Structural Determinants That Target the Hepatitis C Virus Core Protein to Lipid Droplets

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 ␣-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.

Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles

“…Lipids are known to play a vital role in HCV particle production; a number of HCV proteins are known to associate with lipid droplets, notably core (Boulant et al, 2006) and NS5A (Miyanari et al, 2007), and these organelles play a key, albeit uncharacterized, role in virus production (Miyanari et al, 2007;Huang et al, 2007;Ye, 2007). It is possible therefore that ESCRT function might be required to transport nascent virus particles from the site of assembly (assumed to be proximal to lipid droplets) to the cell surface.…”

Cellular Lipid Droplets and Hepatitis C Virus Life Cycle

“…Residues on the hydrophobic face of two amphipathic alpha-helices (HI, aa119-136 and HII, aa148-164) in D2 are critical for LD association from the extensive mutational studies. 43,44) HI and HII are separated by a short hydrophobic loop, and a combination of these may lead to an in-plane membrane interaction of the two helices at the phospholipid layer interface. A family of plant proteins associated with lipid body, called oleosins, the interaction of which with lipid body has been well characterized (reviewed in refs.…”

“…1). [42][43][44] D2 is important for the membrane association activity of the core protein as well as for its folding and stability. Residues on the hydrophobic face of two amphipathic alpha-helices (HI, aa119-136 and HII, aa148-164) in D2 are critical for LD association from the extensive mutational studies.…”

Targeting of the Arf-GEF GBF1 to lipid droplets and Golgi membranes

“…Our mutagenesis analysis of this region supports the conclusion that it forms an amphipathic helix in vitro and in cells, and that this property is important for its targeting to LDs. Interestingly, other proteins, including perilipins (Bulankina et al, 2009), hepatitis C virus (HCV) core protein (Boulant et al, 2006), HCV NS5A protein, the antiviral peptide viperin (Hinson and Cresswell, 2009) and CTP:phosphocholine cytidylyltransferase a (CCTa) (Krahmer et al, 2011), all have amphipathic helices that are necessary and sufficient for targeting to LDs. Because the LD surface is a phospholipid monolayer, amphipathic helices are ideal Fig.…”