The ability to modify responses to type I interferons (IFNs) could alter processes such as hematopoiesis and immunity, which involve endogenous IFNs and responses to exogenous IFNs. The data presented here support a significant role for a recently identified soluble isoform of the murine type I IFN receptor, muIfnar2a, as an efficient regulator of IFN responses. The messenger RNA (mRNA) transcript encoding muIfnar-2a is generally more abundant than that encoding the transmembrane isoform, muIfnar-2c. Furthermore, the ratio of muIfnar-2a:2c transcripts varied from more than 10:1 in the liver and other organs to less than 1:1 in bone-marrow macrophages, indicating independent regulation of the 2 transcripts encoding receptor isoforms and suggesting that the soluble muIfnar-2a levels are biologically relevant in some organs. Western blot analysis showed that soluble muIfnar-2 was present at high levels in murine serum and other biologic fluids and bound type I IFN. Recombinant muIfnar-2a competitively inhibited the activity of both IFN␣ and ␤ in reporter assays using the L929 cell line and in antiproliferative and antiviral assays using primary cells. Surprisingly, IntroductionThe type I interferons (IFNs) are a family of cytokines produced in physiologic and pathologic conditions. Their biologic activities include inhibition of macrophage and lymphocyte proliferation, differentiation of macrophages, activation of natural killer and dendritic cells, and increased survival of memory T cells in addition to their well-characterized antiviral and antitumor activities. [1][2][3][4][5][6] These activities make type I IFNs important mediators of physiologic processes and disease pathogenesis and useful in the treatment of viral infections, some cancers (particularly those of hematopoietic origin), and other diseases, including multiple sclerosis. 7 In both human and mouse, the type I IFNs include multiple IFN␣ subtypes with about 75% to 99% amino acid identity and a single IFN␤ with about 30% amino acid identity to IFN␣. 8 All type I IFNs have similar biologic activities and compete for binding to a common cell surface receptor. 9 The type I IFN receptor is composed of 2 known subunits: IFNAR-1 10 and IFNAR-2. 11 In humans and mice, there is a single form of IFNAR-1. 12 In contrast, multiple isoforms of human IFNAR-2 have been shown to result from alternative splicing of the same gene producing huIFNAR-2a (encoding a putative soluble isoform), huIFNAR-2b (encoding a putative transmembrane receptor with a truncated cytoplasmic domain), and huIFNAR-2c (encoding a putative full-length transmembrane receptor). 13 HuIFNAR-2c reconstituted IFN signaling in the huIFNAR-2-deficient cell line (U5A), which was otherwise unresponsive to IFN, whereas huIFNAR-2b did not, indicating that the latter was a nonfunctional isoform. 13 We recently cloned the murine orthologue of IFNAR-2 (muIfnar-2) and identified complementary DNAs (cDNAs) encoding full-length muIfnar-2c and soluble muIfnar-2a isoforms. 14 Northern blots showed 2 transcript...

Virus reprogramming of cellular metabolism is recognised as a critical determinant for viral growth. While most viruses appear to activate central energy metabolism, different viruses have been shown to rely on alternative mechanisms of metabolic activation. Whether related viruses exploit conserved mechanisms and induce similar metabolic changes is currently unclear. In this work we investigate how two alphaviruses, Semliki Forest virus and Ross River virus, reprogram host metabolism and define the molecular mechanisms responsible. We demonstrate that in both cases the presence of a YXXM motif in the viral protein nsP3 is necessary for binding to the PI3K regulatory subunit p85 and for activating AKT. This leads to an increase in glucose metabolism towards the synthesis of fatty acids, although additional mechanisms of metabolic activation appear to be involved in Ross River virus infection. Importantly, a Ross River virus mutant that fails to activate AKT has an attenuated phenotype in vivo, suggesting that viral activation of PI3K/AKT contributes to virulence and disease.

Intron selection during the formation of prespliceosomes is a critical event in pre-mRNA splicing. Chemical modulation of intron selection has emerged as a route for cancer therapy. Splicing modulators alter the splicing patterns in cells by binding to the U2 snRNP (small nuclear ribonucleoprotein)—a complex chaperoning the selection of branch and 3′ splice sites. Here we report crystal structures of the SF3B module of the U2 snRNP in complex with spliceostatin and sudemycin FR901464 analogs, and the cryo-electron microscopy structure of a cross-exon prespliceosome-like complex arrested with spliceostatin A. The structures reveal how modulators inactivate the branch site in a sequence-dependent manner and stall an E-to-A prespliceosome intermediate by covalent coupling to a nucleophilic zinc finger belonging to the SF3B subunit PHF5A. These findings support a mechanism of intron recognition by the U2 snRNP as a toehold-mediated strand invasion and advance an unanticipated drug targeting concept.

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ABSTRACT:The aim of this study was to report the effect of diabetes mellitus on the pharmacokinetics of verapamil in a route-dependent manner. Diabetes in rats was induced by streptozotocin. Plasma concentrations of verapamil and its metabolite, norverapamil, were measured after oral (10 mg/kg) or intravenous (1 mg/kg) administration. The concentrations of verapamil in portal plasma after oral administration were also determined. Norverapamil formation was used for assessing CYP3A activity in hepatic and intestinal microsomes of diabetic rats. The protein levels of CYP3A1 and CYP3A2 in liver and intestine were measured by Western blot. It was found that diabetes significantly increased the plasma concentration of verapamil and norverapamil after oral administration, which resulted in a 74% increase in the area under the concentration-time curve (AUC) of verapamil, but the ratio of AUC (norverapamil) /AUC (verapamil) was significantly decreased by 38%. In contrast, diabetes significantly decreased the AUC of verapamil by 22% after intravenous administration. Diabetes also resulted in increased AUC of verapamil in portal vein by 3.8-fold compared with that in control rats. The absolute bioavailability of verapamil was higher than that of control rats. An in vitro study showed that increased CYP3A activity in the hepatic microsome and decreased CYP3A activity in the intestinal microsome were accompanied by an increase and decrease in the protein expression of CYP3A1/2 in liver and intestine of diabetic rats, respectively. In conclusion, diabetes mellitus revealed a tissue-specific effect on CYP3A activity and expression (induced in liver and inhibited in intestine), resulting in opposite pharmacokinetic behaviors of verapamil after oral and intravenous administration to diabetic rats.