PAP1 [poly(A) polymerase 1] homozygosity and hyperadenylation are major determinants of increased mRNA stability of CDR1 in azole-resistant clinical isolates of Candida albicans.

Using genetically matched azole-susceptible (AS) and azole-resistant (AR) clinical isolates of Candida albicans, we recently demonstrated that CDR1 overexpression in AR isolates is due to its enhanced transcriptional activation and mRNA stability. This study examines the molecular mechanisms underlying enhanced CDR1 mRNA stability in AR isolates. Mapping of the 3' untranslated region (3' UTR) of CDR1 revealed that it was rich in adenylate/uridylate (AU) elements, possessed heterogeneous polyadenylation sites, and had putative consensus sequences for RNA-binding proteins. Swapping of heterologous and chimeric lacZ-CDR1 3' UTR transcriptional reporter fusion constructs did not alter the reporter activity in AS and AR isolates, indicating that cis-acting sequences within the CDR1 3' UTR itself are not sufficient to confer the observed differential mRNA decay. Interestingly, the poly(A) tail of the CDR1 mRNA of AR isolates was approximately 35-50 % hyperadenylated as compared with AS isolates. C. albicans poly(A) polymerase (PAP1), responsible for mRNA adenylation, resides on chromosome 5 in close proximity to the mating type-like (MTL) locus. Two different PAP1 alleles, PAP1-a/PAP1-alpha, were recovered from AS (MTL-a/MTL-alpha), while a single type of PAP1 allele (PAP1-alpha) was recovered from AR isolates (MTL-alpha/MTL-alpha). Among the heterozygous deletions of PAP1-a (Deltapap1-a/PAP1-alpha) and PAP1-alpha (PAP1-a/Deltapap1-alpha), only the former led to relatively enhanced drug resistance, to polyadenylation and to transcript stability of CDR1 in the AS isolate. This suggests a dominant negative role of PAP1-a in CDR1 transcript polyadenylation and stability. Taken together, our study provides the first evidence, to our knowledge, that loss of heterozygosity at the PAP1 locus is linked to hyperadenylation and subsequent increased stability of CDR1 transcripts, thus contributing to enhanced drug resistance.

Analytical validation of a lymphocytic choriomeningitis virus real-time RT-PCR assay.

Lymphocytic choriomeningitis virus (LCMV) is a rare cause of central nervous system disease in humans. Screening by real-time RT-PCR assay is of interest in the case of aseptic meningitis of unknown etiology. A specific LCMV real-time RT-PCR assay, based on the detection of genomic sequences of the viral nucleoprotein (NP), was developed to assess the presence of LCMV in cerebrospinal fluids (CSF) sent for viral screening to a Swiss university hospital laboratory. A 10-fold dilution series assay using a plasmid containing the cDNA of the viral NP of the LCMV isolate Armstrong (Arm) 53b demonstrated the high sensitivity of the assay with a lowest detection limit of ≤50 copies per reaction. High sensitivity was confirmed by dilution series assays in a pool of human CSF using four different LCMV isolates (Arm53b, WE54, Traub and E350) with observed detection limits of ≤10PFU/ml (Arm53b and WE54) and 1PFU/ml (Traub and E350). Analysis of 130 CSF showed no cases of acute infection. The absence of positive cases was confirmed by a published PCR assay detecting all Old World arenaviruses. This study validates a specific and sensitive real-time RT-PCR assay for the diagnosis of LCMV infections. Results showed that LCMV infections are extremely rare in hospitalized patients western in Switzerland.

Leishmania aethiopica field isolates bearing an endosymbiontic dsRNA virus induce pro-inflammatory cytokine response.

BACKGROUND: Infection with Leishmania parasites causes mainly cutaneous lesions at the site of the sand fly bite. Inflammatory metastatic forms have been reported with Leishmania species such as L. braziliensis, guyanensis and aethiopica. Little is known about the factors underlying such exacerbated clinical presentations. Leishmania RNA virus (LRV) is mainly found within South American Leishmania braziliensis and guyanensis. In a mouse model of L. guyanensis infection, its presence is responsible for an hyper-inflammatory response driven by the recognition of the viral dsRNA genome by the host Toll-like Receptor 3 leading to an exacerbation of the disease. In one instance, LRV was reported outside of South America, namely in the L. major ASKH strain from Turkmenistan, suggesting that LRV appeared before the divergence of Leishmania subgenera. LRV presence inside Leishmania parasites could be one of the factors implicated in disease severity, providing rationale for LRV screening in L. aethiopica. METHODOLOGY/PRINCIPAL FINDINGS: A new LRV member was identified in four L. aethiopica strains (LRV-Lae). Three LRV-Lae genomes were sequenced and compared to L. guyanensis LRV1 and L. major LRV2. LRV-Lae more closely resembled LRV2. Despite their similar genomic organization, a notable difference was observed in the region where the capsid protein and viral polymerase open reading frames overlap, with a unique -1 situation in LRV-Lae. In vitro infection of murine macrophages showed that LRV-Lae induced a TLR3-dependent inflammatory response as previously observed for LRV1. CONCLUSIONS/SIGNIFICANCE: In this study, we report the presence of an immunogenic dsRNA virus in L. aethiopica human isolates. This is the first observation of LRV in Africa, and together with the unique description of LRV2 in Turkmenistan, it confirmed that LRV was present before the divergence of the L. (Leishmania) and (Viannia) subgenera. The potential implication of LRV-Lae on disease severity due to L. aethiopica infections is discussed.

Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

Association of the Epstein-Barr virus latent membrane protein 1 with lipid rafts is mediated through its N-terminal region.

The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus acts like a constitutively activated receptor of the tumor necrosis factor receptor (TNFR) family and is enriched in lipid rafts. We showed that LMP1 is targeted to lipid rafts in transfected HEK 293 cells, and that the endogenous TNFR-associated factor 3 binds LMP1 and is recruited to lipid rafts upon LMP1 expression. An LMP1 mutant lacking the C-terminal 55 amino acids (Cdelta55) behaves like the wild-type (WT) LMP1 with respect to membrane localization. In contrast, a mutant with a deletion of the 25 N-terminal residues (Ndelta25) does not concentrate in lipid rafts but still binds TRAF3, demonstrating that cell localization of LMP1 was not crucial for TRAF3 localization. Moreover, Ndelta25 inhibited WT LMP1-mediated induction of the transcription factors NF-kappaB and AP-1. Morphological data indicate that Ndelta25 hampers WT LMP1 plasma membrane localization, thus blocking LMP1 function.

Glomus intraradices induces changes in root system architecture of rice independently of common symbiosis signaling

? Arbuscular mycorrhizal fungi colonize the roots of most monocotyledons and dicotyledons despite their different root architecture and cell patterning. Among the cereal hosts of arbuscular mycorrhizal fungi, Oryza sativa (rice) possesses a peculiar root system composed of three different types of roots: crown roots; large lateral roots; and fine lateral roots. Characteristic is the constitutive formation of aerenchyma in crown roots and large lateral roots and the absence of cortex from fine lateral roots. Here, we assessed the distribution of colonization by Glomus intraradices within this root system and determined its effect on root system architecture. ? Large lateral roots are preferentially colonized, and fine lateral roots are immune to arbuscular mycorrhizal colonization. Fungal preference for large lateral roots also occurred in sym mutants that block colonization of the root beyond rhizodermal penetration. ? Initiation of large lateral roots is significantly induced by G. intraradices colonization and does not require a functional common symbiosis signaling pathway from which some components are known to be needed for symbiosis-mediated lateral root induction in Medicago truncatula. ? Our results suggest variation of symbiotic properties among the different rice root-types and induction of the preferred tissue by arbuscular mycorrhizal fungi. Furthermore, signaling for arbuscular mycorrhizal-elicited alterations of the root system differs between rice and M. truncatula.

Expression of a functional single chain antibody on the surface of extracellular enveloped vaccinia virus as a step towards selective tumour cell targeting.

Recombinant vaccinia virus with tumour cell specificity may provide a versatile tool either for direct lysis of cancer cells or for the targeted transfer of genes encoding immunomodulatory molecules. We report the expression of a single chain antibody on the surface of extracellular enveloped vaccinia virus. The wild-type haemagglutinin, an envelope glycoprotein which is not required for viral infection and replication, was replaced by haemagglutinin fusion molecules carrying a single chain antibody directed against the tumour-associated antigen ErbB2. ErbB2 is an epidermal growth factor receptor-related tyrosine kinase overexpressed in a high percentage of human adenocarcinomas. Two fusion proteins carrying the single chain antibody at different NH2-terminal positions were expressed and exposed at the envelope of the corresponding recombinant viruses. The construct containing the antibody at the site of the immunoglobulin-like loop of the haemagglutinin was able to bind solubilized ErbB2. This is the first report of replacement of a vaccinia virus envelope protein by a specific recognition structure and represents a first step towards modifying the host cell tropism of the virus.

Targeting mitochondria in the infection strategy of the hepatitis C virus.

Hepatitis C virus (HCV) infection induces a state of oxidative stress more pronounced than that observed in many other inflammatory diseases. Here, we propose a temporal sequence of events in the HCV-infected cell whereby the primary alteration consists of a release of Ca(2+) from the endoplasmic reticulum, followed by uptake into mitochondria. This ensues successive mitochondrial dysfunction leading to the generation of reactive oxygen species and a progressive metabolic adaptive response. Evidence is provided for a positive feed-back mechanism between alterations of calcium and redox homeostasis. This likely involves deregulation of the mitochondrial permeability transition and induces progressive dysfunction of cellular bioenergetics. Pathogenetic implications of the model and new opportunities for therapeutic intervention are discussed. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Molecular determinants for membrane association of the hepatitis C virus NS2 protease domain

Background: Hepatitis C virus (HCV) nonstructural protein 2 (NS2) plays essential roles in particle assembly and polyprotein processing. It harbors an N-terminal membrane domain comprising three putative transmembrane s egments ( amino acids [aa] 1-93) a nd a C-terminal cysteine protease domain (aa 94-217). Given that the latter has been predicted to be membrane-associated, we aimed to identify molecular determinants for membrane association of the NS2 protease domain. Methods: A comprehensive panel of NS2 deletion constructs was analyzed by fluorescence microscopy, selective membrane extraction, and m embrane flotation assays. Candidate aa r esidues involved in membrane association were substituted by site-directed mutagenesis. Results: The NS2 protease domain alone was found to associate with membranes. Two N-terminal α-helices comprising aa 102-114 and aa 123-136 were found to m ediate this a ssociation, w ith c onserved hydrophobic and positively charged aa residues representing the key determinants. I nterestingly, m utagenesis analyses r evealed that electrostatic interactions involving a positively charged aa residue in α-helix aa 123-136 are required for membrane association. Mono- and bicistronic (i.e. NS2 c leavage-independent) HCV constructs were prepared to i nvestigate the effect o f these substitutions on RNA replication and infectious viral particle formation. Conclusions: T he NS2 protease d omain itself harbors m olecular determinants for membrane association within α-helices aa 102-114 and aa 1 23-136 which may contribute to p roper p ositioning of t he active site. These results provide new insights i nto the membrane topology and t he p oorly understood f unction of t his essential viral protease.

Investigation of the hepatitis C virus replication complex.

Formation of a membrane-associated replication complex, composed of viral proteins, replicating RNA, altered cellular membranes, and other host factors, is a hallmark of all positive-strand RNA viruses. In the case of HCV, RNA replication takes place in a likely endoplasmic reticulum-derived membrane alteration referred to as the "membranous web." In vitro transcription-translation, membrane extraction and flotation analyses, immunofluorescence microscopy, fluorescent in situ hybridization, and RNA metabolic labeling followed by confocal laser scanning microscopy have yielded insights into the structure and function of the HCV replication complex. We describe these techniques and highlight selected results.

Cell surface expression of a functional rubella virus E1 glycoprotein by addition of a GPI anchor.

Rubella virus (RV) envelope glycoproteins E1 and E2 are targeted to the Golgi as heterodimers. While E2 contains a transmembrane Golgi retention signal, E1 is arrested in a pre-Golgi compartment in the absence of E2, and appears to require heterodimerization in order to reach the Golgi. Various forms of E1 with deletions in the ectodomain or lacking the cytoplasmic (CT) and transmembrane (TM) domains, as well as the 29 C-terminal amino acid residues of the ectodomain were also retained intracellularly. We therefore investigated the possibility of targetting E1 to the plasma membrane by addition of a glycosylphosphatidylinositol (GPI) anchor. We found that E1GPI was transported to the cell surface where it retained the hemadsorption activity characteristic of the wild-type E1/E2 heterodimer. Furthermore, coexpression of a mammalian GPI-specific phospholipase D (GPI-PLD) resulted in the release of E1GPI and in constitutive expression of a soluble form of E1. This study thus demonstrates that the GPI anchor has a dominant effect over the E1 pre-Golgi retention signal and that E1 is sufficient for hemadsorption.

An mRNA region of the canine distemper virus fusion protein gene lacking AUG codons can promote protein expression.

Canine distemper virus (CDV) produces a glycosylated type I fusion protein (F) with an internal hydrophobic signal sequence beginning around 115 residues downstream of the first AUG used for translation initiation. Cleavage of the signal sequence yields the F0 molecule, which is cleaved into the F1 and F2 subunits. Surprisingly, when all in-frame AUGs located in the first third of the F gene were mutated a protein of the same molecular size as the F0 molecule was still expressed from both the Onderstepoort (OP) and A75/17-CDV F genes. We designated this protein, which is initiated from a non-AUG codon protein Fx. Site-directed mutagenesis allowed to identify codon 85, a GCC codon coding for alanine, as the most likely position from which translation initiation of Fx occurs in OP-CDV. Deletion analysis demonstrated that at least 60 nucleotides upstream of the GCC codon are required for efficient Fx translation. This sequence is GC-rich, suggesting extensive folding. Secondary structure may therefore be important for translation initiation at codon 85.

Functional Characterization of a n NTPase Activity of the Hepatitis C Virus Nonstructural Protein 4B

Background: Nonstructural p rotein 4 B (NS4B) i s the m asterorganizer of hepatitis C virus (HCV) replication complexformation. It is a multispanning membrane protein that has beenreported to p ossess NTPase activity. This enzymatic functionhas been poorly studied so far and its role in the HCV life cycleis u nknown. T he present w ork-in-progress a ims at validatingand functionally c haracterizing this a ctivity a nd its r ole in t heviral life cycle.Methods: B ioinformatic analyses were performed to i dentifykey residues for site-directed mutagenesis, both in t he contextof s ubgenomic r eplicons a s well as recombinant v iruses.Mutants were investigated with respect to R NA replication andinfectious particle p roduction. In p arallel, expression andpurification of recombinant wild-type and mutant NS4B proteinsare being pursued to characterize enzymatic activity in vitro.Results: B ioinformatic a nalyses revealed t hat p redictedNTPase features are conserved only in H CV NS4B b ut n ot i nNS4B from other Flaviviridae f amily m embers. A laninesubstitutions were designed to target predicted key Walker A, Band C motifs. These substitutions affected RNA replication andinfectious virus production to v arying degrees. Optimization ofrecombinant protein production is i n progress both in b acterialas well as mammalian expression systems.Conclusions: These studies should yield new insights into thefunctions of this hitherto poorly characterized viral nonstructuralprotein and may reveal novel targets for antiviral intervention inthe future.

Comparative transcriptomics of rice reveals an ancient pattern of response to microbial colonization.

Glomalean fungi induce and colonize symbiotic tissue called arbuscular mycorrhiza on the roots of most land plants. Other fungi also colonize plants but cause disease not symbiosis. Whole-transcriptome analysis using a custom-designed Affymetrix Gene-Chip and confirmation with real-time RT-PCR revealed 224 genes affected during arbuscular mycorrhizal symbiosis. We compared these transcription profiles with those from rice roots that were colonized by pathogens (Magnaporthe grisea and Fusarium moniliforme). Over 40% of genes showed differential regulation caused by both the symbiotic and at least one of the pathogenic interactions. A set of genes was similarly expressed in all three associations, revealing a conserved response to fungal colonization. The responses that were shared between pathogen and symbiont infection may play a role in compatibility. Likewise, the responses that are different may cause disease. Some of the genes that respond to mycorrhizal colonization may be involved in the uptake of phosphate. Indeed, phosphate addition mimicked the effect of mycorrhiza on 8% of the tested genes. We found that 34% of the mycorrhiza-associated rice genes were also associated with mycorrhiza in dicots, revealing a conserved pattern of response between the two angiosperm classes.