Chronic intermittent hypoxia (CIH) occurs in patients with sleep apnoea and has adverse effects on multiple physiological functions. Previous studies have shown that reflexes arising from carotid bodies mediate CIH‐evoked cardio‐respiratory responses, and reactive oxygen species (ROS) play important roles in eliciting systemic responses to CIH. Very little is known about the molecular mechanisms underlying CIH. The transcriptional activator hypoxia‐inducible factor‐1 (HIF‐1) mediates a broad range of cellular and systemic responses to hypoxia, and HIF‐1 is activated in cell cultures exposed to IH. In the present study we examined whether CIH activates HIF‐1 and if so whether it contributes to cardio‐respiratory responses and ROS generation in mice. Experiments were performed on male littermate wild‐type (WT) and heterozygous (HET) mice partially deficient in HIF‐1α, the O₂ regulated subunit of the HIF‐1 complex. Both groups of mice were exposed to either 10 days of CIH (15 s of hypoxia followed by 5 min of normoxia, 9 episodes h⁻¹, 8 h day⁻¹) or to 10 days of 21% O₂ (controls). Carotid body response to hypoxia was augmented, and acute intermittent hypoxia (AIH) induced sensory long‐term facilitation (sLTF) of the chemoreceptor activity in CIH‐exposed WT mice. In striking contrast, hypoxic sensory response was unaffected and AIH was ineffective in eliciting sLTF in CIH‐exposed HET mice. Analysis of cardio‐respiratory responses in CIH‐exposed WT mice revealed augmented hypoxic ventilatory response, LTF of breathing, elevated blood pressures and increased plasma noradrenaline. In striking contrast these responses were either absent or attenuated in HET mice exposed to CIH. In CIH‐exposed WT mice, ROS were elevated and this response was absent in HET mice. Manganese (III) tetrakis(1‐methyl‐4‐pyridyl) porphyrin pentachloride, a potent scavenger of superoxide, not only prevented CIH‐induced increases in ROS but also CIH‐evoked HIF‐1α up‐regulation in WT mice. These results indicate that: (a) HIF‐1 activation is critical for eliciting CIH‐induced carotid body‐mediated cardio‐respiratory responses; (b) CIH increases ROS; and (c) the effects of CIH involve complex positive interactions between HIF‐1 and ROS.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) has been shown to antagonize numerous cellular pathways, including the antiviral interferon-α response. However, the capacity of this protein to interact with the viral polymerase suggests a more direct role for NS5A in genome replication. In this study, we employed two bacterially expressed, soluble derivatives of NS5A to probe for novel functions of this protein. We find that NS5A has the capacity to bind to the 3′-ends of HCV plus and minus strand RNAs. The high affinity binding site for NS5A in the 3′-end of plus strand RNA maps to the polypyrimidine tract, an element known to be essential for genome replication and infectivity. NS5A has a preference for single-stranded RNA containing stretches of uridine or guanosine. Values for the equilibrium dissociation constants for high affinity binding sites were in the 10 nm range. Two-dimensional gel electrophoresis followed by Western blotting revealed the presence of unphosphorylated NS5A in Huh-7 cells stably expressing the subgenomic replicon. Moreover, RNA immunoprecipitation and NS5A pull-down experiments showed the capacity of replicon-derived NS5A to bind to synthetic RNA and the HCV genome, respectively. Deletion of all of the casein kinase II phosphorylation sites in NS5A supported stable replication of a subgenomic replicon in Huh-7. However, this derivative could not be labeled with inorganic phosphate, suggesting that extensive phosphorylation of NS5A is not required for the replication functions of NS5A. The discovery that NS5A is an RNA-binding protein defines a new functional target for development of agents to treat HCV infection and a new structural class of RNA-binding proteins.

Hepatitis C virus non-structural protein 3 contains a serine protease and an RNA helicase. Protease cleaves the genome-encoded polyprotein and inactivates cellular proteins required for innate immunity. Protease has emerged as an important target for the development of antiviral therapeutics, but drug resistance has turned out to be an obstacle in the clinic. Helicase is required for both genome replication and virus assembly. Mechanistic and structural studies of helicase have hurled this enzyme into a prominent position in the field of helicase enzymology. Nevertheless, studies of helicase as an antiviral target remain in their infancy.

Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2′-C-methyl, 4′-methyl and 4′-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2′-C-methyl, 4′-methyl and 4′-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity. Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by side effects of unknown origin. Here we show in biochemical and cell-based studies that antiviral ribonucleotide analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Analogues that terminate RNA synthesis by viral RNA polymerases also inhibit these cellular RNA polymerase. Importantly, the TFIIS proofreading activity of Pol II is capable of excising these analogues from Pol II transcripts. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity.

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains. A crystal structure of the NS3 helicase domain (NS3h) was generated in the presence of a single-stranded oligonucleotide long enough to accommodate binding of two molecules of enzyme. Several amino acid residues at the interface of the two NS3h molecules were identified that appear to mediate a protein-protein interaction between domains 2 and 3 of adjacent molecules. Mutations were introduced into domain 3 to disrupt the putative interface and subsequently examined using an HCV subgenomic replicon, resulting in significant reduction in replication capacity. The mutations in domain 3 were then examined using recombinant NS3h in biochemical assays. The mutant enzyme showed RNA binding and RNA-stimulated ATPase activity that mirrored wild type NS3h. In DNA unwinding assays under single turnover conditions, the mutant NS3h exhibited a similar unwinding rate and only ∼2-fold lower processivity than wild type NS3h. Overall biochemical activities of the mutant NS3h were similar to the wild type enzyme, which was not reflective of the large reduction in HCV replicative capacity observed in the biological experiment. Hence, the biological results suggest that the known biochemical properties associated with the helicase activity of NS3h do not reveal all of the likely biological roles of NS3 during HCV replication. Domain 3 of NS3 is implicated in protein-protein interactions that are necessary for HCV replication.