In infected cells, hepatitis C virus (HCV) core protein is targeted to lipid droplets, which serve as intracellular storage organelles. Using a tissue culture system to generate infectious HCV, we have shown that the coating of lipid droplets by the core protein occurs in a time-dependent manner and coincides with higher rates of virus production. At earlier times, the protein was located at punctate sites in close proximity to the edge of lipid droplets. Investigations by using Z-stack analysis have shown that many lipid droplets contained a single punctate site that could represent positions where core transfers from the endoplasmic reticulum membrane to droplets. The effects of lipid droplet association on virus production were studied by introducing mutations into the domain D2, the C-terminal region of the core protein necessary for droplet attachment. Alteration of a phenylalanine residue that was crucial for lipid droplet association generated an unstable form of the protein that could only be detected in the presence of a proteasome inhibitor. Moreover, converting two proline residues in D2 to alanines blocked coating of lipid droplets by core, although the protein was directed to punctate sites that were indistinguishable from those observed at early times for wild-type core protein. Neither of these virus mutants gave rise to virus progeny. By contrast, mutation at a cysteine residue positioned 2 aa upstream of the phenylalanine residue did not affect lipid droplet localization and produced wild-type levels of infectious progeny. Taken together, our findings indicate that lipid droplet association by core is connected to virus production. INTRODUCTIONChronic infection by hepatitis C virus (HCV) affects about 170 million individuals worldwide and is a major cause of liver disease (Hoofnagle, 2002). HCV is an enveloped virus belonging to the genus Hepacivirus within the family Flaviviridae (Murphy et al., 1995). The viral genome is a single-stranded, positive-sense RNA molecule approximately 9.6 kb in length that encodes a polyprotein of some 3000 aa. The polyprotein is cleaved both co-and post-translationally at the endoplasmic reticulum (ER) membrane by cellular and viral proteases to yield the mature viral proteins (Bartenschlager & Lohmann, 2000;Penin et al., 2004). The structural proteins are located at the N-terminal end of the polyprotein and consist of the core protein, which forms the viral capsid, and two envelope glycoproteins, E1 and E2. The glycoproteins are released from the polyprotein by signal peptidase (SP) cleavage, whereas maturation of the core protein requires proteolysis by both SP and signal peptide peptidase (SPP) (Hussy et al., 1996;McLauchlan et al., 2002).The mature form of core is a dimeric, a-helical protein that is separable into two domains, D1 and D2 (McLauchlan, 2000;Boulant et al., 2005Boulant et al., , 2006. D1 consists of the Nterminal 117 aa, while D2 begins at amino acid residue 118 and ends between amino acids 171 and 182. D2 is required for correct folding of D1 a...

Attachment of hepatitis C virus (HCV) core protein to lipid droplets (LDs) is linked to release of infectious progeny from infected cells. Core progressively coats the entire LD surface from a unique site on the organelle, and this process coincides with LD aggregation around the nucleus. We demonstrate that LD redistribution requires only core protein and is accompanied by reduced abundance of adipocyte differentiation-related protein (ADRP) on LD surfaces. Using small hairpin RNA technology, we show that knock down of ADRP has a similar phenotypic effect on LD redistribution. Hence, ADRP is crucial to maintain a disperse intracellular distribution of LDs. From additional experimental evidence, LDs are associated with microtubules and aggregate principally around the microtubule-organizing centre in HCV-infected cells. Disrupting the microtubule network or microinjecting anti-dynein antibody prevented core-mediated LD redistribution. Moreover, microtubule disruption reduced virus titres, implicating transport networks in virus assembly and release. We propose that the presence of core on LDs favours their movement towards the nucleus, possibly to increase the probability of interaction between sites of HCV RNA replication and virion assembly.

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.

Neutral lipid is stored in spherical organelles called lipid droplets that are bounded by a coat of proteins. The protein that is most frequently found at the surface of lipid droplets is adipocyte differentiation-related protein (ADRP). In this study, we demonstrate that fusion of either the human or mouse ADRP coding sequences to green fluorescent protein (GFP) does not disrupt the ability of the protein to associate with lipid droplets. Using this system to identify targeting elements, discontinuous segments within the coding region were required for directing ADRP to lipid droplets. GFP-tagged protein was employed also to examine the behavior of lipid droplets in live cells. Time lapse microscopy demonstrated that in HuH-7 cells, which are derived from a human hepatoma, a small number of lipid droplets could move rapidly, indicating transient association with intracellular transport pathways. Most lipid droplets did not show such movement but oscillated within a confined area; these droplets were in close association with the endoplasmic reticulum membrane and moved in concert with the endoplasmic reticulum. Fluorescence recovery analysis of GFP-tagged ADRP in live cells revealed that surface proteins do not rapidly diffuse between lipid droplets, even in conditions where they are closely packed. This system provides new insights into the properties of lipid droplets and their interaction with cellular processes.

The mechanisms involved in hepatitis C virus (HCV) RNA replication are unknown, and this aspect of the virus life cycle is not understood. It is thought that virus-encoded nonstructural proteins and RNA genomes interact on rearranged endoplasmic reticulum (ER) membranes to form replication complexes, which are believed to be sites of RNA synthesis. We report that, through the use of an antibody specific for doublestranded RNA (dsRNA), dsRNA is readily detectable in Huh-7 cells that contain replicating HCV JFH-1 genomes but is absent in control cells. Therefore, as that of other RNA virus genomes, the replication of the HCV genome may involve the generation of a dsRNA replicative intermediate. In Huh-7 cells supporting HCV RNA replication, dsRNA was observed as discrete foci, associated with virus-encoded NS5A and core proteins and identical in morphology and distribution to structures containing HCV RNA visualized by fluorescencebased hybridization methods. Three-dimensional reconstruction of deconvolved z-stack images of virus-infected cells provided detailed insight into the relationship among dsRNA foci, NS5A, the ER, and lipid droplets (LDs). This analysis revealed that dsRNA foci were located on the surface of the ER and often surrounded, partially or wholly, by a network of ER-bound NS5A protein. Additionally, virus-induced dsRNA foci were juxtaposed to LDs, attached to the ER. Thus, we report the visualization of HCV-induced dsRNA foci, the likely sites of virus RNA replication, and propose that HCV genome synthesis occurs at LD-associated sites attached to the ER in virus-infected cells.