Dengue Virus NS4b N-Terminus Disordered Region Interacts with NS3 Helicase C-Terminal Subdomain to Enhance Helicase Activity

Dengue virus replicates its single-stranded RNA genome in membrane-bound complexes formed on the endoplasmic reticulum, where viral non-structural proteins (NS) and RNA co-localize. The NS proteins interact with one another and with the host proteins. The interaction of the viral helicase and protease, NS3, with the RNA-dependent RNA polymerase, NS5, and NS4b proteins is critical for replication. In vitro, NS3 helicase activity is enhanced by interaction with NS4b. We characterized the interaction between NS3 and NS4b and explained a possible mechanism for helicase activity modulation by NS4b. Our bacterial two-hybrid assay results showed that the N-terminal 57 residues region of NS4b is enough to interact with NS3. The molecular docking of the predicted NS4b structure onto the NS3 structure revealed that the N-terminal disordered region of NS4b wraps around the C-terminal subdomain (CTD) of the helicase. Further, NS3 helicase activity is enhanced upon interaction with NS4b. Molecular dynamics simulations on the NS4b-docked NS3 crystal structure and intrinsic tryptophan fluorescence studies suggest that the interaction results in NS3 CTD domain motions. Based on the interpretation of our results in light of the mechanism explained for NS3 helicase, NS4b–NS3 interaction modulating CTD dynamics is a plausible explanation for the helicase activity enhancement

Syncytial Phenotype of C-Terminally Truncated Herpes Simplex Virus Type 1 gB Is Associated with Diminished Membrane Interactions ▿

The cytoplasmic domain of glycoprotein B (gB) from herpes simplex virus type 1 (HSV-1) is an important regulator of membrane fusion. C-terminal truncations of the cytoplasmic domain lead to either hyperfusion or fusion-null phenotypes. Currently, neither the structure of the cytoplasmic domain nor its mechanism of fusion regulation is known. Here we show, for the first time, that the full-length cytoplasmic domain of HSV-1 gB associates stably with lipid membranes, preferentially binding to membranes containing anionic head groups. This interaction involves a large increase in helical content. However, the truncated cytoplasmic domains associated with the hyperfusion phenotype show a small increase in helical structure and a diminished association with lipid membranes, whereas the one associated with the fusion-null phenotype shows no increase in helical structure and only a minimal association with lipid membranes. We hypothesize that stable binding to lipid membranes is an important part of the mechanism by which the cytoplasmic domain negatively regulates membrane fusion. Moreover, our experiments with truncated cytoplasmic domains point to two specific regions that are critical for membrane interactions. Taken together, our work provides several important new insights into the architecture of the cytoplasmic domain of HSV-1 gB and its interaction with lipid membranes