Identification of GBF1 as a Cellular Factor Required for Hepatitis C Virus RNA Replication

In infected cells, hepatitis C virus (HCV) induces the formation of membrane alterations referred to as membranous webs, which are sites of RNA replication. In addition, HCV RNA replication also occurs in smaller membrane structures that are associated with the endoplasmic reticulum. However, cellular mechanisms involved in the formation of HCV replication complexes remain largely unknown. Here, we used brefeldin A (BFA) to investigate cellular mechanisms involved in HCV infection. BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARF), which can lead to a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways. Our data show that HCV RNA replication is highly sensitive to BFA. Individual knockdown of the cellular targets of BFA using RNA interference and the use of a specific pharmacological inhibitor identified GBF1, a guanine nucleotide exchange factor for small GTPases of the ARF family, as a host factor critically involved in HCV replication. Furthermore, overexpression of a BFA-resistant GBF1 mutant rescued HCV replication in BFA-treated cells, indicating that GBF1 is the BFA-sensitive factor required for HCV replication. Finally, immunofluorescence and electron microscopy analyses indicated that BFA does not block the formation of membranous web-like structures induced by expression of HCV proteins in a nonreplicative context, suggesting that GBF1 is probably involved not in the formation of HCV replication complexes but, rather, in their activity. Altogether, our results highlight a functional connection between the early secretory pathway and HCV RNA replication.Hepatitis C virus (HCV) is an important human pathogen. It mainly infects human hepatocytes, and this often leads to chronic hepatitis, cirrhosis, or hepatocarcinoma. HCV studies have been hampered for many years by the difficulty in propagating this virus in vitro. Things have recently changed with the development of a cell culture model referred to as HCVcc (34, 60, 65), which allows the study of the HCV life cycle in cell culture and facilitates studies of the interactions between HCV and the host cell.HCV is an enveloped positive-strand RNA virus belonging to the family Flaviviridae (35). The viral genome contains a single open reading frame, which is flanked by two noncoding regions that are required for translation and replication. All viral proteins that are produced after proteolytic processing of the initially synthesized polyprotein are membrane associated (15, 43). This reflects the fact that virtually all steps of the viral life cycle occur in close association with cellular membranes.Interactions of HCV with cell membranes begin during entry. Several receptors, coreceptors, and other entry factors have been discovered over the years, which link HCV entry to specialized domains of the plasma membrane, such as tetraspanin-enriched microdomains and tight junctions (8, 16, 59). The internalization of the viral particle occurs by clathrin-mediated endocytosis (5, 40). The fusion of the viral envelope with the membrane of an acidic endosome likely mediates the transfer of the viral genome to the cytosol of the cell (5, 40, 57). However, little is known regarding the pre- and postfusion intracellular transport steps of entering viruses in the endocytic pathway.HCV RNA replication is also associated with cellular membranes. Replication begins with the translation of the genomic RNA of an incoming virus. This leads to the production of viral proteins, which in turn initiate the actual replication of the viral RNA. Mechanisms regulating the transition from the translation of the genomic RNA to its replication are not yet known. All viral proteins are not involved in RNA replication. Studies performed with subgenomic replicons demonstrated that proteins NS3-4A, NS4B, NS5A, and NS5B are necessary and sufficient for replication (6, 27, 37). RNA replication proceeds through the synthesis of a cRNA strand (negative strand), catalyzed by the RNA-dependent RNA polymerase activity of NS5B, which is then used as a template for the synthesis of new positive strands.Electron microscopy studies using a subgenomic replicon model suggested that replication takes place in membrane structures made of small vesicles, referred to as “membranous webs,” which are induced by the virus (26). Membranous webs are detectable not only in cells carrying subgenomic replicons but also in infected cells (50). They appear to be associated with the endoplasmic reticulum (ER) (26). In addition to the membranous webs, a second type of ER-associated replicase that is smaller and more mobile has recently been described (63). Cellular mechanisms leading to these membrane alterations are still poorly understood. In cells replicating and secreting infectious viruses effectively, the situation appears to be even more complex, since replicase components appear to be, at least in part, associated with cytoplasmic lipid droplets (41, 50, 56). This association depends on the capsid protein (41) and may reflect a coupling between replication and assembly. Indeed, HCV assembly and secretion show some similarities with very-low-density lipoprotein (VLDL) maturation and secretion (24, 64).Our knowledge of the cellular membrane mechanisms involved in the HCV life cycle is still limited. The expression of NS4B alone induces membrane alterations that are reminiscent of membranous webs (19). However, cellular factors that participate in this process are still unknown. On the other hand, several cellular proteins potentially involved in the HCV life cycle have been identified through their interactions with viral proteins. For some of these proteins, a functional role in infection was recently confirmed using RNA interference (48). It is very likely that other cellular factors critical to HCV infection have yet to be identified.To gain more insight into cellular mechanisms underlying HCV infection, we made use of brefeldin A (BFA), a macrocyclic lactone of fungal origin that exhibits a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways (30). BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARFs). ARFs are small GTP-binding proteins of the Ras superfamily. They function as regulators of vesicular traffic, actin remodeling, and phospholipid metabolism by recruiting effectors to membranes. BFA does not actually interfere directly with ARF GTPases but rather interferes with their activation by regulators known as guanine nucleotide exchange factors (GEFs) (14, 25). We now report the identification of an ARF GEF as a cellular BFA-sensitive factor that is required for HCV replication.     

FliZ induces a kinetic switch in flagellar gene expression

FliZ is an activator of class 2 flagellar gene expression in Salmonella enterica. To understand its role in flagellar assembly, we investigated how FliZ affects gene expression dynamics. We demonstrate that FliZ participates in a positive-feedback loop that induces a kinetic switch in class 2 gene expression.     

Foraging tactics of chick-rearing Crozet shags: individuals display repetitive activity and diving patterns over time

It is in the interest of resident and long-lived benthic foragers to learn to apply efficient foraging tactics throughout their lifetime, thus increasing their individual efficiency. To test whether individuals are capable of applying an individual-specific foraging pattern, we checked for the existence of established foraging routines. Using ventrally attached time-depth recorders, we studied the individual foraging tactics of chick-rearing Crozet shags (Phalacrocorax melanogenis, Blyth 1860), as measured by the consistency in individual daily activity patterns and diving profiles over time. Individuals displayed a fidelity to the time of first daily trip to sea and also a strong fidelity to one, two or three depth ranges day after day. We suggest foraging area fidelity, a behaviour that could help increase foraging efficiency thanks to the memorization of the bottom’s topography and the habits of its fauna, as a hypothesis for explaining some of these patterns. We propose the question of foraging area fidelity should be more specifically addressed in the future.     

Measles virus: a future therapeutic agent in oncology?

Measles is a potential lethal disease, justifying large immunization campaigns. Attenuated strains are used in immunization with very good safety records. Interestingly, following clinical observations of tumor regressions after measles infection, preclinical and clinical studies have highlighted the therapeutic potential of attenuated strains of measles. The aim of this review is to explain how these viruses can selectively infect and kill cancer cells, and how this selectivity can be improved. We will detail the therapeutic strategies under development, in particular the combination of viruses with chemotherapy and radiation therapy. Furthermore, the engineering of measles viruses encoding the sodium/iodide symporter could enable virus-directed radio-isotope therapy. Antiviral immunity could be a limit of measles therapy. We will highlight the promising combinations with immunosuppressive drugs and innovative strategies using infected cell carriers, aiming at circumventing the immune response and paving the way to future clinical trials.     

Expected distance between terminal nucleotides of RNA secondary structures

In "The ends of a large RNA molecule are necessarily close", Yoffe et al. (Nucleic Acids Res 39(1):292-299, 2011) used the programs RNAfold [resp. RNAsubopt] from Vienna RNA Package to calculate the distance between 5' and 3' ends of the minimum free energy secondary structure [resp. thermal equilibrium structures] of viral and random RNA sequences. Here, the 5'-3' distance is defined to be the length of the shortest path from 5' node to 3' node in the undirected graph, whose edge set consists of edges {i, i + 1} corresponding to covalent backbone bonds and of edges {i, j} corresponding to canonical base pairs. From repeated simulations and using a heuristic theoretical argument, Yoffe et al. conclude that the 5'-3' distance is less than a fixed constant, independent of RNA sequence length. In this paper, we provide a rigorous, mathematical framework to study the expected distance from 5' to 3' ends of an RNA sequence. We present recurrence relations that precisely define the expected distance from 5' to 3' ends of an RNA sequence, both for the Turner nearest neighbor energy model, as well as for a simple homopolymer model first defined by Stein and Waterman. We implement dynamic programming algorithms to compute (rather than approximate by repeated application of Vienna RNA Package) the expected distance between 5' and 3' ends of a given RNA sequence, with respect to the Turner energy model. Using methods of analytical combinatorics, that depend on complex analysis, we prove that the asymptotic expected 5'-3' distance of length n homopolymers is approximately equal to the constant 5.47211, while the asymptotic distance is 6.771096 if hairpins have a minimum of 3 unpaired bases and the probability that any two positions can form a base pair is 1/4. Finally, we analyze the 5'-3' distance for secondary structures from the STRAND database, and conclude that the 5'-3' distance is correlated with RNA sequence length.     

BDNF and DYRK1A Are Variable and Inversely Correlated in Lymphoblastoid Cell Lines from Down Syndrome Patients

Down syndrome or trisomy 21 is the most common genetic disorder leading to mental retardation. One feature is impaired short- and long-term spatial memory, which has been linked to altered brain-derived neurotrophic factor (BDNF) levels. Mouse models of Down syndrome have been used to assess neurotrophin levels, and reduced BDNF has been demonstrated in brains of adult transgenic mice overexpressing Dyrk1a, a candidate gene for Down syndrome phenotypes. Given the link between DYRK1A overexpression and BDNF reduction in mice, we sought to assess a similar association in humans with Down syndrome. To determine the effect of DYRK1A overexpression on BDNF in the genomic context of both complete trisomy 21 and partial trisomy 21, we used lymphoblastoid cell lines from patients with complete aneuploidy of human chromosome 21 (three copies of DYRK1A) and from patients with partial aneuploidy having either two or three copies of DYRK1A. Decreased BDNF levels were found in lymphoblastoid cell lines from individuals with complete aneuploidy as well as those with partial aneuploidies conferring three DYRK1A alleles. In contrast, lymphoblastoid cell lines from individuals with partial trisomy 21 having only two DYRK1A copies displayed increased BDNF levels. A negative correlation was also detected between BDNF and DYRK1A levels in lymphoblastoid cell lines with complete aneuploidy of human chromosome 21. This finding indicates an upward regulatory role of DYRK1A expression on BDNF levels in lymphoblastoid cell lines and emphasizes the role of genetic variants associated with psychiatric disorders.     

Exposure of mycobacteria to cell wall-inhibitory drugs decreases production of arabinoglycerolipid related to Mycolyl-arabinogalactan-peptidoglycan metabolism

The "cell wall core" consisting of a mycolyl-arabinogalactan-peptidoglycan (mAGP) complex represents the hallmark of the mycobacterial cell envelope. It has been the focus of intense research at both structural and biosynthetic levels during the past few decades. Because it is essential, mAGP is also regarded as a target for several antitubercular drugs. Herein, we demonstrate that exposure of Mycobacterium bovis Bacille Calmette-Guérin or Mycobacterium marinum to thiacetazone, a second line antitubercular drug, is associated with a severe decrease in the level of a major apolar glycolipid. This inhibition requires MmaA4, a methyltransferase reported to participate in the activation process of thiacetazone. Following purification, this glycolipid was subjected to detailed structural analyses, combining gas-liquid chromatography, mass spectrometry, and nuclear magnetic resonance. This allowed to identify it as a 5-O-mycolyl-β-Araf-(1→2)-5-O-mycolyl-α-Araf-(1→1)-Gro, designated dimycolyl diarabinoglycerol (DMAG). The presence of DMAG was subsequently confirmed in other slow growing pathogenic species, including Mycobacterium tuberculosis. DMAG production was stimulated in the presence of exogenous glycerol. Interestingly, DMAG appears structurally identical to the terminal portion of the mycolylated arabinosyl motif of mAGP, and the metabolic relationship between these two components was provided using antitubercular drugs such as ethambutol or isoniazid known to inhibit the biosynthesis of arabinogalactan or mycolic acid, respectively. Finally, DMAG was identified in the cell wall of M. tuberculosis. This opens the possibility of a potent biological function for DMAG that may be important to mycobacterial pathogenesis.     

Hybridization and barriers to gene flow in an island bird radiation

While reinforcement may play a role in all major modes of speciation, relatively little is known about the timescale over which species hybridize without evolving complete reproductive isolation. Birds have high potential for hybridization, and islands provide simple settings for uncovering speciation and hybridization patterns. Here we develop a phylogenetic hypothesis for a phenotypically diverse radiation of finch-like weaver-birds (Foudia) endemic to the western Indian Ocean islands. We find that unlike Darwin's finches, each island-endemic Foudia population is a monophyletic entity for which speciation can be considered complete. In explaining the only exceptions-mismatches between taxonomy, mitochondrial, and nuclear data-phylogenetic and coalescent methods support introgressive hybridization rather than incomplete lineage sorting. Human introductions of known timing of one island-endemic species, to all surrounding archipelagos provide two fortuitous experiments; (1) population sampling at known times in recent evolutionary history, (2) bringing allopatric lineages of an island radiation into secondary contact. Our results put a minimum time bound on introgression (235 years), and support hybridization between species in natural close contact (parapatry), but not between those in natural allopatry brought into contact by human introduction. Time in allopatry, rather than in sympatry, appears key in the reproductive isolation of Foudia species.     

Development of a genetic tool for activating chromosomal expression of cryptic or tightly regulated loci in Pseudomonas aeruginosa

A method for replacing endogenous promoter by a constitutive promoter in Pseudomonas aeruginosa is described. Plasmid pKNG101, a broadly used shuttle suicide vector in P. aeruginosa, was improved to allow chromosomal introduction of a Plac promoter in front of any kind of gene especially those with unknown function. Using this strategy alleviates the need for cloning difficulties encountered in this bacteria and antibiotic marker selection.