Dengue Virus Capsid Protein Binding to Hepatic Lipid Droplets (LD) Is Potassium Ion Dependent and Is Mediated by LD Surface Proteins

Abstract

Dengue virus (DENV) affects millions of people, causing more than 20,000 deaths annually. No effective treatment for the disease caused by DENV infection is currently available, partially due to the lack of knowledge on the basic aspects of the viral life cycle, including the molecular basis of the interaction between viral components and cellular compartments. Here, we characterized the properties of the interaction between the DENV capsid (C) protein and hepatic lipid droplets (LDs), which was recently shown to be essential for the virus replication cycle. Zeta potential analysis revealed a negative surface charge of LDs, with an average surface charge of ؊19 mV. The titration of LDs with C protein led to an increase of the surface charge, which reached a plateau at ؉13.7 mV, suggesting that the viral protein-LD interaction exposes the protein cationic surface to the aqueous environment. Atomic force microscopy (AFM)-based force spectroscopy measurements were performed by using C proteinfunctionalized AFM tips. The C protein-LD interaction was found to be strong, with a single (un)binding force of 33.6 pN. This binding was dependent on high intracellular concentrations of potassium ions but not sodium. The inhibition of Na ؉ /K ؉ -ATPase in DENV-infected cells resulted in the dissociation of C protein from LDs and a 50-fold inhibition of infectious virus production but not of RNA replication, indicating a biological relevance for the potassium-dependent interaction. Limited proteolysis of the LD surface impaired the C protein-LD interaction, and force measurements in the presence of specific antibodies indicated that perilipin 3 (TIP47) is the major DENV C protein ligand on the surface of LDs. Dengue virus (DENV) causes the most important arthropodborne human viral disease, with 2.5 billion people at risk, 100 million infections, and more than 20,000 deaths annually, primarily in tropical developing countries (20). Four genetically distinct serotypes (DENV1 to DENV4) have been identified, which are transmitted among humans through the bite of an infected mosquito of the genus Aedes. DENV belongs to the family Flaviviridae, together with other important human pathogens, such as yellow fever virus (YFV), West Nile virus (WNV), and hepatitis C virus (HCV). The clinical manifestations of DENV infection range from a mild illness to a severe and potentially life-threatening disease for which no treatment is available so far, due at least in part to the limited understanding of the molecular mechanisms that underlie the interaction between DENV and its host cells.The DENV genome is a single-stranded positive-sense RNA molecule of approximately 11 kb that is translated from a single open reading frame, generating a polyprotein associated with the endoplasmic reticulum (ER) membrane (34). The polyprotein is cleaved co-and posttranslationally by cellular and viral proteases into three structural proteins (capsid [C], premembrane [prM], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B,...

Nucleolin Interacts with the Dengue Virus Capsid Protein and Plays a Role in Formation of Infectious Virus Particles

“…The error bars indicate SD. (13,18,19). The presence of DENV C in the nucleoli of infected cells has also been observed in several prior studies (22-24, 27, 84).…”

“…DENV C has been shown to associate with lipid droplets in the cytoplasm of infected cells (18,19). The localization of DENV C to lipid droplets has been linked to the formation of virus particles infected with DENV (MOI, 3), followed by treatment with AS1411 or the negative control.…”

“…Within the cytoplasm, the DENV C protein not only is localized to sites of virus assembly, but is found in association with lipid droplets, where it may play an important role in the formation of virus particles (18)(19)(20)(21). The DENV C protein is also localized in the nuclei of infected cells, where it is primarily associated with the nucleolus (22)(23)(24)(25).…”

Monotopic topology is required for lipid droplet targeting of ancient ubiquitous protein 1

“…This suggests that the positively charged residues specifi cally support the LD association of AUP1, e.g., by electrostatic interactions. Due to its proximity to the membrane, the arginine motif may bind to negatively charged phospholipids or other components that contribute to the negative potential of the LD surface ( 53 ). In this case, posttranslational modifi cations infl uencing the protein charge, such as phosphorylation and acetylation, could regulate AUP1 targeting to LDs.…”

Rab Proteins and the Compartmentalization of the Endosomal System

“…Because TIP47 is also involved in lipid droplet biogenesis under conditions of rapid fat storage, it remains to be determined if TIP47 might be a shared effector that also interacts with one of the myriad of Rab GTPases involved in lipid droplet formation or, alternatively, promotes interorganellar tethering to lipid droplets to allow access to critical lipid regulators of endocytosis Bulankina et al 2009;Hynson et al 2012). Indeed, Tip47 and Rab9 are important targets in viral infectivity and have been associated with lipid droplet consumption, suggesting that resolving this open question will be of significant interest (Murray et al 2005;Chen et al 2009b;Carvalho et al 2012;Vogt et al 2013). Based on the cumulative evidence, it is speculated that Rab-mediated cargo selection and vesicle budding will involve specific lipid recruitment through cis or trans membrane interactions, signal-dependent cargo binding, cooperativity between Arf and Rab GTPases, and GTPase regulatory factors for spatiotemporal control of protein -protein interactions.…”