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The aryl hydrocarbon receptor nuclear translocator (ARNT) is a basic helix-loop-helix Period/ARNT/Single-minded (bHLH-PAS) protein that controls various biological pathways as part of dimeric transcriptional regulator complexes with other bHLH-PAS proteins. The two PAS domains within ARNT, PAS-A and PAS-B, are essential for the formation of these complexes because they mediate protein-protein interactions via residues located on their β-sheet surfaces. While investigating the importance of residues in ARNT PAS-B involved in these interactions, we uncovered a point mutation (Y456T) on the solvent-exposed β-sheet surface that allowed this domain to inter-convert with a second, stable conformation. Although both conformations are present in equivalent quantities in the Y456T mutant, this can be shifted almost completely to either end point by additional mutations. A high-resolution solution structure of a mutant ARNT PAS-B domain stabilized in the new conformation revealed a 3-residue slip in register and accompanying inversion of the central Iβ-strand. We have demonstrated that the new conformation has >100-fold lower in vitro affinity for its heterodimerization partner, hypoxia-inducible factor 2α PAS-B. We speculate that the pliability in β-strand register is related to the flexibility required of ARNT to bind to several partners and, more broadly, to the abilities of some PAS domains to regulate their activities in response to small-molecule cofactors. [ABSTRACT FROM AUTHOR]

The study of the hepatitis C virus (HCV) has been hindered by the lack of in vitro model systems. The recent development of HCV subgenomic RNA replicons has permitted the study of viral RNA replication in cell culture; however, the requirements for efficient replication of replicons in this system are poorly understood. Many viral isolates do not function as replicons and most require conserved changes, termed adaptive mutations, to replicate efficiently. In this report, we focus on the HCV nonstructural protein 5A (NS5A), a frequent locus for adaptive mutation. We found the interaction between NS5A and human vesicle-associated membrane protein-associated protein A (hVAP-A), a cellular target N-ethylmaleimide-sensitive factor attachment protein receptor, to be required for efficient RNA replication: NS5A mutations that blocked interaction with hVAP-A strongly reduced HCV RNA replication. Further analyses revealed an inverse correlation between NS5A phosphorylation and hVAP-A interaction. A subset of the previously identified adaptive mutations suppressed NS5A hyper- phosphorylation and promoted hVAP-A binding. Our results support a model in which NS5A hyperphosphorylation disrupts interaction with hVAP-A and negatively regulates viral RNA replication, suggesting that replicon-adaptive mutations act by preventing the phosphorylation-dependent dissociation of the RNA replication complex. [ABSTRACT FROM AUTHOR]

The authors discuss the replication of miR-122 independent hepatitis C virus (HCV). They observe the impact of miR-122 on the stability of HCV RNA and its replication. They note that miR-122 shields HCV RNA from exoribonuclease enzyme (Xrn1) degradation and plays a significant role in the turnover of HCV RNA in cell replication.

In the core protein-coding region of hepatitis C virus (HCV), evidence exists for both phylogenetically conserved RNA structures and a +1 alternative reading frame (ARF). To investigate its role in HCV infection, we introduced four stop codons into the ARF of a genotype 1 a H77 molecular clone. The changes did not alter the core protein sequence, but were predicted to disrupt RNA secondary structures. An attenuated infection was established after inoculation of the mutant HCV RNA into an HCV naïve chimpanzee. The acute infection was atypical with low peak viremia, minimal alanine aminotransferase elevation, and early virus control by a diverse adaptive immune response. Sequencing circulating virus revealed progressive reversions at the third and then fourth stop codon. In cell culture, RNA replication of a genome with four stop codons was severely impaired. In contrast, the revertant genome exhibited only a 5-fold reduction in replication. Genomes harboring only the first two stop codons replicated to WT levels. Similarly, reversions at stop codons 3 and 4, which improved replication, were selected with recombinant, infectious HCV in cell culture. We conclude that ARF-encoded proteins initiating at the polyprotein AUG are not essential for HCV replication in cell culture or in vivo. Rather, our results provide evidence for a functionally important RNA element in the ARF region. [ABSTRACT FROM AUTHOR]

To determine the role of glucagon action in diet-induced and genetic type 2 diabetes (T2D), we studied high-fat-diet–induced obese (DIO) and leptin receptor-defective (LepR–/–) rodents with and without glucagon receptors (GcgRs). DIO and LepR–/–,GcgR+/+ mice both developed hyperinsulinemia, increased liver sterol response element binding protein 1c, and obesity. DIO GcgR+/+ mice developed mild T2D, whereas LepR–/–,GcgR+/+ mice developed severe T2D. High-fat–fed (HFF) glucagon receptor-null mice did not develop hyperinsulinemia, increased liver sterol response element binding protein 1c mRNA, or obesity. Insulin treatment of HFF GcgR–/– to simulate HFF-induced hyperinsulinemia caused obesity and mild T2D. LepR–/–,GcgR–/– did not develop hyperinsulinemia or hyperglycemia. Adenoviral delivery of GcgR to GcgR–/–,LepR–/– mice caused the severe hyperinsulinemia and hyperglycemia of LepR–/– mice to appear. Spontaneous disappearance of the GcgR transgene abolished the hyperinsulinemia and hyperglycemia. In conclusion, T2D hyperglycemia requires unsuppressible hyperglucagonemia from insulin-resistant α cells and is prevented by glucagon suppression or blockade. [ABSTRACT FROM AUTHOR]