Levels of Circulating NS1 Impact West Nile Virus Spread to the Brain

Abstract

Dengue (DENV) and West Nile (WNV) viruses are arthropod-transmitted flaviviruses that respectively cause systemic vascular leakage and encephalitis syndromes in humans. However, the viral factors contributing to these specific clinical disorders are not completely understood. Flavivirus nonstructural protein 1 (NS1) is required for replication, expressed on the cell surface, and secreted as a soluble glycoprotein, reaching high levels in the blood of infected individuals. Extracellular DENV and WNV NS1 interact with host proteins and cells, have immune evasion functions, and promote endothelial dysfunction in a tissue-specific manner. To characterize how differences in DENV and WNV NS1 might function in pathogenesis, we generated WNV NS1 variants with substitutions corresponding to residues found in DENV NS1. We discovered that the substitution NS1-P101K led to reduced WNV infectivity of the brain and attenuated lethality in infected mice, although the virus replicated efficiently in cell culture and peripheral organs and bound at wild-type levels to brain endothelial cells and complement components. The P101K substitution resulted in reduced NS1 antigenemia in mice, and this was associated with reduced WNV spread to the brain. As exogenous administration of NS1 protein rescued WNV brain infectivity in mice, we conclude that circulating WNV NS1 facilitates viral dissemination into the central nervous system and impacts disease outcome. IMPORTANCE Flavivirus NS1 serves as an essential scaffolding molecule during virus replication but also is expressed on the cell surface and secreted as a soluble glycoprotein that circulates in the blood of infected individuals. Although extracellular forms of NS1 are implicated in immune modulation and in promoting endothelial dysfunction at blood-tissue barriers, it has been challenging to study specific effects of NS1 on pathogenesis without disrupting its key role in virus replication. Here we assessed West Nile virus (WNV) NS1 variants that do not affect virus replication and evaluated their effects on pathogenesis in mice. Our characterization of WNV NS1-P101K suggests that the levels of NS1 in circulation facilitate WNV dissemination to the brain and disease outcome. Our findings help understand the role of NS1 during flavivirus infection and support antiviral strategies for targeting circulating forms of NS1.

Molecular Determinants of Tissue Specificity of Flavivirus Nonstructural Protein 1 Interaction with Endothelial Cells

“…It is important to note that while WT WNV NS1 bound to HPMEC less efficiently than DENV NS1, it did bind at detectable levels, although this level of binding was not sufficient to trigger endothelial hyperpermeability (Fig. 3) (14). In the TEER model used to measure endothelial hyperpermeability in vitro, cells are treated with NS1 and left undisturbed over time under static conditions, in contrast to DENV infection in vivo, where NS1 travels through the bloodstream with constant blood flow.…”

“…Like DENV NS1, the NS1 proteins of WNV and ZIKV have also been shown to modulate endothelial barriers. WNV NS1 also has been shown to contribute to WNV infection in the brain (14), while ZIKV NS1 has been shown to modulate the barrier integrity of placental explants as well as the testis (10), contributing to ZIKV infection of Sertoli cells (9,15). NS1 is a ~55-kDa glycoprotein that is highly conserved across the flaviviruses (16).…”

SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling

“…Previous data indicate that NS1-mediated vascular leak can exacerbate a sublethal DENV infection, providing direct evidence that soluble NS1 can promote DENV pathogenesis 29 , 65 . In addition to inflammatory responses directly triggered by DENV NS1 65 , one hypothesis by which NS1 promotes pathogenesis is through facilitating dissemination of blood-borne flaviviruses from the blood into distal tissues where the virus can replicate to high titers 32 , 42 , 55 , 66 . Thus, we speculate that a potential contribution of S-mediated barrier dysfunction to COVID-19 pathogenesis is to promote dissemination of SARS-CoV-2 from the lung to the blood, and then into distal organs where virus-permissive cells reside.…”

Intrinsic antiviral immunity of barrier cells revealed by an iPSC-derived blood-brain barrier cellular model

“…However, the fact that only two amino acid changes in the envelope protein of an alphavirus can completely change the infection outcome suggests that it should be possible for certain isolates of nonneuroinvasive viruses to gain the ability to infect BMECs. Clinically, a high level of viremia in severe dengue cases may also lead to a high level of NS1 in blood, which can compromise endothelial integrity (Beatty et al, 2015;Biering et al, 2021;Pan et al, 2021;Puerta-Guardo et al, 2019;Wessel et al, 2021). Of the five human IFITM family members, IFITM1, IFITM2, and IFITM3 have broad antiviral activities both in vitro and in vivo (Liao et al, 2019;Perreira et al, 2013;Zhao et al, 2018).…”