigrama por difusão em disco utilizou-se: anfotericina B, cetoconazol, voriconazol, itraconazol e miconazol. Resultados: C. gattii destaca-se por maior desenvolvimento da cápsula além da melhor capacidade de sobreviver a fagocitose em relação ao C. neoformans. No antifungigrama, ambos os isolados se apresentam sensíveis às drogas estudadas. Conclusão: Esses achados contribuem para a compreensão das diferentes patogêneses entre C. gattii e C. neoformans.

Cryptococcosis is a serious disease that can affect both immunocompromised and immunocompetent individuals, thus the virulence analysis is fundamental for the development of new treatments. Objective: To analyze the virulence and susceptibility of Cryptococcus spp. isolated from cerebrospinal fluid of patients from a hospital in the north of Paraná. Methods: From two clinical isolates, C. neoformans and C. gattii were confirmed and identified. For virulence, capsule size, survival capacity after exposure to neutrophils, melanin production and urease were evaluated. In the disc-diffusion method, the following antifungals were used: amphotericin B, ketoconazole, voriconazole, itraconazole and miconazole Results: It was observed that C. gattii presents greater results for development of the capsule beside presenting the best ability to survive phagocytosis in relation to C. neoformans. In the disc-diffusion method, both isolates presented sensitivity to the studied drugs. Conclusion: These findings contribute to the understanding of the different pathogens between C. gattii and C. neoformans.

The ecology of killer yeasts: Interference competition in natural habitats.

Killer yeasts are ubiquitous in the environment: They have been found in diverse habitats ranging from ocean sediment to decaying cacti to insect bodies and on all continents including Antarctica. However, environmental killer yeasts are poorly studied compared with laboratory and domesticated killer yeasts. Killer yeasts secrete so-called killer toxins that inhibit nearby sensitive yeasts, and the toxins are frequently assumed to be tools for interference competition in diverse yeast communities. The diversity and ubiquity of killer yeasts imply that interference competition is crucial for shaping yeast communities. Additionally, these toxins may have ecological functions beyond use in interference competition. This review introduces readers to killer yeasts in environmental systems, with a focus on what is and is not known about their ecology and evolution. It also explores how results from experimental killer systems in laboratories can be extended to understand how competitive strategies shape yeast communities in nature. Overall, killer yeasts are likely to occur everywhere yeasts are found, and the killer phenotype has the potential to radically shape yeast diversity in nature.

Isolation and Characterization of Extrachromosomal Double-Stranded RNA Elements from Carotenogenic Yeasts.

Double-stranded RNA (dsRNA) molecules are widely found in yeasts and filamentous fungi. It has been suggested that these molecules may play an important role in the evolution of eukaryote genomes and could be a valuable tool in yeast typing. The characterization of these extrachromosomal genetic elements is usually a laborious process, especially when trying to analyze a large number of samples. In this chapter, we describe a simple method to isolate dsRNA elements from yeasts using low amounts of starting material and their application to different Xanthophyllomyces dendrorhous strains and other psychrotolerant carotenogenic yeasts. Furthermore, the methodologies for enzymatic and hybridization characterizations and quantification of relative dsRNA abundance are detailed.

Recombinant Expression of Tandem-HBc Virus-Like Particles (VLPs).

The hepatitis B virus (HBV) core protein (HBc) has formed the building block for virus-like particle (VLP) production for more than 30 years. The ease of production of the protein, the robust ability of the core monomers to dimerize and assemble into intact core particles, and the strong immune responses they elicit when presenting antigenic epitopes all demonstrate its promise for vaccine development (reviewed in Pumpens and Grens (Intervirology 44: 98-114, 2001)). HBc has been modified in a number of ways in attempts to expand its potential as a novel vaccine platform. The HBc protein is predominantly α-helical in structure and folds to form an L-shaped molecule. The structural subunit of the HBc particle is a dimer of monomeric HBc proteins which together form an inverted T-shaped structure. In the assembled HBc particle the four-helix bundle formed at each dimer interface appears at the surface as a prominent "spike." The tips of the "spikes" are the preferred sites for the insertion of foreign sequences for vaccine purposes as they are the most highly exposed regions of the assembled particles. In the tandem-core modification two copies of the HBc protein are covalently linked by a flexible amino acid sequence which allows the fused dimer to fold correctly and assemble into HBc particles. The advantage of the modified structure is that the assembly of the dimeric subunits is defined and not formed by random association. This facilitates the introduction of single, larger sequences at the tip of each surface "spike," thus overcoming the conformational clashes contingent on insertion of large structures into monomeric HBc proteins.Differences in inserted sequences influence the assembly characteristics of the modified proteins, and it is important to optimize the design of each novel construct to maximize efficiency of assembly into regular VLPs. In addition to optimization of the construct, the expression system used can also influence the ability of recombinant structures to assemble into regular isometric particles. Here, we describe the production of recombinant tandem-core particles in bacterial, yeast and plant expression systems.

Translation, modification and cellular distribution of two AC4 variants of African cassava mosaic virus in yeast and their pathogenic potential in plants.

Plant infecting geminiviruses encode a small (A)C4 protein within the open reading frame of the replication-initiator protein. In African cassava mosaic virus, two in-frame start codons may be used for the translation of a longer and a shorter AC4 variant. Both were fused to green fluorescent protein or glutathione-S-transferase genes and expressed in fission yeast. The longer variant accumulated in discrete spots in the cytoplasm, whereas the shorter variant localized to the plasma membrane. A similar expression pattern was found in plants. A myristoylation motif may promote a targeting of the shorter variant to the plasma membrane. Mass spectrometry analysis of the yeast-expressed shorter variant detected the corresponding myristoylation. The biological relevance of the second start codon was confirmed using mutated infectious clones. Whereas mutating the first start codon had no effect on the infectivity in Nicotiana benthamiana plants, the second start codon proved to be essential.

Detection of Hepatitis C core antibody by dual-affinity yeast chimera and smartphone-based electrochemical sensing.

Yeast cell lines were genetically engineered to display Hepatitis C virus (HCV) core antigen linked to gold binding peptide (GBP) as a dual-affinity biobrick chimera. These multifunctional yeast cells adhere to the gold sensor surface while simultaneously acting as a "renewable" capture reagent for anti-HCV core antibody. This streamlined functionalization and detection strategy removes the need for traditional purification and immobilization techniques. With this biobrick construct, both optical and electrochemical immunoassays were developed. The optical immunoassays demonstrated detection of anti-HCV core antibody down to 12.3pM concentrations while the electrochemical assay demonstrated higher binding constants and dynamic range. The electrochemical format and a custom, low-cost smartphone-based potentiostat ($20 USD) yielded comparable results to assays performed on a state-of-the-art electrochemical workstation. We propose this combination of synthetic biology and scalable, point-of-care sensing has potential to provide low-cost, cutting edge diagnostic capability for many pathogens in a variety of settings.

The Phospholipid:Diacylglycerol Acyltransferase Lro1 Is Responsible for Hepatitis C Virus Core-Induced Lipid Droplet Formation in a Yeast Model System.

Chronic infection with the hepatitis C virus frequently induces steatosis, which is a significant risk factor for liver pathogenesis. Steatosis is characterized by the accumulation of lipid droplets in hepatocytes. The structural protein core of the virus induces lipid droplet formation and localizes on the surface of the lipid droplets. However, the precise molecular mechanisms for the core-induced formation of lipid droplets remain elusive. Recently, we showed that the expression of the core protein in yeast as a model system could induce lipid droplet formation. In this study, we probed the cellular factors responsible for the formation of core-induced lipid-droplets in yeast cells. We demonstrated that one of the enzymes responsible for triglyceride synthesis, a phospholipid:diacylglycerol acyltransferase (Lro1), is required for the core-induced lipid droplet formation. While core proteins inhibit Lro1 degradation and alter Lro1 localization, the characteristic localization of Lro1 adjacent to the lipid droplets appeared to be responsible for the core-induced lipid droplet formation. RNA virus genomes have evolved using high mutation rates to maintain their ability to replicate. Our observations suggest a functional relationship between the core protein with hepatocytes and yeast cells. The possible interactions between core proteins and the endoplasmic reticulum membrane affect the mobilization of specific proteins.

The hop-like stress-induced protein 1 cochaperone is a novel cell-intrinsic restriction factor for mitochondrial tombusvirus replication.

UNLABELLED: Recent genome-wide screens reveal that the host cells express an arsenal of proteins that inhibit replication of plus-stranded RNA viruses by functioning as cell-intrinsic restriction factors of viral infections. One group of cell-intrinsic restriction factors against tombusviruses contains tetratricopeptide repeat (TPR) domains that directly interact with the viral replication proteins. In this paper, we find that the TPR domain-containing Hop-like stress-inducible protein 1 (Sti1p) cochaperone selectively inhibits the mitochondrial membrane-based replication of Carnation Italian ringspot tombusvirus (CIRV). In contrast, Sti1/Hop does not inhibit the peroxisome membrane-based replication of the closely related Tomato bushy stunt virus (TBSV) or Cucumber necrosis virus (CNV) in a yeast model or in plants. Deletion of STI1 in yeast leads to up to a 4-fold increase in CIRV replication, and knockdown of the orthologous Hop cochaperone in plants results in a 3-fold increase in CIRV accumulation. Overexpression of Sti1p derivatives in yeast reveals that the inhibitory function depends on the TPR1 domain known to interact with heat shock protein 70 (Hsp70), but not on the TPR2 domain interacting with Hsp90. In vitro CIRV replication studies based on isolated mitochondrial preparations and purified recombinant proteins has confirmed that Sti1p, similar to the TPR-containing Cyp40-like Cpr7p cyclophilin and the Ttc4 oncogene-like Cns1 cochaperone, is a strong inhibitor of CIRV replication. Sti1p interacts and colocalizes with the CIRV replication proteins in yeast. Our findings indicate that the TPR-containing Hop/Sti1 cochaperone could act as a cell-intrinsic virus restriction factor of the mitochondrial CIRV, but not against the peroxisomal tombusviruses in yeast and plants. IMPORTANCE: The host cells express various cell-intrinsic restriction factors that inhibit the replication of plus-stranded RNA viruses. In this paper, the authors find that the Hop-like stress-inducible protein 1 (Sti1p) cochaperone selectively inhibits the mitochondrial membrane-based replication of Carnation Italian ringspot tombusvirus (CIRV) in yeast. Deletion of STI1 in yeast or knockdown of the orthologous Hop cochaperone in plants leads to increased CIRV replication. In addition, overexpression of Sti1p derivatives in yeast reveals that the inhibitory function depends on the TPR1 domain known to interact with heat shock protein 70 (Hsp70), but not on the TPR2 domain interacting with Hsp90. In vitro CIRV replication studies based on isolated mitochondrial preparations and purified recombinant proteins have confirmed that Sti1p is a strong inhibitor of CIRV replication. The authors' findings reveal that the Hop/Sti1 cochaperone could act as a cell-intrinsic restriction factor against the mitochondrial CIRV, but not against the related peroxisomal tombusviruses.

Inferring host gene subnetworks involved in viral replication.

Systematic, genome-wide loss-of-function experiments can be used to identify host factors that directly or indirectly facilitate or inhibit the replication of a virus in a host cell. We present an approach that combines an integer linear program and a diffusion kernel method to infer the pathways through which those host factors modulate viral replication. The inputs to the method are a set of viral phenotypes observed in single-host-gene mutants and a background network consisting of a variety of host intracellular interactions. The output is an ensemble of subnetworks that provides a consistent explanation for the measured phenotypes, predicts which unassayed host factors modulate the virus, and predicts which host factors are the most direct interfaces with the virus. We infer host-virus interaction subnetworks using data from experiments screening the yeast genome for genes modulating the replication of two RNA viruses. Because a gold-standard network is unavailable, we assess the predicted subnetworks using both computational and qualitative analyses. We conduct a cross-validation experiment in which we predict whether held-aside test genes have an effect on viral replication. Our approach is able to make high-confidence predictions more accurately than several baselines, and about as well as the best baseline, which does not infer mechanistic pathways. We also examine two kinds of predictions made by our method: which host factors are nearest to a direct interaction with a viral component, and which unassayed host genes are likely to be involved in viral replication. Multiple predictions are supported by recent independent experimental data, or are components or functional partners of confirmed relevant complexes or pathways. Integer program code, background network data, and inferred host-virus subnetworks are available at http://www.biostat.wisc.edu/~craven/chasman_host_virus/.

Yeast screens for host factors in positive-strand RNA virus replication based on a library of temperature-sensitive mutants.

RNA viruses exploit host cells by altering cellular pathways, recruiting host factors, remodeling intracellular membranes and escaping host antiviral responses. Model hosts, such as Saccharomyces cerevisiae (yeast), are valuable to identify host factors involved in viral RNA replication. The many advantages of using yeast include the availability of various yeast mutant libraries, such as (i) single gene-deletion library; (ii) the essential gene library (yTHC); and (iii) the yeast ORF over-expression library. Here, we have used a novel temperature-sensitive (ts) mutant library of essential yeast genes to identify 118 host proteins affecting replication of Tomato bushy stunt virus, in yeast model host. Testing 787 ts mutants led to the identification of host factors, of which 72 proteins facilitated TBSV replication in yeast and 46 proteins were inhibitory. Altogether, ~85% of the identified proteins are novel host factors affecting tombusvirus replication. The ts mutant library screen also led to the identification of 17 essential genes, which have been documented before, thus confirming the importance of these genomic screens. Overall, we show the power of ts mutant library in identification of host factors for RNA virus replication.

Host acyl coenzyme A binding protein regulates replication complex assembly and activity of a positive-strand RNA virus.

All positive-strand RNA viruses reorganize host intracellular membranes to assemble their replication complexes. Similarly, brome mosaic virus (BMV) induces two alternate forms of membrane-bound RNA replication complexes: vesicular spherules and stacks of appressed double-membrane layers. The mechanisms by which these membrane rearrangements are induced, however, remain unclear. We report here that host ACB1-encoded acyl coenzyme A (acyl-CoA) binding protein (ACBP) is required for the assembly and activity of both BMV RNA replication complexes. ACBP is highly conserved among eukaryotes, specifically binds to long-chain fatty acyl-CoA, and promotes general lipid synthesis. Deleting ACB1 inhibited BMV RNA replication up to 30-fold and resulted in formation of spherules that were ∼50% smaller but ∼4-fold more abundant than those in wild-type (wt) cells, consistent with the idea that BMV 1a invaginates and maintains viral spherules by coating the inner spherule membrane. Furthermore, smaller and more frequent spherules were preferentially formed under conditions that induce layer formation in wt cells. Conversely, cellular karmella structures, which are arrays of endoplasmic reticulum (ER) membranes formed upon overexpression of certain cellular ER membrane proteins, were formed normally, indicating a selective inhibition of 1a-induced membrane rearrangements. Restoring altered lipid composition largely complemented the BMV RNA replication defect, suggesting that ACBP was required for maintaining lipid homeostasis. Smaller and more frequent spherules are also induced by 1a mutants with specific substitutions in a membrane-anchoring amphipathic α-helix, implying that the 1a-lipid interactions play critical roles in viral replication complex assembly.

A Co-Opted DEAD-Box RNA helicase enhances tombusvirus plus-strand synthesis.

Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV). To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3'-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3'-end of the TBSV (-)RNA, rendering the RNA compatible for initiation of (+)-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is another host factor for TBSV, play non-overlapping functions to enhance (+)-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (-)RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV), a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells.

Perfil de sensibilidad in vitro y contenido en ergosterol en levaduras de origen clínico / In vitro antifungal susceptibility pattern and ergosterol content in clinical yeast strains

Antecedentes. La ausencia o disminución en la cantidad de ergosterol, así como su sustitución por otros esteroles en la membrana, se ha considerado como un posible mecanismo de resistencia de la célula fúngica. Objetivos y métodos. En este trabajo hemos evaluado la cantidad de ergosterol de una colección de 51 aislamientos clínicos de levaduras, incluyendo cepas sensibles y resistentes a antifúngicos, mediante un sencillo método cromatográfico (HPLC-UV). Resultados. Algunas cepas de Candida glabrata, Candida tropicalis y Pichia membranifaciens mostraron mayor contenido en ergosterol que el resto, mientras que las de Cryptococcus neoformans y Dipodascus capitatus presentaron el contenido más bajo. No se observó ninguna relación con suficiente potencia estadística entre el patrón de sensibilidad in vitro y el contenido de ergosterol. Conclusiones. Podemos concluir de este estudio que el contenido en ergosterol no se relaciona sistemáticamente con un patrón de resistencia definido(AU)

Background. Absence or severe reduction in the amount of ergosterol in the fungal membrane and its replacement with other sterols have been described as potential antifungal resistance mechanisms in fungi. Aims and methods. The ergosterol content in a collection of 51 clinical yeast isolates, including susceptible and resistant strains to amphotericin B and azoles, was estimated by a simple chromatographic method (HPLC-UV). Results. A high content of ergosterol was detected for several strains of Candida glabrata, Candida tropicalis or Pichia membranifaciens. In contrast, strains of Cryptococcus neoformans and Dipodascus capitatus had the lowest ergosterol concentrations. No significant correlation was observed between antifungal susceptibility patterns and ergosterol content. Conclusions. We conclude from this study that ergosterol content on yeasts may not be associated with specific resistant patterns(AU)

Methods to construct recombinant adenovirus vectors.

The most efficient system to introduce genes of interest within the adenovirus genome is by homologous recombination in microorganisms. In this chapter, the most popular procedures are described: two for homologous recombination in Escherichia coli, and one in yeast. Main differences between procedures are found in the plasmids needed as well as in the selection system used to rapidly identify newly generated recombinant adenovirus. The adenovirus genomes are then analyzed to confirm their identity and integrity, and further linearized to generate a viral pre-stock in permissive human cells. Finally, as a previous step before its amplification at medium or large scale, the viral pre-stock must be analyzed to quantify its potency and infectivity as well as to exclude the presence of unwanted replication competent particles.

A molecular clamp ensures allosteric coordination of peptidyltransfer and ligand binding to the ribosomal A-site.

Although the ribosome is mainly comprised of rRNA and many of its critical functions occur through RNA-RNA interactions, distinct domains of ribosomal proteins also participate in switching the ribosome between different conformational/functional states. Prior studies demonstrated that two extended domains of ribosomal protein L3 form an allosteric switch between the pre- and post-translocational states. Missing was an explanation for how the movements of these domains are communicated among the ribosome's functional centers. Here, a third domain of L3 called the basic thumb, that protrudes roughly perpendicular from the W-finger and is nestled in the center of a cagelike structure formed by elements from three separate domains of the large subunit rRNA is investigated. Mutagenesis of basically charged amino acids of the basic thumb to alanines followed by detailed analyses suggests that it acts as a molecular clamp, playing a role in allosterically communicating the ribosome's tRNA occupancy status to the elongation factor binding region and the peptidyltransferase center, facilitating coordination of their functions through the elongation cycle. The observation that these mutations affected translational fidelity, virus propagation and cell growth demonstrates how small structural changes at the atomic scale can propagate outward to broadly impact the biology of cell.

Wine yeast molecular typing using a simplified method for simultaneously extracting mtDNA, nuclear DNA and virus dsRNA.

Quick and accurate methods are required for the identification of industrial, environmental, and clinical yeast strains. We propose a rapid method for the simultaneous extraction of yeast mtDNA, nuclear DNA, and virus dsRNA. It is simpler, cheaper, and faster than the previously reported methods. It allows one to choose among a broad range of molecular analysis approaches for yeast typing, avoiding the need to use of several different methods for the separate extraction of each nucleic acid type. The application of this method followed by the combined analysis of mtDNA and dsRNA (ScV-M and W) is a highly attractive option for fast and efficient wine yeast typing.

Ubiquitination of tombusvirus p33 replication protein plays a role in virus replication and binding to the host Vps23p ESCRT protein.

Post-translational modifications of viral replication proteins could be widespread phenomena during the replication of plus-stranded RNA viruses. In this article, we identify two lysines in the tombusvirus p33 replication co-factor involved in ubiquitination and show that the same lysines are also important for the p33 to interact with the host Vps23p ESCRT-I factor. We find that the interaction of p33 with Vps23p is also affected by a "late-domain"-like sequence in p33. The combined mutations of the two lysines and the late-domain-like sequences in p33 reduced replication of a replicon RNA of Tomato bushy stunt virus in yeast model host, in plant protoplasts, and plant leaves, suggesting that p33-Vps23p ESCRT protein interaction affects tombusvirus replication. Using ubiquitin-mimicking p33 chimeras, we demonstrate that high level of p33 ubiquitination is inhibitory for TBSV replication. These findings argue that optimal level of p33 ubiquitination plays a regulatory role during tombusvirus infections.