Dengue virus NS1 protein does not activate TLR4 and has a modest effect on endothelial monolayer integrity compared to TNF

Severe dengue virus (DENV) infection is characterised by vascular leak and can lead to shock and death. The virus-derived non-structural protein 1 (NS1), which is released into the circulation as a lipoparticle, has been implicated in exacerbation of inflammation through activation of Toll-like receptor 4 (TLR4), and in disruption of endothelial monolayer integrity. We sought to identify the component of the NS1 lipoparticle that activates TLR4. Native and C-terminally His-tagged recombinant NS1 were produced in suspension HEK293 cells and formed hexamers indicating structural integrity. However, neither the recombinant NS1 nor NS1 secreted by DENV-infected Vero or K562 cells activated TLR4. Thus, the NS1 apoprotein itself does not activate TLR4, although it could potentially associate with inflammatory lipids as a lipoparticle. Notably, the lack of TLR4 stimulation by NS1 protein does not exclude a role for TLR4 in dengue disease as it may be activated by LPS influx from the gut or by putative endogenous stimulatory lipids. Furthermore, purified His-tagged NS1, at a biologically relevant concentration, only had a modest effect on endothelial monolayer integrity as assessed by changes in the electrical impedance of an endothelial monolayer. In contrast TNF, a cytokine with an established role in vascular leak, had a potent effect when used at a concentration seen in severe dengue disease. Medium from DENV-infected human macrophages also had a potent disruptive effect. This medium had low NS1, compared to that encountered in some patients, but high TNF concentration and its effect on endothelial monolayers was mostly ablated by an anti-TNF antibody. While multiple factors are likely to contribute to DENV-associated vascular leak in vivo, our data support a key role for TNF. Importantly, our data indicate that while NS1 has a modest effect on endothelial monolayer integrity there is no evidence for it activating TLR4.