Dengue virus nonstructural 3 protein interacts directly with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and reduces its glycolytic activity

Emiliana M Silva,Jonas N Conde,Kíssila Rabelo,Ronaldo Mohana-Borges,Manuela L Silva,Marciano V Paes,Paulo Mascarello Bisch,Gustavo T Ventura,Diego Allonso,Pedro Henrique Carneiro,Diego R Coelho,Gilberto Weissmuller

doi:10.1038/s41598-019-39157-7

Abstract

Dengue is an important mosquito-borne disease and a global public health problem. The disease is caused by dengue virus (DENV), which is a member of the Flaviviridae family and contains a positive single-stranded RNA genome that encodes a single precursor polyprotein that is further cleaved into structural and non-structural proteins. Among these proteins, the non-structural 3 (NS3) protein is very important because it forms a non-covalent complex with the NS2B cofactor, thereby forming the functional viral protease. NS3 also contains a C-terminal ATPase/helicase domain that is essential for RNA replication. Here, we identified 47 NS3-interacting partners using the yeast two-hybrid system. Among those partners, we highlight several proteins involved in host energy metabolism, such as apolipoprotein H, aldolase B, cytochrome C oxidase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH directly binds full-length NS3 and its isolated helicase and protease domains. Moreover, we observed an intense colocalization between the GAPDH and NS3 proteins in DENV2-infected Huh7.5.1 cells, in NS3-transfected BHK-21 cells and in hepatic tissue from a fatal dengue case. Taken together, these results suggest that the human GAPDH-DENV NS3 interaction is involved in hepatic metabolic alterations, which may contribute to the appearance of steatosis in dengue-infected patients. The interaction between GAPDH and full-length NS3 or its helicase domain in vitro as well as in NS3-transfected cells resulted in decreased GAPDH glycolytic activity. Reduced GAPDH glycolytic activity may lead to the accumulation of metabolic intermediates, shifting metabolism to alternative, non-glycolytic pathways. This report is the first to identify the interaction of the DENV2 NS3 protein with the GAPDH protein and to demonstrate that this interaction may play an important role in the molecular mechanism that triggers hepatic alterations.

Highlights

Dengue virus (DENV) belongs to the Flaviviridae family, which includes 70 other viruses, such as yellow fever virus (YFV), Zika virus, Japanese encephalitis virus (JEV) and West Nile virus[1]
Forty-seven putative NS3-interacting partners were selected by HIS3, ADE2 and lacZ reporter gene activation, as visualized by colony growth on triple (SD-His-Leu-Trp) and quadruple (SD-Ade-His-Leu-Trp) drop-out plates and by blue staining following colony-lift filter assays to detect β-galactosidase expression (Fig. 1)
We identified 47 different cellular proteins from a liver library that potentially interact with DENV2 NS3 in vivo

Summary

Introduction

Dengue virus (DENV) belongs to the Flaviviridae family, which includes 70 other viruses, such as yellow fever virus (YFV), Zika virus, Japanese encephalitis virus (JEV) and West Nile virus[1]. The chimeric yellow fever-DENV tetravalent dengue vaccine (CYD-TDV) is a live-attenuated vaccine that expresses the structural antigens of the four DENV serotypes, the membrane protein (prM) and envelope protein (E), which act as targets for the host immune response[8,9]. Several factors, such as age, host physiology and repeated exposure to DENV, have been observed to affect vaccine efficacy[9]. The NS3 N-terminal region contains a protease catalytic domain that forms a non-covalent complex with the NS2B cofactor for its optimum proteolytic activity. Because of its ability to cleave different parts of the polyprotein precursor and its participation in viral replication, NS3 is considered an important target for screening drug candidates and evaluating their efficacy

Methods

Results

Conclusion