Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1

Abstract

Hepatitis C virus (HCV) infection is closely tied to the lipid metabolism of liver cells. Here, we identify the triglyceride-synthesizing enzyme DGAT1 as an important host factor for HCV infection; DGAT1 interacts with the viral nucleocapsid core and is required for the trafficking of core to lipid droplets. Inhibition of DGAT1 activity or RNAi-mediated knockdown severely impairs infectious virion production, implicating DGAT1 as a new target for antiviral therapy.Over 160 million individuals are infected with HCV, and in ~80% of cases, infection persists and can lead to severe liver disease. No vaccine is available, and current treatments are not effective against the strains most prevalent in the US and Europe. After the virus enters liver cells, a viral polyprotein is translated at the endoplasmic reticulum (ER) from a single positive-stranded RNA genome and processed into structural and nonstructural proteins 1 . Nonstructural proteins autonomously replicate viral RNA, which together with structural proteins, the nucleocapsid core and E1 and E2 envelope proteins, is packaged into progeny virions. Both RNA replication and infectious particle assembly are thought to occur at ER membranes. However, recent reports showed a critical involvement of lipid droplets Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms * To whom correspondence should be addressed: Melanie Ott, MD, PhD, Gladstone Institute of Virology and Immunology, 1650 Owens Street, San Francisco, CA 94158, Tel: (415) Fax: (415) Competing financial interestsThe authors declare no competing financial interests. LDs form when triglycerides and cholesterol esters accumulate between the bilayer of the ER membrane and are surrounded by a phospholipid monolayer and associated proteins 8 . DGAT1 and DGAT2 enzymes catalyze the final step in triglyceride biosynthesis and are critical in LD biogenesis 9 . Both enzymes are ER-resident, and upon uptake of fatty acids, DGAT2, but not DGAT1, localizes to LDs 9 . Both enzymes have similar activities in vitro; however, DGAT1 has broader substrate specificity 9 , and only DGAT2 is essential in vivo in vivo 10,11 . HHS Public AccessTo determine if DGAT enzymes influence the HCV life cycle, shRNAs directed against DGAT1 or DGAT2 were introduced into HCV-permissive hepatoma cells [12][13][14] . Transduced cultures were inoculated with low concentrations of infectious HCV expressing eGFP as a marker for infection (eGFP-Jc1), and viral spread was monitored by flow cytometry (Fig. 1a). Spreading infection was suppressed by 65-90% with two DGAT1 shRNAs (day 9 post infection). Expression of shRNA-resistant DGAT1 rescued the suppressive effect of the DGAT1 knockdown on viral infection ( Supplementary Fig. S1). No change was seen with DGAT2 shRNA pointing to a unique role of DGAT1 in HCV infection. Knockdown of DGAT expre...

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

“…mDGAT1 point mutants and small hairpin RNA vectors targeting DGAT1 and DGAT2 were also generated as described (11). Luc-Jc1 reporter virus was described (14).…”

“…We recently showed that the HCV core protein interacts with DGAT1 and depends on DGAT1 activity for access to LDs. Inhibition of DGAT1 activity or RNAi-mediated knockdown of DGAT1 severely impaired HCV infectious particle production, demonstrating DGAT1 is critical to HCV infection (11,12).…”

Hepatitis c virus and host cell lipids: An intimate connection

“…155 NS5A is also targeted to the surface of LDs and presumably via a core-NS5A interaction the early step of assembly is triggered. 59 Indeed, point mutations interfering with core-NS5A co-localization at LDs strongly impair virus production.…”

Evidence That Diacylglycerol Acyltransferase 1 (DGAT1) Has Dual Membrane Topology in the Endoplasmic Reticulum of HepG2 Cells

“…The structural formula of inhibitor compound A (iA) is 2-((1s,4s)-4-(4-(4-amino-7,7-dimethyl-7H-pyrimido [4,5-b] [1,4]oxazin-6-yl)phenyl)cyclohexyl)acetic acid, and its use as a DGAT1 inhibitor has been described in Ref. 22 …”