Abstract
Hepatitis C virus core protein is targeted to lipid droplets, which serve as intracellular storage organelles, by its C-terminal domain, termed D2. From circular dichroism and nuclear magnetic resonance analyses, we demonstrate that the major structural elements within D2 consist of two amphipathic alpha-helices (Helix I and Helix II) separated by a hydrophobic loop. Both helices require a hydrophobic environment for folding, indicating that lipid interactions contribute to their structural integrity. Mutational studies revealed that a combination of Helix I, the hydrophobic loop, and Helix II is essential for efficient lipid droplet association and pointed to an in-plane membrane interaction of the two helices at the phospholipid layer interface. Aside from lipid droplet association, membrane interaction of D2 is necessary for folding and stability of core following maturation at the endoplasmic reticulum membrane by signal peptide peptidase. These studies identify critical determinants within a targeting domain that enable trafficking and attachment of a viral protein to lipid droplets. They also serve as a unique model for elucidating the specificity of protein-lipid interactions between two membrane-bound organelles.
Highlights
Lipid droplets (LDs)3 are intracellular storage organelles, composed primarily of a hydrophobic core of cholesterol ester and triacylglycerol, which is bounded by a phospholipid leaflet encased in a layer of proteins [1]
To determine whether domain D2 could target a heterologous protein to LDs, the coding region for GFP was linked upstream from sequences encoding amino acids 118 –171 from Hepatitis C virus (HCV) core, generating a construct that produced chimeric GFP/D2 (Fig. 1)
The locations of LDs were established by incubating cells with a BODIPY-labeled fatty acid, which accumulates in these structures following cellular uptake [10]
Summary
Construction of Plasmids—Plasmids pgHCV/CE1E2Gla and pSFV/CE1E2Gla have been described previously [20, 24]. Indirect Immunofluorescence, Staining of LDs, and GFP Fluorescence—Analyses of the intracellular localization of proteins and LDs in live and fixed cells were performed as described previously [10]. To provide additional biochemical evidence that GFP/D2 behaved to LD-associated proteins, extracts from transfected cells were fractionated by flotation centrifugation on sucrose gradients (Fig. 2B). Individual fractions were probed with antibodies against GFP, ADRP, a marker for LDs [10], and calnexin, a marker for ER membranes [30], to determine the mobility profiles of these various proteins in gradients. Compared with GFP/D2 and ADRP, calnexin was detected only in the lower portion of the gradient These data provided further conclusive evidence that GFP/D2 associated with LDs. D2 of core contains targeting determinants that are sufficient for directing proteins to LDs
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