Class III Phosphatidylinositol 4-Kinase Alpha and Beta Are Novel Host Factor Regulators of Hepatitis C Virus Replication

Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase. Knockdown of PI4KA could inhibit the replication and/or HCV RNA levels of the two subgenomic genotype 1b clones (SG-1b and Luc-1b), two subgenomic genotype 1a clones (SG-1a and Luc-1a), JFH-1 genotype 2a infectious virus (JFH1-2a), and the genomic genotype 1a (FL-1a) replicon. In contrast, PI4KB knockdown inhibited replication and/or HCV RNA levels of Luc-1b, SG-1b, and Luc-1a replicons. The small molecule inhibitor, PIK93, was found to block subgenomic genotype 1b (Luc-1b), subgenomic genotype 1a (Luc-1a), and genomic genotype 2a (JFH1-2a) infectious virus replication in the nanomolar range. PIK93 was characterized by using quantitative chemical proteomics and in vitro biochemical assays to demonstrate PIK93 is a bone fide PI4KA and PI4KB inhibitor. Our data demonstrate that genetic or pharmacological modulation of PI4KA and PI4KB inhibits multiple genotypes of HCV and represents a novel druggable class of therapeutic targets for HCV infection.Hepatitis C virus (HCV) causes liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (52). The HCV genome is a single-stranded RNA molecule where both the 5′ and the 3′ untranslated region (UTR) contain highly conserved RNA structures necessary for polyprotein translation and genome replication (43). The processed polyprotein yields at least three structural proteins and six nonstructural proteins. The structural proteins include the core, which forms the viral nucleocapsid, and the envelope glycoproteins E1 and E2. The viral proteins processed by signal peptidases form viral particles that assemble at the endoplasmic reticulum (ER) and/or Golgi bodies and are released from the host cell by viral budding. The structural protein coding regions are separated from nonstructural proteins by the short membrane peptide p7, thought to function as an ion channel (43, 53). The nonstructural proteins NS2, NS3/4A, NS5A, and NS5B are involved in coordinating the intracellular processes of the virus life cycle, including polyprotein processing and viral RNA replication (34).The Luc-1b cell is a human hepatoma cell line (Huh7) that contains a genotype 1b HCV subgenomic replicon, a luciferase reporter, and a neomycin selection marker, allowing HCV replication to be studied both in vitro and in vivo (8, 36). This subgenomic replicon lacks the coding regions for NS2 and the structural proteins but contains the nonstructural proteins in cis, which are required for replication of the viral RNA. Expression of the luciferase gene acts as a surrogate marker for levels of HCV RNA produced in the cell. The goal of the present study was to use this subgenomic HCV replicon to screen siRNA libraries and identify novel host proteins that are involved in HCV replication.A number of cellular pathways and proteins that play critical roles in HCV replication have recently been described (41, 42, 46). In particular, replication of HCV is tied closely to its localization and transport to various internal membranes and to lipid metabolism (2). Most of the HCV proteins appear to be targeted to the surface of the ER and replication complexes appear to be transported to lipid rafts, where RNA replication can occur (2). Infectious virus particle formation occurs in association with lipid droplets, and this process requires the core and NS5A proteins. In addition, cholesterol pathway production of geranylgeranyl-PP is important to geranylate the FBL2 protein, which serves as a membrane anchor for NS5A (62). The hVAP proteins involved in the localization and trafficking between internal membranous structures are known to be associated with the HCV proteins NS5A and NS5B (59). Thus, host factor lipid metabolism and intracellular protein transport are necessary for HCV replication in cells.Targeting host factors that are required for viral replication offers a strategy to overcome viral resistance and may allow treatment for more than one genotype of HCV and/or a related Flaviviridae virus such as Dengue, West Nile, or yellow fever virus. The current standard-of-care treatment for the genotype 1 strain of HCV infection is pegylated interferon alpha plus ribavirin over a 6-month time course with more than half of infected patients being refractory to this treatment (57). In addition to genotype 1, there are at least five naturally occurring genotype variants of HCV that can complicate a patient''s response to therapy when infected with more than one genotype. As well as the development of mutations, the presence of multiple variants coexisting in patients is thought to contribute to the rapid development of resistance (40). A variety of antiviral therapeutic strategies aim to inhibit viral proteins directly with small molecules or siRNAs (13, 31, 33). Although some small molecule approaches have been successful in preclinical studies, small-molecule strategies directed against the viral targets can still be rendered ineffective due to the development of mutant, treatment-resistant viral strains (13, 40). Thus, combination therapies are a necessary approach to treat the many variants of HCV that exist in the patient population.In the present study, a set of 779 SMARTpool small interfering RNAs (siRNAs) targeting the kinome and 4 siRNAs targeting 5,000 druggable genes (20,000 siRNAs) were tested for their ability to block replication of the Luc-1b HCV subgenomic replicon. siRNAs targeting CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase), a tripartite enzyme that catalyzes the first three steps of pyrimidine biosynthesis, inhibited both the Luc-1b replicon and JFH1-2a virus expression. This activity is consistent with the known inhibitor of this enzyme, leflunomide, which has been shown previously to inhibit both respiratory syncytial virus and HCV (12, 54). siRNAs targeting the mevalonate (diphospho) decarboxylase (MVD) enzyme, which catalyzes the formation of mevalonate, were found to inhibit Luc-1b replication (19). Inhibition of the cholesterol biosynthesis pathway and host cell geranylation has been previously reported to inhibit HCV subgenomic replication (3, 24, 51, 62, 67). siRNA-mediated knockdown of the class III phosphatidylinositol 4-kinases PI4KA and PI4KB inhibited luciferase expression not only for the genotype 1b subgenomic replicons (Luc-1a and Luc-1b) but also for the viral RNA levels of SG-1b, Luc-1b, and Luc-1a. PI4KA knockdown also inhibited Renilla expression in the JFH-1 genotype 2a infectious virus (JFH1-2a), genotype 2a subgenomic replicon (SG-1a), and a genomic and subgenomic genotype 1a replicon (FL-1a and SG-1a). Using the small-molecule inhibitor PIK93 in compound affinity competition experiments and in vitro biochemical assays, we demonstrated PIK93 could bind and inhibit both PI4KA and PI4KB enzymatic activity (58). PIK93 could inhibit luciferase expression in the Luc-1b, Luc-1a, and JFH1-2a infectious virus assays in the submicromolar range. Together, our data suggest that PI4KA and PI4KB regulate HCV replication and that pharmacological inhibition of these enzymes represents a new class of antiviral agents for multiple genotypes of HCV. Finally, since PI4KA and PI4KB are known to regulate protein and lipid transport to and from the ER and Golgi bodies, their function may hold clues as to how movement of HCV replication complexes throughout different organelles is regulated.     

A systems approach to prion disease

Prions cause transmissible neurodegenerative diseases and replicate by conformational conversion of normal benign forms of prion protein (PrP^C) to disease‐causing PrP^Sc isoforms. A systems approach to disease postulates that disease arises from perturbation of biological networks in the relevant organ. We tracked global gene expression in the brains of eight distinct mouse strain–prion strain combinations throughout the progression of the disease to capture the effects of prion strain, host genetics, and PrP concentration on disease incubation time. Subtractive analyses exploiting various aspects of prion biology and infection identified a core of 333 differentially expressed genes (DEGs) that appeared central to prion disease. DEGs were mapped into functional pathways and networks reflecting defined neuropathological events and PrP^Sc replication and accumulation, enabling the identification of novel modules and modules that may be involved in genetic effects on incubation time and in prion strain specificity. Our systems analysis provides a comprehensive basis for developing models for prion replication and disease, and suggests some possible therapeutic approaches.     

What determines a species' geographical range? Thermal biology and latitudinal range size relationships in European diving beetles (Coleoptera: Dytiscidae)

1.  The geographical range sizes of individual species vary considerably in extent, although the factors underlying this variation remain poorly understood, and could include a number of ecological and evolutionary processes. A favoured explanation for range size variation is that this result from differences in fundamental niche breadths, suggesting a key role for physiology in determining range size, although to date empirical tests of these ideas remain limited.
2.  Here we explore relationships between thermal physiology and biogeography, whilst controlling for possible differences in dispersal ability and phylogenetic relatedness, across 14 ecologically similar congeners which differ in geographical range extent; European diving beetles of the genus Deronectes Sharp (Coleoptera, Dytiscidae). Absolute upper and lower temperature tolerance and acclimatory abilities are determined for populations of each species, following acclimation in the laboratory.
3.  Absolute thermal tolerance range is the best predictor of both species' latitudinal range extent and position, differences in dispersal ability (based on wing size) apparently being less important in this group. In addition, species' northern and southern range limits are related to their tolerance of low and high temperatures respectively. In all cases, absolute temperature tolerances, rather than acclimatory abilities are the best predictors of range parameters, whilst the use of independent contrasts suggested that species' thermal acclimation abilities may also relate to biogeography, although increased acclimatory ability does not appear to be associated with increased range size.
4.  Our study is the first to provide empirical support for a relationship between thermal physiology and range size variation in widespread and restricted species, conducted using the same experimental design, within a phylogenetically and ecologically controlled framework.     

Steady-state kinetic behaviour of two- or n-enzyme systems made of free sequential enzymes involved in a metabolic pathway

The overall rate of functioning of a set of free sequential enzymes of the Michaelis–Menten type involved in a metabolic pathway has been computed as a function of the concentration of the initial substrate under steady-state conditions. Curves monotonically increasing up to a saturation plateau have been obtained in all cases. The shape of these curves is sometimes, but not usually, close to that of a hyperbola. Cases exist in which the overall rate of reaction becomes quasi proportional to the concentration of initial substrate almost up to the saturation plateau, which never occurs with individual enzymes. Increasing the number of enzymes sequentially involved in a metabolic pathway does not seem to generate any particularly original behaviour compared with that of two-enzyme systems. To cite this article: G. Legent et al., C. R. Biologies 329 (2006).     

Steady-state kinetic behaviour of two- or n-enzyme systems made of free sequential enzymes involved in a metabolic pathway

The overall rate of functioning of a set of free sequential enzymes of the Michaelis-Menten type involved in a metabolic pathway has been computed as a function of the concentration of the initial substrate under steady-state conditions. Curves monotonically increasing up to a saturation plateau have been obtained in all cases. The shape of these curves is sometimes, but not usually, close to that of a hyperbola. Cases exist in which the overall rate of reaction becomes quasi proportional to the concentration of initial substrate almost up to the saturation plateau, which never occurs with individual enzymes. Increasing the number of enzymes sequentially involved in a metabolic pathway does not seem to generate any particularly original behaviour compared with that of two-enzyme systems.     

Ovarian function in nutritionally induced anoestrous cows: effect of exogenous gonadotrophin-releasing hormone in vivo and effect of insulin and insulin-like growth factor I in vitro

Ovarian function of nutritionally induced anoestrus cows was evaluated in vivo (Expt 1) and in vitro (Expt 2). In Expt 1, 32 nutritionally induced anoestrous beef cows were divided into four treatment groups receiving: (1) saline infusions at one pulse every 4 h for 13 days (control); (2) 2 micrograms GnRH at one pulse every 4 h (2 micrograms infused in 1.8 ml saline over 5 min) for 13 days (GnRH-4); (3) 2 micrograms GnRH at one pulse every 1 h for 13 days (GnRH-1); and (4) continuous infusion of 2 micrograms GnRH (a total of 2 micrograms in 34 ml h-1) for 13 days (GnRH-C). On the last day of treatment, cows were killed, ovaries were removed and follicular fluid samples (n = 149) were collected. The percentage of cows with luteal activity on day 13 was significantly different (P < 0.01) among treatments (0, 25, 75 and 25% for control, GnRH-4, GnRH-1 and GnRH-C cows, respectively). Owing to the large percentage of ovulatory cows in the GnRH-1 group (n = 6), anovulatory cows (n = 2) were removed from this treatment group for statistical analysis, as were cows with luteal tissue from the GnRH-4 (n = 2) and GnRH-C (n = 2) groups. The numbers of small (1.0-4.9 mm) and medium plus large (> or = 5 mm) follicles were not affected (P > 0.10) by treatment. However, GnRH-4 cows (n = 6) had greater (P < 0.05) concentrations of oestradiol in follicular fluid than did control (n = 8) but not GnRH-1 (n = 6) or GnRH-C (n = 6) cows. Concentrations of insulin-like growth factor I were greater (P < 0.05) in the follicular fluid of GnRH-1 cows than in all other treatment groups. Concentrations of androstenedione and progesterone in follicular fluid were not affected (P > 0.10) by treatment or follicle size. The binding activity of insulin-like growth factor binding proteins was not affected by GnRH treatment. However, the binding activity of insulin-like growth factor binding protein 2, 29-32 kDa and 22 kDa insulin-like growth factor binding proteins were greater (P < 0.05) in small versus medium plus large follicles. In Expt 2, granulosa cells were collected from nutritionally anoestrous cows to determine whether ovarian cells from anoestrous cows have the capacity to respond to insulin-like growth factor I or insulin in vitro. Both insulin-like growth factor I (20 and 200 ng ml-1) and insulin (10, 100 and 1000 ng ml-1) increased (P < 0.05) granulosa cell proliferation and progesterone production. In conclusion, pulsatile infusion of 2 micrograms GnRH (every 1 or 4 h) for 13 days into nutritionally induced anoestrous cows results in increased intrafollicular oestradiol and insulin-like growth factor I concentrations and can stimulate ovulation without markedly affecting concentrations of androstenedione or progesterone, or the binding activity of insulin-like growth factor binding proteins, in follicular fluid. In addition, granulosa cells from nutritionally induced anoestrous cows have the capacity to respond to insulin-like growth factor I and insulin in vitro, indicating that the decrease in trophic factors observed with restricted feeding does not reduce the response of the ovary to insulin-like growth factor I and insulin.