Dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis

Abstract

Dengue virus (DENV) modifies cellular membranes to establish its sites of replication. Although the 3D architecture of these structures has recently been described, little is known about the cellular pathways required for their formation and expansion. In this report, we examine the host requirements for DENV replication using a focused RNAi analysis combined with validation studies using pharmacological inhibitors. This approach identified three cellular pathways required for DENV replication: autophagy, actin polymerization, and fatty acid biosynthesis. Further characterization of the viral modulation of fatty acid biosynthesis revealed that a key enzyme in this pathway, fatty acid synthase (FASN), is relocalized to sites of DENV replication. DENV nonstructural protein 3 (NS3) is responsible for FASN recruitment, inasmuch as (i) NS3 expressed in the absence of other viral proteins colocalizes with FASN and (ii) NS3 interacts with FASN in a two-hybrid assay. There is an associated increase in the rate of fatty acid biosynthesis in DENV-infected cells, and de novo synthesized lipids preferentially cofractionate with DENV RNA. Finally, purified recombinant NS3 stimulates the activity of FASN in vitro. Taken together, these experiments suggest that DENV co-opts the fatty acid biosynthetic pathway to establish its replication complexes. This study provides mechanistic insight into DENV membrane remodeling and highlights the potential for the development of therapeutics that inhibit DENV replication by targeting the fatty acid biosynthetic pathway.

Fig 1.

Effects of pharmacological inhibitors on DENV replication. DENV replicon RNAs were introduced into Huh-7.5 cells. At 24 h postelectroporation, the cells were treated with the indicated concentrations of Cerulenin (A) and C75 (B), maintained for 24 h, and assayed for luciferase activity. RLU, relative luciferase units. Huh-7.5 cells were DENV-infected [multiplicity of infection (MOI) = 1] for 4 h and then treated with the indicated C75 concentrations. Twenty-four hours postinfection, viral RNA levels (C) or released virus (D) was quantified along with cellular ATP levels. The dotted line indicates the limit of detection. Baby hamster kidney cells were infected with YFV 17D (E; MOI = 1), or Vero cells were infected with WNV NY99 (F; MOI = 1) and treated as above. Data represent the average ± SEM.

Fig. 2.

FASN is relocalized to sites of DENV replication. (A) Huh-7.5 cells were mock- or DENV-infected at a multiplicity of infection of 1–5, fixed 36–48 h postinfection, and probed with a monoclonal antibody against FASN. Insets reflect zoomed areas of the boxed regions. (Scale bar: 30 μm.) ImageJ quantitation of the fluorescent intensity of FASN staining in DENV-infected cells (B) compared with mock-infected cell size and cell size in DENV-infected or mock-infected cells (C) (average ± SEM). ns, not significant. Infected cells were probed with antibodies against FASN and the DENV NS1 (D) or dsRNA (E), the DENV replication intermediate. Insets reflect zoomed areas of the boxed regions. (Scale bar: 30 μm.) (F) 3D reconstruction of a z-stack taken through a DENV-infected cell stained with antibodies to dsRNA (green) and FASN (red). (G) Two-photon microscopy of a DENV-infected cell probed for dsRNA and FASN. Intensities plots represent the fluorescent intensity of red, green, and blue pixels along the white line in the inset panels. (Scale bar: 1,500 nm.)

Fig. 3.

FASN levels are not dramatically changed during DENV infection. Huh-7.5 cells were DENV-infected at a multiplicity of infection of 5. At the indicated times postinfection, cells were lysed and protein was extracted. Western blot analysis was performed to determine the relative levels of FASN protein. The blot was stripped and reprobed with actin antibodies as a loading control or with NS3 antibodies to indicate DENV infection.

Fig. 4.

DENV NS3 interacts with FASN. HEK 293T cells were transfected with a V5-tagged NS3 (A) or V5-tagged NS2B-3 (B) expression construct. Seventy-two hours posttransfection, cells were fixed and stained with antibodies to detect the tagged NS3 (green) and FASN (red). The intensity plots represent the fluorescent intensities along the line indicated in the merge panel. (Scale bar: 30 μm.) (C) Ten-fold serial dilutions of diploid yeast transformed with AD-FASN and BD-NS3 constructs (or their respective controls) were plated on yeast two-hybrid selection media or diploid selection media. Growth on the yeast two-hybrid selection media indicates an interaction between the proteins.

Fig. 5.

DENV infection stimulates fatty acid biosynthesis, and de novo synthesized lipids are incorporated into sites of DENV replication. HEL cells were pulsed with ¹⁴C-acetate for 4 h, rinsed, DENV- or mock-infected for ∼36 h, and lysed, and the postnuclear fraction of the cells was recovered. The lysate was lipid-extracted and quantified (A) or applied to an OptiPrep gradient (B–E). (B) Lipid extraction of the fractions to determine the amount of radiolabel incorporated into lipids. (C) Percent increase in radiolabel in each fraction from DENV-infected cells compared with mock infection. (D) DENV RNA in each fraction as determined by RT-PCR. The dotted line at 500 copies represents the detection limit of the assay. (E) Cell lysates from DENV- or mock-infected cells were incubated with ¹⁴C-malonyl–CoA as a direct measure of fatty acid biosynthesis. The FASN inhibitor C75 was applied to verify that the radiolabel incorporation was attributable to FASN activity. Protein was extracted from the DENV and mock reactions and blotted for the presence of FASN and actin. (F) Huh-7.5 cell lysates were incubated with ¹⁴C-malonyl–CoA and increasing concentrations of purified recombinant NS3 (2, 4, or 8 μg) or a corresponding BSA control. C75 (1 mM) was added in parallel to a sample containing NS3. Lipids were extracted, and the radiolabel incorporation was quantified via scintillation counting. The buffer-only control indicates the background of the assay.