Recombinant Dengue virus protein NS2B alters membrane permeability in different membrane models

Abstract

BackgroundOne of the main phenomena occurring in cellular membranes during virus infection is a change in membrane permeability. It has been observed that numerous viral proteins can oligomerize and form structures known as viroporins that alter the permeability of membranes. Previous findings have identified such proteins in cells infected with Japanese encephalitis virus (JEV), a member of the same family that Dengue virus (DENV) belongs to (Flaviviridae). In the present work, we investigated whether the small hydrophobic DENV protein NS2B serves a viroporin function.MethodsWe cloned the DENV NS2B sequence and expressed it in a bacterial expression system. Subsequently, we evaluated the effect of DENV NS2B on membranes when NS2B was overexpressed, measured bacterial growth restriction, and evaluated changes of permeability to hygromycin. The NS2B protein was purified by affinity chromatography, and crosslinking assays were performed to determine the presence of oligomers. Hemolysis assays and transmission electron microscopy were performed to identify structures involved in permeability changes.ResultsThe DENV-2 NS2B protein showed similitude with the JEV viroporin. The DENV-2 NS2B protein possessed the ability to change the membrane permeability in bacteria, to restrict bacterial cell growth, and to enable membrane permeability to hygromycin B. The NS2B protein formed trimers that could participate in cell lysis and generate organized structures on eukaryotes membranes.ConclusionsOur data suggest that the DENV-2 NS2B viral protein is capable of oligomerizing and organizing to form pore-like structures in different lipid environments, thereby modifying the permeability of cell membranes.

A New Israeli Tobamovirus Isolate Infects Tomato Plants Harboring Tm-22 Resistance Genes

“…If indeed this is the case, a dire result might occur in which double infection of ToMMV and the currently studied isolate will spread. Recombination events between tobamovirus strains have been reported [49, 50]. In this potential scenario of double tobamovirus infection (the new tobamovirus isolate described in this current study and ToMMV), unknown consequences might occur regarding host range and symptom severity.…”

Structure of the NS2B-NS3 protease from Zika virus after self-cleavage

“…The dimerization may be further driven by the membrane association of the polyprotein in the in vivo membrane-rich environment where the effective local concentration of the replicative enzymes is very high. Indeed, full length transmembrane protein NS2B dimerizes in a cell-based assay19. N-terminal residues V49-D58 from the NS2B N-terminal region form a β-strand within the N-terminal lobe of the NS3 protease, while C-terminal residues S71-V87 of NS2B fold into a β-hairpin that inserts into the catalytic site of the protease, and participate in binding the substrate.…”

Finding a helix in a haystack: nucleic acid cytometry with droplet microfluidics

“…This approach has recently been demonstrated by enriching SV40 genomes from a heterogeneous sample 14 and T4 genomes from a background of ΦX174. 78 …”

“…It is possible to couple the advantages of droplets with the ease of use and availability of fluorescence-activated cell sorting (FACS) instruments using double emulsions. 36,78,101 These water-in-oil-in-water (W/O/W) emulsions can be loaded onto FACS instruments directly. FACS is a mature technology and instruments are often located in core facilities where they are calibrated daily and well maintained.…”