Interferon alpha (IFNα)-induced TRIM22 interrupts HCV replication by ubiquitinating NS5A

TRIM22, a tripartite-motif (TRIM) protein, is upregulated upon interferon alpha (IFNα) administration to hepatitis C virus (HCV)-infected patients. However, the physiological role of TRIM22 upregulation remains unclear. Here, we describe a potential antiviral function of TRIM22''s targeting of the HCV NS5A protein. NS5A is important for HCV replication and for resistance to IFNα therapy. During the first 24 h following the initiation of IFNα treatment, upregulation of TRIM22 in the peripheral blood mononuclear cells (PBMCs) of HCV patients correlated with a decrease in viral titer. This phenomenon was confirmed in the hepatocyte-derived cell line Huh-7, which is highly permissive for HCV infection. TRIM22 over-expression inhibited HCV replication, and Small interfering RNA (siRNA)-mediated knockdown of TRIM22 diminished IFNα-induced anti-HCV function. Furthermore, we determined that TRIM22 ubiquitinates NS5A in a concentration-dependent manner. In summary, our results suggest that TRIM22 upregulation is associated with HCV decline during IFNα treatment and plays an important role in controlling HCV replication in vitro.     

Evaluation of sequencing of HCV core/E1, NS5A and NS5B as a genotype predictive tool in comparison with commercial assays targeting 5′UTR

BackgroundHepatitis C virus (HCV) genotyping is required for tailoring the dose and duration of antiviral therapy, predicting virological response rates, and selecting future treatment options.ObjectiveTo establish whether baseline genotypes, performed by INNO-LiPA Version 1.0 (v1.0), before 2008, were valid for making treatment decisions now or whether genotypic determination should be repeated. Furthermore, to evaluate concordance between Abbott RealTime genotype II assay (RT) and genotyping by sequencing HCV C/E1, NS5A, NS5B.Study designGenotyping by RT and sequencing was performed on paired historic and current specimens from 50 patients previously baseline genotyped using INNO-LiPA.ResultsOf 100 samples from 50 patients, ≥2 of HCV genomic target regions yielded a sequence that was suitable for genotyping, with 100% concordance, providing no evidence of recombination events. Genotype and subtype prediction based on RT and sequencing agreed in 62.8% historic and 72.7% current specimens, with a kappa coefficient score of 0.48 and 0.76, respectively.LiPA could not subtype 46% of HCV gt1 infections, and LiPA subgenotype was only in agreement with RT and sequencing in 28.6% cases, where matched baseline and historic specimens were available.Three patients were indeterminate by RT, and five patients with HCV gt1 infections could not be subtyped by RT. However, RT revealed mixed infections in five patients where sequencing detected only single HCV infection at 20% threshold.ConclusionGenotyping by sequencing, exhibited excellent concordance, with moderate to good agreement with RT, and could resolve RT indeterminates and subtype HCV-gt1 infections not possible by LiPA.     

The hepatitis C virus NS5A protein and response to interferon α: mutational analyses in patients with chronic HCV genotype 3a infection from India

The hepatitis C virus (HCV) non-structural (NS)5A protein is linked to interferon α resistance in vitro and higher numbers of NS5A amino acid (aa) variations in HCV 1a/b isolates are associated with virologic response to interferon α-based therapy in vivo. Here, we aimed to study NS5A aa variations in Indian patients undergoing interferon α/ribavirin treatment infected with HCV 3a. The NS5A region [aa 2194–2401, comprising interferon sensitivity determining region, protein kinase resource (PKR) binding domain, V3 region] was sequenced from pre-treatment sera of 24 patients with HCV 3a infection. Mean number and physicochemical properties of aa variations (conserved vs. non-conserved) were assessed. Additionally, published NS5A sequences [NS5A region (n = 61), PKR binding domain (n = 111)] of characterized HCV 3a isolates were analyzed. The mean number of NS5A aa variations was not correlated with treatment response in our cohort. When all available NS5A sequences were included, a higher number of non-conserved aa variations within PKR binding domain and an extended V3 region of NS5A was associated with virologic response (P = 0.004 and 0.05, respectively). Mutational analyses of a large number of NS5A sequences suggest, that a higher number of non-conserved aa variations within the PKR binding domain and the extended V3 region is correlated with virologic response in HCV 3a infected patients. Ankur Goyal and Wolf P. Hofmann contributed equally to this work.     

Serum levels of IgG to the peptide of HCV1b core at positions 35–44 correlated with persistent infection,while levels of IgG to the peptide of NS5A at positions 2132–2140 correlated with better prognosis in HCV-infected patients

We previously reported that two IgG Abs to the hepatitis C virus (HCV), anti-core 35–44 (C35) and anti-NS5A 2132–2140 (NS5A2132), existed in the sera of the majority of patients with HCV infection. This study investigated if measuring the two Abs would facilitate the prediction of a patient’s prognosis. The serum levels of anti-C35 were found to correlate with persistent infection, while those of anti-NS5A2132 correlated with a better prognosis in HCV-infected patients. These results suggest that sequential measurement of the two Abs together may provide new information for the prediction of prognosis.     

A putative α-glucoside transporter gene BbAGT1 contributes to carbohydrate utilization,growth, conidiation and virulence of filamentous entomopathogenic fungus Beauveria bassiana

Carbohydrate transporters are critical players mediating nutrient uptake during saprophytic and pathogenic growth for most filamentous fungi. For entomopathogenic fungi, such as Beauveria bassiana, assimilation of α-glucosides, in particular, trehalose, the major carbohydrate constituent of the insect haemolymph, has been hypothesized to represent an important ability for infectious growth within the insect hemocoel. In this study, a B. bassiana α-glucoside transporter homolog was identified and genetically characterized via generation of a targeted gene disruption mutant. Trehalose utilization was compromised in the mutant strain. In addition, inactivation of the α-glucoside transporter resulted in decreased conidial germination, growth, and yield on various carbohydrates (α-glucosides, monosaccharides and polyols) as compared to the wild-type strain. Insect bioassays revealed decreased mean lethal mortality time using both topical and intrahemocoel injection assays, although final mortality levels were comparable in both the mutant and wild type. Gene expression profiles showed altered expression of other putative transporters in the knockout mutant as compared to the wild type. These results highlighted complex sugar utilization and responsiveness in B. bassiana and the potential role for trehalose assimilation during fungal pathogenesis of insects.     

Regulation of respiratory-related hypoglossal motor output by α₁ adrenergic and serotonin 5-HT₃ receptor activation in isolated adult turtle brainstems

The effects of brainstem α(1) adrenergic receptor activation on respiratory control in reptiles are poorly understood. Isolated adult turtle brainstems were exposed to phenylephrine (α(1) adrenergic agonist) and respiratory motor bursts were recorded on hypoglossal nerves. Phenylephrine acutely increased burst frequency, amplitude (low concentrations only), and regularity of the time interval between the start of respiratory events (single or clustered bursts), and decreased bursts/respiratory event. Burst frequency and timing changes persisted during a 2.0 h washout. Acute increases in burst frequency and amplitude were blocked by prazosin (α(1) adrenergic antagonist). Pretreatment with prazosin and tropisetron (5-HT(3) antagonist) blocked the increase in respiratory event regularity, but did not alter the decrease in bursts/respiratory event. Intermittent phenylephrine application (4 × 5.0 min separated by 20 min) did not produce long-lasting changes in burst frequency and amplitude, bursts/respiratory event, or respiratory event regularity. Thus, sustained α(1) adrenergic receptor activation in turtle brainstems produces acute and long-lasting changes in respiratory burst frequency and pattern.     

Effects of the C-terminal Peptide of the αs subunit of the G protein on the regulation of Adenylyl Cyclase and Protein Kinase A activities by Biogenic Amines and Glucagon in Mollusk and Rat Muscles

The C-terminal parts of the subunits of heteromeric G proteins play an important role in the functional linkage of G proteins with receptors of the serpentine type. The present report describes studies of the effects of the C-terminal octapeptide 387–394 of the _s subunit of the mammalian G protein on the transmission of the hormonal signal via the hormone-sensitive adenylyl cyclase signal system, whose major components are receptors of the serpentine type, G proteins, and the enzymes adenylyl cyclase and protein kinase A. The peptide synthesized here, 387–394 amide (10^–7 - 10^–4 M), dose-dependently decreased adenylyl cyclase and protein kinase A activities stimulated by serotonin and glucagon in smooth muscle from the freshwater bivalve mollusk Anodonta cygnea and by the agonist isoproterenol in rat skeletal muscle. At a concentration as low as 10^–7 M, the peptide released potentiation of the stimulatory effects of hormones on adenylyl cyclase activity due to the non-hydrolyzable guanine nucleotide analog Gpp[NH]p. At the same time, it had almost no effect on the stimulation of adenylyl cyclase activity by non-hormonal agents (NaF, Gpp[NH]p, and forskolin). The inhibitory effects of hormones on adenylyl cyclase and protein kinase A activities persisted in the presence of the peptide. Our data demonstrate the importance of the C-terminal part of the _s subunit of the stimulatory G protein for its functional linkage with receptors of the serpentine type and throw light on the molecular mechanisms of the interactions between G proteins and receptors.Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 89, No. 7, pp. 837–850, July, 2003.     

Regulation of the L-type calcium channel α-1 subunit by chronic depolarization in the neuron-like PC12 and aortic smooth muscle A7r5 cell lines

The regulation of L-type voltage-dependent Ca²⁺ channels by chronic depolarization was studied in the aortic smooth muscle A7r5 and neuron-type PC12 cell lines, by probing the expression and the functional state of their constitutive -1 subunits. PC12 cells showed, after prolonged exposure to a high-K⁺ depolarizing solution, a 25% reduction of the functional Ca²⁺ channel density which was accompanied by a decrease of the -1 subunit mRNA expression. In A7r5 cells submitted to a similar protocol of depolarization, ⁴⁵Ca²⁺ uptake measurements revealed a fall in the functional activity of L-type Ca²⁺ channels which was not related to a modulation of their mRNA expression, but arose from a long-term voltage-dependent channel inactivation. Accordingly, the lag time and the mechanisms of recovery were different in the two cell types. In PC12 cells, when restoring physiological culture conditions, de novo synthesis of -1 subunits allowed the recovery of the original density of L-type Ca²⁺ channels at the membrane surface. As for the A7r5 cells, we showed that after chronic depolarization, the complete restoration of the resting membrane potential and the related Ca²⁺ channel activity required a 2-day incubation in physiological medium and could probably be related to a normalization of the increased intracellular Ca²⁺ concentration. In contrast, it is noteworthy that, in PC12 cells, the only transient increase of intracellular Ca²⁺ content in the first hours of depolarization could account for the long-term down-regulation of L-type Ca²⁺ channels.     

Effects of N-methyl-d-aspartate (dizocilpine) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (LY215490) receptor antagonists on the voiding reflex induced by perineal stimulation in the neonatal rat

The present study was undertaken to examine the role of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-d-aspartate glutamate receptors in the regulation of voiding reflexes induced by perineal stimulation in the neonatal rat. Four-, six- and 10-day-old awake rats were used in the experiments and perineal stimulation was applied using the tip of a 1-ml syringe to evoke voiding. Voided volume and residual volume were measured. In 10-day-old rats, LY215490 (3–10 mg/kg, i.p.), a competitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor antagonist, significantly inhibited reflex voiding, increasing the residual volume 34–53-fold. A 3 mg/kg dose decreased the urine release by 55%, whereas 10 mg/kg totally suppressed the voiding reflex induced by the perineal stimulation. LY215490 (10 mg/kg, i.p.) produced similar effects in four- and six-day-old rats. Dizocilpine (1–3 mg/kg, i.p.), a non-competitive N-methyl-d-aspartate receptor antagonist, also significantly decreased the urine release (62–82%) and increased residual volume (180–230-fold). Combined administration of LY215490 (1 mg/kg, i.p.) and dizocilpine (0.3 mg/kg, i.p.) to 10-day-old rats, in doses that individually had no effect on perineal stimulation-evoked voiding, depressed voided volume by 65%.These results indicate that, in neonatal rats, glutamatergic transmission in the spinal cord has an essential role in reflex micturition induced by perineal stimulation, and that facilitatory interactions between α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-d-aspartate glutamatergic mechanisms are important for voiding, as noted previously in adult rats.