Dengue virus NS1 leads to downregulation of HNF4 alpha in liver cells resulting in a decrease in coagulation factors I, V, X, and XIII, contributing to coagulopathy.

Abstract

Dengue virus NS1 protein is a major pathogenic protein. In this study, we examined the role of NS1 in coagulopathy associated with Dengue infection, a common feature of Dengue virus pathogenesis. Since most coagulation factors are produced by hepatocytes and liver is key organ affected during infection, we conducted transcriptomics using total-RNA extracted from Huh7 cells overexpressing NS1 protein. Coagulation factors 1, 5, 10, and 13 were downregulated and was confirmed using quantitative real-time polymerase chain reaction (RT-PCR) and western blot assays in both adherent and non-adherent cell culture systems across all four serotypes of Dengue. We also determined that downregulation of coagulation factors is a result of reduced expression of transcription activator HNF4α. Furthermore, we demonstrated that phosphorylation of extracellular signal-regulated kinase (ERK) leads to HNF4α downregulation and subsequent downregulation of coagulation factors. The downregulation of HNF4α and the downregulation of subsequent coagulation factors were validated in BALB/c mice by hydrodynamic tail vein injection of NS1 expression plasmids. Western blot assays using plasma from Dengue patients indicated that at least two coagulation factors of the common pathway of coagulation cascade are downregulated during the febrile phase, with levels improving toward the convalescent phase. NS1-mediated downregulation of coagulation factors was observed for both intracellular and secreted NS1. The hypothesis was also validated using virus infection assays. Overall, our study highlights the role of NS1 in mediating coagulopathy by modulating the expression of coagulation factors through transcriptional suppression of HNF4α by elevated phosphorylated ERK. This signaling cascade could be targeted for therapeutic intervention against virus-related coagulopathies.

Importance: Thrombocytopenia has been linked to coagulopathy of Dengue infection, and Dengue patients with coagulopathies are often administered platelet transfusion. For coagulopathies without thrombocytopenia, platelet transfusion might not help. We demonstrated the role of NS1 in coagulopathy by downregulating coagulation factors themselves. When thrombocytopenia does not exist or when thrombocytopenia as well as reduced levels of coagulation factors are the causative factors for coagulopathies, only platelet transfusion might not be effective. Alternative strategies, like administration of coagulation factor cocktails or platelet transfusion along with coagulation factor cocktail, might be promising. Our work also leads to a signaling pathway of NS1-mediated downregulation of coagulation factors via phosphorylated ERK and HNF4α. HNF4α is a transcription regulator for many other liver-based metabolic factors and pathways like lipid metabolism, carbohydrate metabolism, etc, and thus, therapeutic targeting of NS1-based downregulation of HNF4α can lead to designing therapeutic candidates for managing other Dengue-based liver dysfunction.