HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway.

The HIV-1 dimerization initiation sequence (DIS) is a conserved palindrome in the apical loop of a conserved hairpin motif in the 5′-untranslated region of its RNA genome. DIS hairpin plays an important role in genome dimerization by forming a ‘kissing complex’ between two complementary hairpins. Understanding the kinetics of this interaction is key to exploiting DIS as a possible human immunodeficiency virus (HIV) drug target. Here, we present a single-molecule Förster resonance energy transfer (smFRET) study of the dimerization reaction kinetics. Our data show the real-time formation and dissociation dynamics of individual kissing complexes, as well as the formation of the mature extended duplex complex that is ultimately required for virion packaging. Interestingly, the single-molecule trajectories reveal the presence of a previously unobserved bent intermediate required for extended duplex formation. The universally conserved A272 is essential for the formation of this intermediate, which is stabilized by Mg(2+), but not by K(+) cations. We propose a 3D model of a possible bent intermediate and a minimal dimerization pathway consisting of three steps with two obligatory intermediates (kissing complex and bent intermediate) and driven by Mg(2+) ions

Storage of Factor VIII Variants with Impaired von Willebrand Factor Binding in Weibel-Palade Bodies in Endothelial Cells

BACKGROUND: Point mutations resulting in reduced factor VIII (FVIII) binding to von Willebrand factor (VWF) are an important cause of mild/moderate hemophilia A. Treatment includes desmopressin infusion, which concomitantly increases VWF and FVIII plasma levels, apparently from storage pools containing both proteins. The source of these VWF/FVIII co-storage pools and the mechanism of granule biogenesis are not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: We studied intracellular trafficking of FVIII variants implicated in mild/moderate hemophilia A together with VWF in HEK293 cells and primary endothelial cells. The role of VWF binding was addressed using FVIII variants displaying reduced VWF interaction. Binding studies using purified FVIII proteins revealed moderate (Arg2150His, Del2201, Pro2300Ser) to severe (Tyr1680Phe, Ser2119Tyr) VWF binding defects. Expression studies in HEK293 cells and primary endothelial cells revealed that all FVIII variants were present within VWF-containing organelles. Quantitative studies showed that the relative amount of FVIII storage was independent of various mutations. Substantial amounts of FVIII variants are co-stored in VWF-containing storage organelles, presumably by virtue of their ability to interact with VWF at low pH. CONCLUSIONS: Our data suggest that the potential of FVIII co-storage with VWF is not affected in mild/moderate hemophilia A caused by reduced FVIII/VWF interaction in the circulation. These data support the hypothesis that Weibel-Palade bodies comprise the desmopressin-releasable FVIII storage pool in vivo

Convalescent plasma therapy with high titers of neutralizing antibodies to reduce mortality in patients with COVID-19 associated ARDS: the Belgian randomized CONFIDENT trial.

peer reviewe

Biogeography and taxonomy of Apodemus sylvaticus (the woodmouse) in the Tyrrhenian region: Enzymatic variations and mitochondrial DNA restriction pattern analysis

In the western Mediterranean area, the taxonomic status of the various forms of Apodemus sylvaticus is quite unclear. Moreover, though anthropogenic, the origins of the island populations remain unknown in geographical terms. In order to examine the level of genetic relatedness of insular and continental woodmice, 258 animals were caught in 24 localities distributed in Belgium, France, mainland Italy, Sardinia, Corsica and Elba. Electrophoresis of 33 allozymes and mtDNA restriction fragments were performed and a UPGMA dendrogram built from the indices of genetic divergence. The dendrogram based on restriction patterns shows two main groups: 'Tyrrhenian', comprising all the Italian and Corsican animals and 'North-western', corresponding to all the other mice trapped from the Pyrenees to Belgium. Since all the Tyrrhenian mice are similar and well isolated from their relatives living on the western edge of the Alpine chain, they must share a common origin. The insular populations are consequently derived from peninsular Italian ones. From a taxonomic point of view and taking the priority rules into account, we have to invalidate A. s. clanceyi Harrison, 1948 and to consider the Tyrrhenian woodmice as belonging to A. s. milleri de Beaux, 1926, whereas the North-western ones must be referred to as the nominal subspecies. As far as the Elban woodmouse is concerned, at the moment we prefer to keep its present subspecific status because we only studied one animal