Hemagglutinin esterases (HEs), closely related envelope glycoproteins in influenza C and corona-and toroviruses, mediate reversible attachment to O-acetylated sialic acids (Sias). They do so by acting both as lectins and as receptor-destroying enzymes, functions exerted by separate protein domains. HE divergence was accompanied by changes in quaternary structure and in receptor and substrate specificity. The selective forces underlying HE diversity and the molecular basis for Sia specificity are poorly understood. Here we present crystal structures of porcine and bovine torovirus HEs in complex with receptor analogs. Torovirus HEs form homodimers with sialate-Oacetylesterase domains almost identical to corresponding domains in orthomyxo-and coronavirus HEs, but with unique lectin sites. Structure-guided biochemical analysis of the esterase domains revealed that a functionally, but not structurally conserved arginine-Sia carboxylate interaction is critical for the binding and positioning of glycosidically bound Sias in the catalytic pocket. Although essential for efficient de-O-acetylation of Sias, this interaction is not required for catalysis nor does it affect substrate specificity. In fact, the distinct preference of the porcine torovirus enzyme for 9-mono-over 7,9-di-O-acetylated Sias can be explained from a single-residue difference with HEs of more promiscuous specificity. Apparently, esterase and lectin pockets coevolved; also the porcine torovirus HE receptorbinding site seems to have been designed to use 9-mono-and exclude di-O-acetylated Sias, possibly as an adaptation to replication in swine. Our findings shed light on HE evolution and provide fundamental insight into mechanisms of substrate binding, substrate recognition, and receptor selection in this important class of virion proteins.glycobiology ͉ influenza ͉ nidovirus ͉ sialate-O-acetylesterase ͉ X-ray crystallography
ABSTRACT:A split plot experiment with 72 male pigs weighing 52.9 ± 0.39 kg (mean ± SEM) was conducted to examine AA partitioning and body protein deposition (PD) in response to increasing dietary sulfur amino acids (SAA) with or without immune system (IS) activation. The main plot was with and without IS activation, and 4 diets containing different amounts of standardized ileal digestible (SID) SAA (SAA to Lys ratios of 0.45, 0.55, 0.65 and 0.75) were the subplots. Activation of IS was achieved by intramuscular injection of Escherichia coli lipopolysaccharides (LPS; serotype 055:B5, Sigma; 30 μg/kg BW) every Monday and Thursday, with control pigs injected with sterile saline. Maximum body PD, measured by dual-energy X-ray absorptiometry (DXA), and minimum plasma urea content were achieved at SID SAA:Lys ratio of 0.55 in saline-injected pigs but were achieved at a SID SAA:Lys ratio of 0.75 in IS-activated pigs. Immune system activation increased rectal temperature (P < 0.05), plasma haptoglobin (1.1 vs. 2.0 mg/mL; P < 0.001), and the proportion of neutrophils (0.39 vs. 0.42; P < 0.05) and decreased serum albumin content (38.4 vs. 36.8 g/L; P < 0.01). Increasing dietary SAA had no effects on these variables. Immune systemactivated pigs had lower levels of homocysteine (Hcy; P < 0.001) and a lower Ser content (P < 0.05). Results showed that increasing dietary SAA as DL-methionine in growing and/or fi nishing pigs altered plasma AA contents, and that use effi ciency of the AA was improved when greater levels of SAA were supplemented in ISactivated pigs.
Nucleoside analogues have long been recognized as prospects for the discovery of direct acting antivirals (DAAs) to treat hepatitis C virus because they have generally exhibited crossgenotype activity and a high barrier to resistance. C-Nucleosides have the potential for improved metabolism and pharmacokinetic properties over their N-nucleoside counterparts due to the presence of a strong carbon−carbon glycosidic bond and a non-natural heterocyclic base. Three 2′CMe-C-adenosine analogues and two 2′CMe-guanosine analogues were synthesized and evaluated for their anti-HCV efficacy. The nucleotide triphosphates of four of these analogues were found to inhibit the NS5B polymerase, and adenosine analogue 1 was discovered to have excellent pharmacokinetic properties demonstrating the potential of this drug class.
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