Characterization of the Fur regulon in Pseudomonas syringae pv. tomato DC3000.

The plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000) is found in a wide variety of environments and must monitor and respond to various environmental signals such as the availability of iron, an essential element for bacterial growth. An important regulator of iron homeostasis is Fur (ferric uptake regulator), and here we present the first study of the Fur regulon in DC3000. Using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq), 312 chromosomal regions were highly enriched by coimmunoprecipitation with a C-terminally tagged Fur protein. Integration of these data with previous microarray and global transcriptome analyses allowed us to expand the putative DC3000 Fur regulon to include genes both repressed and activated in the presence of bioavailable iron. Using nonradioactive DNase I footprinting, we confirmed Fur binding in 41 regions, including upstream of 11 iron-repressed genes and the iron-activated genes encoding two bacterioferritins (PSPTO_0653 and PSPTO_4160), a ParA protein (PSPTO_0855), and a two-component system (TCS) (PSPTO_3382 to PSPTO_3380).

Replication fitness and NS5B drug sensitivity of diverse hepatitis C virus isolates characterized by using a transient replication assay.

The innate genetic variability characteristic of chronic hepatitis C virus (HCV) infection makes drug resistance a concern in the clinical development of HCV inhibitors. To address this, a transient replication assay was developed to evaluate the replication fitness and the drug sensitivity of NS5B sequences isolated from the sera of patients with chronic HCV infection. This novel assay directly compares replication between NS5B isolates, thus bypassing the potential sequence and metabolic differences which may arise with independent replicon cell lines. Patient-derived NS5B sequences were similar to those of the established HCV genotypes, but isolates from each patient shared genetic variability specific to that patient, with additional genetic variability observed across the individual isolates. Every sample provided functional NS5B isolates which supported subgenomic replication, frequently to levels comparable to that of laboratory-optimized replicons. All isolates were equivalently sensitive to an active-site nucleoside inhibitor, but the sensitivities to a panel of nonnucleoside inhibitors which targeted three distinct sites on NS5B varied among the isolates. In con1, the original laboratory-optimized replicon, the NS5B S282T substitution confers resistance to the nucleoside inhibitor but impairs replication. This substitution was engineered into both genotype 1a and genotype 1b isolates. Replication was severely debilitated, demonstrating that no compensatory residues were encoded within these genetically diverse sequences to increase the replication fitness of the mutated replicons. This work describes a transient replicon-based assay that can support the clinical development of compounds which target NS5B and demonstrates its utility by examining several patient-derived NS5B isolates for replication fitness and differential sensitivity to NS5B inhibitors.

A 7-deaza-adenosine analog is a potent and selective inhibitor of hepatitis C virus replication with excellent pharmacokinetic properties.

Improved treatments for chronic hepatitis C virus (HCV) infection are needed due to the suboptimal response rates and deleterious side effects associated with current treatment options. The triphosphates of 2'-C-methyl-adenosine and 2'-C-methyl-guanosine were previously shown to be potent inhibitors of the HCV RNA-dependent RNA polymerase (RdRp) that is responsible for the replication of viral RNA in cells. Here we demonstrate that the inclusion of a 7-deaza modification in a series of purine nucleoside triphosphates results in an increase in inhibitory potency against the HCV RdRp and improved pharmacokinetic properties. Notably, incorporation of the 7-deaza modification into 2'-C-methyl-adenosine results in an inhibitor with a 20-fold-increased potency as the 5'-triphosphate in HCV RdRp assays while maintaining the inhibitory potency of the nucleoside in the bicistronic HCV replicon and with reduced cellular toxicity. In contrast, while 7-deaza-2'-C-methyl-GTP also displays enhanced inhibitory potency in enzyme assays, due to poor cellular penetration and/or metabolism, the nucleoside does not inhibit replication of a bicistronic HCV replicon in cell culture. 7-Deaza-2'-C-methyl-adenosine displays promising in vivo pharmacokinetics in three animal species, as well as an acute oral lethal dose in excess of 2,000 mg/kg of body weight in mice. Taken together, these data demonstrate that 7-deaza-2'-C-methyl-adenosine is an attractive candidate for further investigation as a potential treatment for HCV infection.

Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase.

Efficient replication of hepatitis C virus (HCV) replicons in cell culture is associated with specific sequences not generally observed in vivo. These cell culture adaptive mutations dramatically increase the frequency with which replication is established in vitro. However, replicons derived from HCV isolates that have been shown to replicate in chimpanzees do not replicate in cell culture even when these adaptive mutations are introduced. To better understand this apparent paradox, we performed a gain-of-function screen to identify sequences that could confer cell culture replication competence to replicons derived from chimpanzee infectious HCV isolates. We found that residue 470 in domain II of the NS3 helicase is a critical determinant in cell culture adaptation. Substitutions in residue 470 when combined with the NS5A-S232I adaptive mutation are both necessary and sufficient to confer cell culture replication to otherwise inactive replicons, including those derived from genotype 1b HCV-BK and genotype 1a HCV-H77 isolates. The specific substitution at residue 470 required for replication is context-dependent, with R470M and P470L being optimal for the activity of HCV-BK and HCV-H77 replicons, respectively. Together these data indicate that mutations in the NS3 helicase domain II act in concert with previously identified adaptive mutations and predict that introduction of compatible residues at these positions can confer cell culture replication activity to diverse HCV isolates.

Persistent replication of hepatitis C virus replicons expressing the beta-lactamase reporter in subpopulations of highly permissive Huh7 cells.

Progress toward development of better therapies for the treatment of hepatitis C virus (HCV) infection has been hampered by poor understanding of HCV biology and the lack of biological assays suitable for drug screening. Here we describe a powerful HCV replication system that employs HCV replicons expressing the beta-lactamase reporter (bla replicons) and subpopulations of Huh7 cells that are more permissive (or "enhanced") to HCV replication than naïve Huh7 cells. Enhanced cells represent a small fraction of permissive cells present among naïve Huh7 cells that is enriched during selection with replicons expressing the neomycin phosphotransferase gene (neo replicons). The level of permissiveness of cell lines harboring neo replicons can vary greatly, and the enhanced phenotype is usually revealed upon removal of the neo replicon with inhibitors of HCV replication. Replicon removal is responsible for increased permissiveness, since this effect could be reproduced either with alpha interferon or with an HCV NS5B inhibitor. Moreover, adaptive mutations present in the replicon genome used during selection do not influence the permissiveness of the resulting enhanced-cell population, suggesting that the mechanisms governing the permissiveness of enhanced cells are independent from viral adaptation. Because the beta-lactamase reporter allows simultaneous quantitation of replicon-harboring cells and reporter activity, it was possible to investigate the relationship between genome replication activity and the frequency with which transfected genomes can establish persistent replication. Our study demonstrates that differences in the replication potential of the viral genome are manifested primarily in the frequency with which persistent replication is established but modestly affect the number of replicons observed per replicon-harboring cell. Replicon copy number was found to vary over a narrow range that may be defined by a minimal number required for persistent maintenance and a maximum that is limited by the availability of essential host factors.