Rab18 Facilitates Dengue Virus Infection by Targeting Fatty Acid Synthase to Sites of Viral Replication

Abstract

Positive-sense RNA viruses, such as dengue virus (DENV), hijack the intracellular membrane machinery for their own replication. The Rab18 protein, a member of the Rab GTPase family, key regulators of membrane trafficking, is located on the organelles involved in DENV infection, such as the endoplasmic reticulum (ER) and lipid droplets (LDs). In this study, we addressed the potential involvement of Rab18 in DENV infection by using cells overexpressing the wild-type, GTP-bound active form, or GDP-bound inactive form of Rab18 and cells with Rab18 knockdown. DENV replication, measured by viral protein, viral RNA, and viral progeny production, as well as LD induction, was reduced in cells with inactive Rab18 and in cells deprived of Rab18 expression, suggesting a positive role of Rab18 in the DENV life cycle. Interestingly, the interaction of fatty acid synthase (FASN), a key lipogenic enzyme in lipid biosynthesis, with DENV NS3 protein relied on the conversion of the GDP-bound to the GTP-bound form of Rab18. Furthermore, the targeting of FASN to sites participating in DENV infection, such as the ER and LDs, depends on functional Rab18. Thus, Rab18-mediated membrane trafficking of FASN and NS3 facilitates DENV replication, probably by ensuring a sufficient and coordinated lipid supply for membrane proliferation and arrangement. IMPORTANCE Infection by dengue virus (DENV), an important mosquito-borne virus threatening ϳ40% of the world's population, can cause mild dengue fever or severe dengue hemorrhagic fever and dengue shock syndrome. The pathogenesis mechanisms of DENVrelated diseases are not clear, but high viral replication is believed to be a risk factor for the severe form of DENV infection. Thus, understanding the detailed mechanism of DENV replication might help address this devastating virus. Here, we found that Rab18, a small GTPase involved in vesicle trafficking and located in the endoplasmic reticulum network and on the surfaces of lipid droplets, positively regulates DENV replication. The functional machinery of Rab18 is required to recruit the enzyme fatty acid synthase to sites of DENV replication and to interact with DENV NS3 protein to promote fatty acid biosynthesis. Thus, DENV usurps Rab18 to facilitate its own replication. Dengue virus (DENV), a member of the genus Flavivirus of the family Flaviviridae, is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome (1). Other members of the family, such as hepatitis C virus (HCV), yellow fever virus, West Nile virus, and Japanese encephalitis virus, are also involved in human diseases. DENV is enveloped and contains a single-stranded positive-sense RNA genome. Like other positive-sense RNA viruses, DENV modifies host cytoplasmic membranes to form platforms for viral replication (2, 3). DENV replication occurs in close association with virus-induced membrane structures derived from the endoplasmic reticulum (ER) network, with spatial coupling between sites of viral replication and virion assembly (4). The memb...

Non-Canonical Roles of Dengue Virus Non-Structural Proteins

“…In addition, interaction between recombinant NS3 and FASN was shown to stimulate FASN activity in vitro, suggesting that this would be a means of increasing de novo FA biosynthesis during DENV infection. The role of NS3 in redistributing FASN to sites of viral replication was later found to be dependent on NS3 interaction with the GTPase Rab18 [88], a protein localized on lipid droplets (LD) which mediate the apposition of these organelles and the ER-derived membranes, enhancing the formation of LD-associated membranes (LAM) [89]. On the other hand, in agreement with the increased requirement of energy supplies during virus replication, DENV infection was shown to increase fatty acid (FA) β-oxidation [84].…”

Coupling of replication and assembly in flaviviruses

“…Reabsorption of LDs, the natural reservoirs and processing centers of lipids in the cell by the ER membrane was observed in DENV-infected cells [51]. Redistribution of fatty acid synthase (FASN), in a Rab18-dependent manner, by DENV NS3 at the sites of replication appears to be a mechanism utilized by the virus to maintain the supply of fatty acids [52,53]. FASN is a multienzyme protein that catalyzes the synthesis of palmitate from acetyl-CoA and malonyl-CoA in the presence of NADH into long chain saturated fatty acids.…”

Endoplasmic Reticulum Protein SCAP Inhibits Dengue Virus NS2B3 Protease by Suppressing Its K27-Linked Polyubiquitylation

“…DENV infection is known to induce host ER stress responses which help to complete its life cycle (30). NS4A of DENV can induce the rearrangement of the ER membrane, leading to the formation of vesicle pockets and convoluted membranes that assist virion budding (31). DENV also adapts the autophagy processes to enhance its replication.…”