Membrane association of the RNA-dependent RNA polymerase is essential for hepatitis C virus RNA replication

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), belongs to a class of integral membrane proteins termed tail-anchored proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane protein-protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored proteins.     

Fungal macrolides: Structure determination and biosynthesis of achaetolide,a lactone from Achaetomium cristalliferum

The structure of a new ten-membered lactone, achaetolide, isolated from cultures of Achaetomium cristalliferum is deduced from its mass and NMR spectra and from the study ofsomederivatives. The ¹³C NMR spectra of achaetolide enriched with [1-¹³C], [2-¹³C] and [1, 2-¹³C] acetate established its formation from eight intact acetate units via a precursor octaketide chain.     

Xanthones and C-glucosylflavones from Gentiana corymbifera

A new xanthone and twelve known compounds were isolated from the aerial parts of Gentiana corymbifera. The new xanthone was shown to be 3-methylcorymbiferin (1,8-dihydroxy-3,4,5-trimethoxy-9H-xanthen-9-one) by spectral and chemical procedures. The chemotaxonomic implications are discussed.     

Field assessment in land of origin of host specificity,infestation rate and impact of <Emphasis Type="Italic">Ceratapion basicorne</Emphasis> a prospective biological control agent of yellow starthistle

Yellow starthistle, Centaurea solstitialis (Asteraceae), is an important invasive alien weed in the western United States. Currently established biological control agents attack only the capitula (flowerheads), and are not effectively controlling the plant in much of its range. The geographic center of diversity for the plant appears to be in Turkey, but no agents have been introduced from this country. Ceratapion basicorne (Coleoptera: Apionidae) is common in Central Turkey, attacking 25–100% of yellow starthistle plants. In a field experiment, Ceratapion spp. attacked 90% of yellow starthistle plants and 88% of milk thistle plants (Silybum marianum) but not seven other plant species, including artichoke and safflower. We suspect that a different species of insect attacked milk thistle, but they emerged before the plants were sampled. Laboratory tests showed that C. basicorne does not oviposit in milk thistle. Ceratapion basicorne appears to be more host specific than was suggested by previous studies of a population in Italy (Clement etal. 1989. Ann. Entomol. Soc. Am. 82: 741–747). The insect is gregarious, and the number of larvae per plant was positively correlated to root diameter. The level of damage to individual plants was positively correlated to the proportion of plants attacked, indicating aggregation both among plants and within plants. Field data did not show any impact of the insect on plant size or number of capitula, but germination rate of seeds produced by infested plants was 15% lower than for uninfested plants at two of three sites studied.     

Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a membrane-associated, essential component of the viral replication complex. Here, we report the three-dimensional structure of the membrane anchor domain of NS5A as determined by NMR spectroscopy. An alpha-helix extending from amino acid residue 5 to 25 was observed in the presence of different membrane mimetic media. This helix exhibited a hydrophobic, Trprich side embedded in detergent micelles, while the polar, charged side was exposed to the solvent. Thus, the NS5A membrane anchor domain forms an in-plane amphipathic alpha-helix embedded in the cytosolic leaflet of the membrane bilayer. Interestingly, mutations affecting the positioning of fully conserved residues located at the cytosolic surface of the helix impaired HCV RNA replication without interfering with the membrane association of NS5A. In conclusion, the NS5A membrane anchor domain constitutes a unique platform that is likely involved in specific interactions essential for the assembly of the HCV replication complex and that may represent a novel target for antiviral intervention.     

Mechanism of the delay phenomenon: tissue protection is mediated by heme oxygenase-1

Induction of the "delay phenomenon" by chronic ischemia is an established clinical procedure, but the mechanisms conferring tissue protection are still incompletely understood. To elucidate the role of heme oxygenase-1 [HO-1 or heat shock protein-32 (HSP-32)] in delay, we examined in the skin-flap model of the ear of the hairless mouse, 1) whether chronic ischemia (delay) is capable to induce expression of HO-1, and 2) whether delay-induced HO-1 affects skin-flap microcirculation and survival by either its carbon monoxide-associated vasodilatory action or its biliverdin-associated anti-oxidative mechanism. Chronic ischemia was induced by transsection of the central feeding vessel of the ear 7 days before flap creation. The flap was finally raised by an incision through four-fifths of the base of the ear. Microcirculatory dysfunction and tissue necrosis were studied with the use of laser Doppler fluxmetry and intravital fluorescence microscopy. HO-1 protein expression was determined with Western blot analysis. Seven days of chronic ischemia (delay) induced a marked expression of HO-1. This was paralleled by a significant improvement (P <0.05) of microvascular perfusion and a reduction (P <0.05) of flap necrosis when compared with nondelayed controls. Importantly, blockade of HO-1 activity by tin protoporhyrin-IX completely blunted the protection of microcirculation and the improvement of tissue survival. Additional administration of the vitamin E analog trolox after blockade of HO-1 to mimic exclusively the anti-oxidative action of the heat shock protein did not restore the HO-1-associated microcirculatory improvement and only transiently attenuated the manifestation of flap necrosis. Thus our data indicate that the delay-induced protection from tissue necrosis is mediated by HO-1, predominantly through its carbon monoxide-associated action of adequately maintaining nutritive capillary perfusion.     

Identification of GBF1 as a cellular factor required for hepatitis E virus RNA replication

The hepatitis E virus (HEV) genome is a single‐stranded, positive‐sense RNA that encodes three proteins including the ORF1 replicase. Mechanisms of HEV replication in host cells are unclear, and only a few cellular factors involved in this step have been identified so far. Here, we used brefeldin A (BFA) that blocks the activity of the cellular Arf guanine nucleotide exchange factors GBF1, BIG1, and BIG2, which play a major role in reshuffling of cellular membranes. We showed that BFA inhibits HEV replication in a dose‐dependent manner. The use of siRNA and Golgicide A identified GBF1 as a host factor critically involved in HEV replication. Experiments using cells expressing a mutation in the catalytic domain of GBF1 and overexpression of wild type GBF1 or a BFA‐resistant GBF1 mutant rescuing HEV replication in BFA‐treated cells, confirmed that GBF1 is the only BFA‐sensitive factor required for HEV replication. We demonstrated that GBF1 is likely required for the activity of HEV replication complexes. However, GBF1 does not colocalise with the ORF1 protein, and its subcellular distribution is unmodified upon infection or overexpression of viral proteins, indicating that GBF1 is likely not recruited to replication sites. Together, our results suggest that HEV replication involves GBF1‐regulated mechanisms.     

Stimulation of ACC-dependent ethylene production in citrus leaf discs by light

The influence of light and darkness incubation on in vivo ethylene forming enzyme (EFE) activity in citrus ( Citrus sinensis L. Osbeck cv. Salustiana) mature leaf discs was studied. Leaf discs incubated in light produced higher amounts of ethylene than in darkness. Transfer of discs from light to the dark resulted in a marked inhibition of EFE activity, whereas transfer of discs from the dark to light enhanced ethylene forming activity considerably. Light did not affect 1-aminocyclopropane-l-carboxylie acid (ACC) uptake. Incubation in a CO₂-eniiched atmosphere enhanced EFE activity both in light and in darkness, but light stimulation of EFE activity was apparently not affected by CO₂. Effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, inhibitor of photosynthetic electron flow) and KCN (inhibitor of cytochrome oxidase) were studied. DCMU at 0.2 m M inhibited EFE activity in light, whereas no effect was detected in the dark. On the other hand 1 m M KCN stimulated EFE activity in the light, and no significant effect was observed in the dark. CoCl₂ at 1 m M inhibited ACC-dependent ethylene production, suggesting that ethylene production from ACC is mediated by EFE in citrus leaf discs both in light and in the dark. Cycloheximide also inhibited EFE activity in the light and no effects were detected in the dark. Therefore protein synthesis in light (perhaps EFE synthesis) could be required for the light stimulation of the in vivo EFE activity.