Development of promiscous rhizobia for diverse rabi legumes (Chickpea, Pea and Lentil)

Conjugation between microsymbiont was used to create genetic variations in rhizobia for diverse rabi legumes (chickpea, pea and lentil) with better characteristics in nodulation and nitrogen fixation process. Ten antibiotics were used as selectable markers for the screening of twenty four bacterial strains to be used in mating experiments for obtaining transconjugants. All bacterial strains were sensitive to gentamycin and resistant to streptomycin, kanamycin and sulphanilamide. Total five fusants were obtained from each rhizobial cross combination with the help of electro-poration. Modified transconjugants, rhizobial strains had promiscuous infection with 50-122% more nodules showed significant increase in shoot fresh weight, dry weight and total nitrogen content in chickpea, pea and lentil plants. Electrofusantsrhizobial strains improved shoot nitrogen content up to 67% in lentil and 54% in pea and chickpea plants. The amount of nitrogen fixed in chickpea was highest (3.71gm) by transconjugants DP-C6- HLN followed by DP-C6-HP14 (3.56gm). Transconjugants DP-HP14-HLN fixed the highest amount of nitrogen (3.92gm) in pea and 4.06gm in lentil plants. Plasmids were also analyzed in order to characterize their role in the evolution of rhizobial symbionts and their involvement in symbiotic behaviour. The developed Rhizobium strains with improved symbiotic association and ability to infect across strict specificity for host legumes would be of great help for the farming community at large

Statistical media optimization studies for growth and polydroxybutyrate (PHB) production by Pseudomonas spp.

Using glucose as carbon source and mustard cake and yeast extract as nitrogen sources bacterial isolate Pseudomonas B2 exhibited a maximum PHB recovery of 0.620 (in terms of O.D.) and PHB weight of 0.27g/L in 96 h. To determine the possibility of growth potential of Pseudomonas spp., it was grown in different carbon sources like fructose, glucose, maltose, mannitol etc. and it was found that glucose yielded good growth and PHB production. In order to incorporate cost effective nitrogen and carbon source, mustard cake and cotton cake as nitrogen source and molasses as carbon were used in medium. Statistical media optimization design was used to optimize the culture conditions for maximizing the PHB production. A maximum of 0.37 g/L of PHB and 0.746 (O.D.) PHB recoveries were obtained using optimized concentrations. Batch kinetics can be used for model development, which will make possible simulation of nutrient limited cultivation(s) for over accumulation of PHB. FTIR studies confirmed the presence of PHB

Base Pairing between Hepatitis C Virus RNA and MicroRNA 122 3' of Its Seed Sequence Is Essential for Genome Stabilization and Production of Infectious Virus

MicroRNA 122 (miR-122) facilitates hepatitis C virus (HCV) replication by recruiting an RNA-induced silencing complex (RISC)-like complex containing argonaute 2 (Ago2) to the 5′ end of the HCV genome, thereby stabilizing the viral RNA. This requires base pairing between the miR-122 “seed sequence” (nucleotides [nt] 2 to 8) and two sequences near the 5′ end of the HCV RNA: S1 (nt 22 to 28) and S2 (nt 38 to 43). However, recent reports suggest that additional base pair interactions occur between HCV RNA and miR-122. We searched 606 sequences from a public database (genotypes 1 to 6) and identified two conserved, putatively single-stranded RNA segments, upstream of S1 (nt 2 and 3) and S2 (nt 30 to 34), with potential for base pairing to miR-122 (nt 15 and 16 and nt 13 to 16, respectively). Mutagenesis and genetic complementation experiments confirmed that HCV nt 2 and 3 pair with nt 15 and 16 of miR-122 bound to S1, while HCV nt 30 to 33 pair with nt 13 to 16 of miR-122 at S2. In genotype 1 and 6 HCV, nt 4 also base pairs with nt 14 of miR-122. These 3′ supplementary base pair interactions of miR-122 are functionally important and are required for Ago2 recruitment to HCV RNA by miR-122, miR-122-mediated stabilization of HCV RNA, and production of infectious virus. However, while complementary mutations at HCV nt 30 and 31 efficiently rescued the activity of a 15C,16C miR-122 mutant targeting S2, similar mutations at nt 2 and 3 failed to rescue Ago2 recruitment at S1. These data add to the current understanding of miR-122 interactions with HCV RNA but indicate that base pairing between miR-122 and the 5′ 43 nt of the HCV genome is more complex than suggested by existing models

Stabilization of hepatitis C virus RNA by an Ago2-miR-122 complex

MicroRNAs (miRNAs) are small noncoding RNAs that regulate eukaryotic gene expression by binding to regions of imperfect complementarity in mRNAs, typically in the 3′ UTR, recruiting an Argonaute (Ago) protein complex that usually results in translational repression or destabilization of the target RNA. The translation and decay of mRNAs are closely linked, competing processes, and whether the miRNA-induced silencing complex (RISC) acts primarily to reduce translation or stability of the mRNA remains controversial. miR-122 is an abundant, liver-specific miRNA that is an unusual host factor for hepatitis C virus (HCV), an important cause of liver disease in humans. Prior studies show that it binds the 5′ UTR of the messenger-sense HCV RNA genome, stimulating translation and promoting genome replication by an unknown mechanism. Here we show that miR-122 binds HCV RNA in association with Ago2 and that this slows decay of the viral genome in infected cells. The stabilizing action of miR-122 does not require the viral RNA to be translationally active nor engaged in replication, and can be functionally substituted by a nonmethylated 5′ cap. Our data demonstrate that a RISC-like complex mediates the stability of HCV RNA and suggest that Ago2 and miR-122 act coordinately to protect the viral genome from 5′ exonuclease activity of the host mRNA decay machinery. miR-122 thus acts in an unconventional fashion to stabilize HCV RNA and slow its decay, expanding the repertoire of mechanisms by which miRNAs modulate gene expression

The antiviral protein viperin inhibits HCV replication via interaction with NS5A

The interferon-stimulated gene viperin has been shown to have antiviral activity against hepatitis C virus (HCV) in the context of the HCV replicon, although the molecular mechanisms responsible are not well understood. Here we demonstrate that viperin plays an integral part in the ability of interferon to limit replication of cell culture derived HCV (JFH-1) that accurately reflects the complete viral life cycle. Using confocal microscopy and Fluorescence Resonance Energy Transfer (FRET) analysis we demonstrate that viperin localizes and interacts with HCV NS5A at the lipid droplet interface. In addition viperin also associates with NS5A and the pro-viral cellular factor, VAP-A at the HCV replication complex. The ability of viperin to limit HCV replication was dependent on residues within the C-terminus as well as an N-terminal amphipathic helix. Removal of the amphipathic helix redirected viperin from the cytosolic face of the ER and the lipid droplet to a homogenous cytoplasmic distribution, coinciding with a loss of antiviral effect. C-terminal viperin mutants still localized to the lipid droplet interface and replication complexes but did not interact with NS5A proteins as determined by FRET analysis. In conclusion we propose that viperin interacts with NS5A and the host factor VAP-A to limit HCV replication at the replication complex. This highlights the complexity of host control of viral replication by interferon stimulated gene expression

Cyclosporin A Associated Helicase-Like Protein Facilitates the Association of Hepatitis C Virus RNA Polymerase with Its Cellular Cyclophilin B

BACKGROUND: Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. PRINCIPAL FINDINGS: Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. CONCLUSIONS: We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology

The DEAD-box RNA Helicase DDX6 is Required for Efficient Encapsidation of a Retroviral Genome

Viruses have to encapsidate their own genomes during the assembly process. For most RNA viruses, there are sequences within the viral RNA and virion proteins needed for high efficiency of genome encapsidation. However, the roles of host proteins in this process are not understood. Here we find that the cellular DEAD-box RNA helicase DDX6 is required for efficient genome packaging of foamy virus, a spumaretrovirus. After infection, a significant amount of DDX6, normally concentrated in P bodies and stress granules, re-localizes to the pericentriolar site where viral RNAs and Gag capsid proteins are concentrated and capsids are assembled. Knockdown of DDX6 by siRNA leads to a decreased level of viral nucleic acids in extracellular particles, although viral protein expression, capsid assembly and release, and accumulation of viral RNA and Gag protein at the assembly site are little affected. DDX6 does not interact stably with Gag proteins nor is it incorporated into particles. However, we find that the ATPase/helicase motif of DDX6 is essential for viral replication. This suggests that the ATP hydrolysis and/or the RNA unwinding activities of DDX6 function in moderating the viral RNA conformation and/or viral RNA-Gag ribonucleoprotein complex in a transient manner to facilitate incorporation of the viral RNA into particles. These results reveal a unique role for a highly conserved cellular protein of RNA metabolism in specifically re-locating to the site of viral assembly for its function as a catalyst in retroviral RNA packaging

Human, Nonhuman Primate, and Bat Cells Are Broadly Susceptible to Tibrovirus Particle Cell Entry

In 2012, the genome of a novel rhabdovirus, Bas-Congo virus (BASV), was discovered in the acute-phase serum of a Congolese patient with presumed viral hemorrhagic fever. In the absence of a replicating virus isolate, fulfilling Koch’s postulates to determine whether BASV is indeed a human virus and/or pathogen has been impossible. However, experiments with vesiculoviral particles pseudotyped with Bas-Congo glycoprotein suggested that BASV particles can enter cells from multiple animals, including humans. In 2015, genomes of two related viruses, Ekpoma virus 1 (EKV-1) and Ekpoma virus 2 (EKV-2), were detected in human sera in Nigeria. Isolates could not be obtained. Phylogenetic analyses led to the classification of BASV, EKV-1, and EKV-2 in the same genus, Tibrovirus, together with five biting midge-borne rhabdoviruses [i.e., Beatrice Hill virus (BHV), Bivens Arm virus (BAV), Coastal Plains virus (CPV), Sweetwater Branch virus (SWBV), and Tibrogargan virus (TIBV)] not known to infect humans. Using individual recombinant vesiculoviruses expressing the glycoproteins of all eight known tibroviruses and more than 75 cell lines representing different animal species, we demonstrate that the glycoproteins of all tibroviruses can mediate vesiculovirus particle entry into human, bat, nonhuman primate, cotton rat, boa constrictor, and Asian tiger mosquito cells. Using four of five isolated authentic tibroviruses (i.e., BAV, CPV, SWBV, and TIBV), our experiments indicate that many cell types may be partially resistant to tibrovirus replication after virion cell entry. Consequently, experimental data solely obtained from experiments using tibrovirus surrogate systems (e.g., vesiculoviral pseudotypes, recombinant vesiculoviruses) cannot be used to predict whether BASV, or any other tibrovirus, infects humans

Qualification study of SiPMs on a large scale for the CMVD Experiment

A Cosmic Muon Veto (CMV) detector using extruded plastic scintillators is being designed around the mini-Iron Calorimeter (mini-ICAL) detector at the transit campus of the India based Neutrino Observatory, Madurai for the feasibility study of shallow depth underground experiments. The scintillation signals that are produced in the plastic due to muon trajectories are absorbed by wavelength shifting (WLS) fibres. The WLS fibres re-emit photons of longer wavelengths and propagate those to silicon photo-multipliers (SiPMs). The SiPMs detect these photons, producing electronic signals. The CMV detector will use more than 700 scintillators to cover the mini-ICAL detector and will require around 3000 SiPMs. The design goal for the cosmic muon veto efficiency of the CMV is >99.99%. Hence, every SiPM used in the detector needs to be tested and characterised to satisfy the design goal of CMV. A mass testing system was developed for the measurement of gain and choice of the overvoltage ($V_{ov}$) of each SiPMs using an LED driver. The $V_{ov}$ is obtained by studying the noise rate, the gain of the SiPM. This paper describes the experimental setup used to test the SiPMs characteristics along with detailed studies of those characteristics as a function of temperature.Comment: 16 pages, 20 figure

An Integrated Transcriptomic and Meta-Analysis of Hepatoma Cells Reveals Factors That Influence Susceptibility to HCV Infection

Hepatitis C virus (HCV) is a global problem. To better understand HCV infection researchers employ in vitro HCV cell-culture (HCVcc) systems that use Huh-7 derived hepatoma cells that are particularly permissive to HCV infection. A variety of hyper-permissive cells have been subcloned for this purpose. In addition, subclones of Huh-7 which have evolved resistance to HCV are available. However, the mechanisms of susceptibility or resistance to infection among these cells have not been fully determined. In order to elucidate mechanisms by which hepatoma cells are susceptible or resistant to HCV infection we performed genome-wide expression analyses of six Huh-7 derived cell cultures that have different levels of permissiveness to infection. A great number of genes, representing a wide spectrum of functions are differentially expressed between cells. To focus our investigation, we identify host proteins from HCV replicase complexes, perform gene expression analysis of three HCV infected cells and conduct a detailed analysis of differentially expressed host factors by integrating a variety of data sources. Our results demonstrate that changes relating to susceptibility to HCV infection in hepatoma cells are linked to the innate immune response, secreted signal peptides and host factors that have a role in virus entry and replication. This work identifies both known and novel host factors that may influence HCV infection. Our findings build upon current knowledge of the complex interplay between HCV and the host cell, which could aid development of new antiviral strategies