Structure of a Murine Norovirus NS6 Protease-Product Complex Revealed by Adventitious Crystallisation

Murine noroviruses have emerged as a valuable tool for investigating the molecular basis of infection and pathogenesis of the closely related human noroviruses, which are the major cause of non-bacterial gastroenteritis. The replication of noroviruses relies on the proteolytic processing of a large polyprotein precursor into six non-structural proteins (NS1–2, NS3, NS4, NS5, NS6pro, NS7pol) by the virally-encoded NS6 protease. We report here the crystal structure of MNV NS6pro, which has been determined to a resolution of 1.6 Å. Adventitiously, the crystal contacts are mediated in part by the binding of the C-terminus of NS6pro within the peptide-binding cleft of a neighbouring molecule. This insertion occurs for both molecules in the asymmetric unit of the crystal in a manner that is consistent with physiologically-relevant binding, thereby providing two independent views of a protease-peptide complex. Since the NS6pro C-terminus is formed in vivo by NS6pro processing, these crystal contacts replicate the protease-product complex that is formed immediately following cleavage of the peptide bond at the NS6-NS7 junction. The observed mode of binding of the C-terminal product peptide yields new insights into the structural basis of NS6pro specificity

Effect of intravenous calcitonin gene-related peptide antagonist on the laryngeal chemoreflex in piglets

The laryngeal chemoreflex (LCR) is characterized by mixed apnea and cardiovascular instability and is elicited by applying water to the laryngeal mucosa of developing animals. The LCR may be fatal in very young animals, and the reflex has been postulated as a possible mechanism of sudden infant death syndrome. Several antagonists have been found to alter the severity of the LCR, but the primary neurotransmitters involved in mediating the reflex response are not yet well understood. This study investigates the effect, on the LCR, of the pharmacologic antagonism of calcitonin gene-related peptide (αCGRP), a neurochemical found in abundance in the mammalian laryngeal mucosa and its innervating system. The LCR was elicited in 10 mixed breed piglets, 17.7 days of age (12 to 22 days), before and during infusion of αCGRP 8-37, a pharmacologic inhibitor of αCGRP, and cardiorespiratory and laryngeal responses were compared. The duration of obstructive apnea decreased from 17.9 to 9.8 seconds (P \u3c 0.03) in the presence of αCGRP 8- 37. Mean central apnea did not change for the group (P \u3e 0.05), although it was completely inhibited in 2 animals. Cardiovascular changes were not significantly altered by the αCGRP inhibitor. αCGRP appears to play a regulatory role in the apneic response of the LCR, particularly its obstructive component, but not the cardiovascular response