Functional analysis of dengue virus nonstructural protein 1

Abstract

Dengue virus NS1 is a glycoprotein that plays different roles in different stages of virus life cycle. It exists in multiple forms: intracellular membraneassociated form which is co-localized with dsRNA and essential in viral replication; cell surface-associated form which is associated with lipid raft at the plasma membrane and mediated signaling and complement activation pathways; secreted form which eventually released into the blood stream. In the first part of this study, we investigate the interaction between intracellular form NS1 and dsRNA by EMSA. Our results show that intracellular form NS1 binds dsRNA in vitro and has multiple dsRBDs. Results of the first part implicate a novel role of intracellular form NS1 in viral replication. In the second part of this study, we investigate the binding partner of secreted form NS1. We use affinity pull-down assay to isolate NS1 complex from the supernatant of transfected AD293 cells, and analyze the components by mass spectrometry. We find that ApoA-I, the major structural protein of HDL in serum, is co-immunoprecipitated with NS1. In addition, NS1 is also associated with ApoA-I that is produced and secreted from human HepG2 cells. Delipidation of ApoA-I disrupts its interaction with NS1. Formation of NS1-ApoA-I complex modulates VCAM-1 expression in HMEC-1 cells. Results of the second part indicate a novel role of secreted form NS1 in modulating endothelial cell functions, which may contribute to dengue pathogenesis. In the third part of this study, we investigate the factor that presents in human serum leading to an enhanced DV infectivity in various types of cells. Through co-immunoprecipitation, we reveal that ApoA-I is associated with DV particles and is able to promote DV infection. We further find that siRNA knockdown of SR-BI, the cell receptor of ApoA-I, abolishes the activity of ApoA-I in enhancement of DV infection. FACS analysis of cell surface dengue antigen after virus absorption further confirms that ApoA-I enhanced DV infection via promoting initial attachment of virus to cells. Results of the third part illustrate a novel entry route of DV into cells, which may provide insights into the functional importance of lipoproteins in dengue pathogenesis.