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 O 2 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% O 2 (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-␣ 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.

Chronic intermittent hypoxia (CIH) augments physiological responses to low partial pressures of O 2 in the arterial blood. Adrenal medullae from adult rats, however, are insensitive to direct effects of acute hypoxia. In the present study, we examined whether CIH induces hypoxic sensitivity in the adult rat adrenal medulla and, if so, by what mechanism(s). Experiments were performed on adult male rats exposed to CIH (15 s of 5% O 2 followed by 5 min of 21% O 2 ; 9 episodes h −1 ; 8 h d −1 ; for 3 or 10 days) or to comparable, cumulative durations of continuous hypoxia (CH; 4 h of 7% O 2 followed by 20 h of 21% O 2 for 1 or 10 days). Noradrenaline (NA) and adrenaline (ADR) effluxes were monitored from ex vivo adrenal medullae. In adrenal medullae of rats exposed to CIH, acute hypoxia evoked robust NA and ADR effluxes, whereas these responses were absent in control rats or in those exposed to CH for 1 or 10 days. Hypercapnia (10% CO 2 ; either acidic, pH 6.8, or isohydric, pH 7.4) was ineffective in eliciting catecholamine (CA) efflux from control, CIH or CH rats. Nicotine (100 μM) evoked NA and ADR effluxes in control rats, and this response was abolished in CIH but not in CH rats. Systemic administration of 2-deoxyglucose depleted ADR content in control rats, and CIH attenuated this response, indicating downregulation of neurally regulated CA secretion. Cytosolic and mitochondrial aconitase enzyme activities decreased in CIH adrenal medullae, suggesting increased generation of superoxide anions. Systemic administration of antioxidants reversed the effect of CIH on the adrenal medulla. Rats exposed to CIH exhibited increased blood pressures and elevated plasma CA, and antioxidants abolished these responses. These observations demonstrate that CIH induces hypoxic sensing in the adult rat adrenal medulla via mechanisms involving increased generation of superoxide anions and suggest that hypoxia-evoked CA efflux from the adrenal medulla contributes, in part, to elevated blood pressure and plasma CA.

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.

Hepatitis C virus non-structural protein 3 contains a serine protease and an RNA helicase. Protease cleaves the genomeencoded 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. Hepatitis C virus (HCV)3 infection is a leading cause of chronic liver disease and hepatocellular carcinoma. HCV is the founding member of the Hepacivirus genus of the family Flaviviridae (1). The HCV genome is a single-stranded RNA of positive polarity that is on the order of 9000 nucleotides (nt) in length (Fig. 1a). The 5Ј-non-translated region (NTR) contains a terminal stem-loop that is a required cis-acting replication element and the internal ribosome entry site. The 3Ј-NTR contains a cis-acting replication element that consists of an RNA stem-loop of variable sequence, a polyuridine/polypyrimidine tract of variable length, and a highly conserved "X-tail. " The variability observed in the 3Ј-NTR and elsewhere in the genome is sufficient to permit classification of HCV into six distinct genotypes.The HCV genome encodes a single open reading frame, the translation of which is directed by the internal ribosome entry site. The HCV polyprotein is on the order of 3000 amino acids long and can be divided into a structural region (C-p7 proteins) and a non-structural (NS) region (NS2-NS5B proteins) (Fig. 1a). Cleavage of the HCV polyprotein occurs co-and posttranslationally by host (structural region) and viral (non-structural region) proteases. Only the NS3-NS5B region of the polyprotein is required for genome replication in cell culture (2). NS5B is the viral RNA-dependent RNA polymerase (3). NS5A is a phosphoprotein (4) capable of specifically interacting with the 3Ј-NTR of the HCV genome (5), other non-structural proteins (6), and numerous cellular proteins (7,8). NS5A also functions in virus assembly (9, 10). NS4B is an integral membrane protein that is required for assembly of the "membranous web," the organelle used for RNA replication (11,12). NS4A is a cofactor for NS3 that directs the localization of NS3 and modulates its enzymatic activities (13). The N-terminal one-third of NS3 contains the protease activity responsible for processing of the non-structural region of the polyprotein (Fig. 1b) (14 -16) and some cellular proteins (17-19). The C-terminal two-thirds of NS3 is an RNA helicase of the DExH family (Fig. 1c) (20). The biological function of the RNA helicase activity is not known but may include 1) RNA folding/remodeling (21), 2) enhancement of polymerase processivity (22), and/or 3) genome encapsidation (23).The significance, if any, of having the major viral p...

No abstract

The primary oligomerization domain of poliovirus polymerase, 3Dpol, is stabilized by the interaction of the back of the thumb subdomain of one molecule with the back of the palm subdomain of a second molecule, thus permitting the head-to-tail assembly of 3Dpol monomers into long fibers. The interaction of Arg-455 and Arg-456 of the thumb with Asp-339, Ser-341, and Asp-349 of the palm is key to the stability of this interface. We show that mutations predicted to completely disrupt this interface do not produce equivalent growth phenotypes. Virus encoding a polymerase with changes of both residues of the thumb to alanine is not viable; however, virus encoding a polymerase with changes of all three residues of the palm to alanine is viable. Biochemical analysis of 3Dpol derivatives containing the thumb or palm substitutions revealed that these derivatives are both incapable of forming long fibers, suggesting that polymerase fibers are not essential for virus viability. The RNA binding activity, polymerase activity, and thermal stability of these derivatives were equivalent to that of the wild-type enzyme. The two significant differences observed for the thumb mutant were a modest reduction in the ability of the altered 3CD proteinase to process the VP0/VP3 capsid precursor and a substantial reduction in the ability of the altered 3Dpol to catalyze oriI-templated uridylylation of VPg. The defect to uridylylation was a result of the inability of 3CD to stimulate this reaction. Because 3C alone can substitute for 3CD in this reaction, we conclude that the lethal replication phenotype associated with the thumb mutant is caused, in part, by the disruption of an interaction between the back of the thumb of 3Dpol and some undefined domain of 3C. We speculate that this interaction may also be critical for assembly of other complexes required for poliovirus genome replication.The RNA-dependent RNA polymerase (RdRP) 1 is the key component of the replication machinery of RNA viruses. The RdRP from poliovirus (3Dpol) serves as a paradigm for this class of nucleic acid polymerases. The crystal structure for 3Dpol (1) revealed that this polymerase has the typical topology observed for other nucleic acid polymerases and can be compared with a cupped, right hand with fingers, palm, and thumb subdomains (Fig. 1A). A unique feature of this polymerase, however, is the presence of two extensive regions of polymerase-polymerase interactions, referred to as interface I and interface II (1). Polymerase molecules interact in a "headto-tail" fashion to form long, extended fibers via interface I, and these fibers interact with each other via interface II. Interface I is formed by an interaction between the back of the thumb of one polymerase molecule and the back of the palm of a second polymerase molecule (Fig. 1B). A few of the critical interactions required for integrity of interface I are shown in Fig. 1C. Specifically, Arg-455 and Arg-456 of the thumb subdomain of one polymerase molecule interact with Asp-339, Ser-341, and Asp-349 of...

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 proteinprotein 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 ϳ2-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. Hepatitis C virus (HCV),5 a member of the family Flaviviridae, is a leading cause of liver cirrhosis and hepatocellular carcinoma and has infected more than 170 million people worldwide (1-3). Standard treatment with interferon-␣ and ribavirin is frequently ineffective or toxic, and no vaccine for HCV is currently available (4). Identification of a novel viral target for therapeutic intervention could lead to the development of a more effective treatment.The HCV genome is a 9.6-kb positive, single-stranded RNA that supports translation of a 3000-amino acid polyprotein, which is subsequently cleaved to produce both structural (C, E1, and E2) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) viral proteins (5). Two viral proteases are encoded by the NS2 and NS3 genes (6, 7). In addition to its protease domain, the NS3 protein also contains a DEXH/ D-box helicase domain (8). NS4A encodes a peptide co-factor for NS3 protease (9), and NS5B encodes an RNA-dependent RNA polymerase (10). The functions of NS4B and NS5A have not been as well characterized, but NS5A is involved in viral interferon sensitivity (11) and in adaptation of subgenomic replicons for growth in cultured hepatoma cells (12).One obstacle to studying viral protein interactions is that HCV cannot be maintained efficiently in cell culture, although recent progress may overcome this difficulty (13). In order to circumvent this problem, selectable replicons carrying the neomycin phosphotransferase gene in add...