Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core

Abstract

Background:The NS5A protein regulates HCV assembly by localizing to lipid droplets. Results: The triglyceride-synthesizing enzyme DGAT1 binds NS5A, enhances interactions of NS5A with the viral capsid core, and enables lipid droplet localization of NS5A. Conclusion: DGAT1 functions as a cellular "hub" for HCV proteins to access lipid droplets. Significance: DGAT1 inhibitors suppress HCV assembly by preventing NS5A access to DGAT1-generated lipid droplets.

The Hepatitis C Virus Core Protein Inhibits Adipose Triglyceride Lipase (ATGL)-mediated Lipid Mobilization and Enhances the ATGL Interaction with Comparative Gene Identification 58 (CGI-58) and Lipid Droplets

“…Lipolysis of Radiolabeled Lipid Droplets-To generate intracellular radiolabeled TG stores, Cos-7 cells either transfected with core or a control plasmid were incubated with 0.6 Ci/ml [ 3 H]oleic acid (American Radiolabeled Chemicals) and 560 M oleic acid complexed to BSA (Sigma) for 22 h. Thereafter, LDs were isolated as described (20). An aliquot of the LD fraction (200,000 cpm) was incubated with Cos-7 lysates (20 g of protein) overexpressing ATGL, CGI-58, or ␤-gal as the control in the presence of 4% BSA (essentially FFA free) in a total volume of 200 l for 1 h at 37°C.…”

“…The HA-core construct is described in Camus et al (20). N-terminal His-tagged ATGL, CGI-58, G0S2, and HSL were generated by inserting the human or mouse cDNA of these genes in the pcDNA4/HisMax C vector (Invitrogen) (21).…”

Vinexin β Interacts with Hepatitis C Virus NS5A, Modulating Its Hyperphosphorylation To Regulate Viral Propagation

“…Previous studies have demonstrated that NS5A hyperphosphorylation induced by CK1␣ regulates the interaction of NS5A with lipid droplets (LDs) and core protein. Moreover, it has been confirmed that the recruitment of hyperphosphorylated NS5A to LDs and the interaction of NS5A with core protein are essential for HCV assembly (10,11,13,14,59). To further evaluate the contribution of vinexin ␤ to the hyperphosphorylation of NS5A catalyzed by CK1␣, we performed co-IP assays to determine the effect of vinexin ␤ on the CK1␣-NS5A association.…”

“…They can be separated by SDS-PAGE on the basis of their sizes; the molecular mass of the former is 56 kDa, and that of the latter is 58 kDa (9). Previous studies have shown that phosphorylation of NS5A is crucial to parts of the HCV life cycle, such as viral genome replication complex formation and infectious particle production (10)(11)(12). Although the details of the mechanism regulating NS5A phosphorylation are still not clear, various host factors (HFs) involved in NS5A phosphorylation, such as casein kinase 1␣ (CK1␣) and CK2␣, have been identified (6,13,14).…”

Cell death-inducing DFFA-like effector B contributes to assembly of hepatitis C virus (HCV) particles and interacts with HCV NS5A

“…HCV assembly begins on the ER-lipid droplet (LD) surface, where the viral core protein, surface glycoproteins (E1 and E2), and viral RNA are assembled and packaged in a temporally and spatially regulated manner [2][3][4] . Nascent HCV particles then form by budding into the ER and fuse with pre-very-low-density lipoprotein (VLDL) particles to form lipo-viro particles (LVPs) 2,5 , or nascent HCV particles then form by budding of viral capsids into the ER lumen, incorporate cholesterol and triglycerides (TGs), and further bind ApoB and exchangeable apolipoproteins in a manner similar to lipoproteins to form LVPs 6 .…”