From analysis of the primary sequence of the hepatitis C virus (HCV) core protein, we have identified three separable regions based on hydrophobicity and clustering of basic amino acids within the protein. Comparison with capsid proteins of related pesti-and flaviviruses suggested that HCV core has a unique central domain (domain 2). Previous findings have revealed that core protein can associate with lipid droplets which are intracellular storage sites for triacylglycerols and cholesterol esters. Confocal analysis of variant forms lacking regions of core indicated that most residues within the unique region are necessary for association of the protein with lipid droplets. A segment within domain 2 (from residues 125 to 144) also was required for stability of the protein and a polypeptide lacking these sequences was degraded apparently by the proteasome. In cells depleted of lipid droplets, core protein remained located in the cytoplasm. Moreover, cleavage of the protein at the maturation site and stability were not affected by inability to bind to lipid droplets.

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.

In mammalian tissue culture cells, the core protein of hepatitis C virus (HCV) is located at the surface of lipid droplets, which are cytoplasmic structures that store lipid. The critical amino acid sequences necessary for this localization are in a region of core protein that is absent in flavi-and pestiviruses, which are related to HCV. From our sequence comparisons, this region in HCV core was present in the corresponding protein of GBV-B, another virus whose genomic sequence has significant similarity to HCV. Expression of the putative GBV-B core protein revealed that it also was directed to lipid droplets. By extending the comparisons to cellular proteins, there were amino acid sequence similarities between the domains for lipid droplet association in HCV core and plant oleosin proteins. To determine whether these similarities were related functionally, an oleosin encoded by the Brassica napus bniii gene was expressed in different mammalian cell lines, where it retained the capacity to bind to lipid droplets. Analysis of deletion mutants indicated that the critical region within the protein required for this localization was the same for both plant and mammalian cells. A common feature in the viral and plant sequences was a motif containing proline residues. Mutagenesis of these residues in HCV core and plant oleosin abolished lipid droplet association. Finally, the domain within HCV core required for binding to lipid droplets could substitute for the equivalent domain in oleosin, further indicating the functional relatedness between the viral and plant sequences. These studies identify common features in disparate proteins that are required for lipid droplet localization.

The possible identity of the herpes simplex virus type 1 (HSV-1) 65K (65,000-Mr) virion protein which stimulates transcription from immediate-early genes with the HSV-1 65K DNA-binding protein was investigated. The two proteins were found to be distinct by the three separate criteria of immunological reactivity, tryptic peptide fingerprinting, and mobility in two-dimensional gels. Using HSV-1/HSV-2 intertypic recombinants and a serotype-specific antiserum, we located the gene encoding the 65K DNA-binding protein between coordinates 0.574 and 0.682 on the HSV-1 genome. The protein is posttranslationally modified by phosphorylation. In crude extracts of HSV-1-infected cells the 65K trans-inducing protein did not detectably bind to double-stranded calf thymus DNA under the conditions of our assay. Expression of herpes simplex virus (HSV) genes is temporally regulated and is considered to occur in at least three phases termed immediate-early (IE), early, and late or (x, P, and-y (13, 27, 28, 31, 50). IE genes are expressed in the absence of de novo protein synthesis (27) but their expression is stimulated by a component of the virus particle (5, 42). This trans-inducing factor was identified as polypeptide Vmw65 (11) and is designated 65KTIF in the present study. The mechanism by which 65KTIF stimulates transcription from IE genes is not understood; however, one possibility is that it interacts with some IE-gene-regulatory DNA sequence. Interestingly, a responder element located several hundred bases upstream of the IE mRNA 5' terminus has been identified and corresponds to the consensus sequence TAATGARATTC (R is purine) (10, 35, 44, 53). Previously, we identified a major DNA-binding protein in HSV type 1 (HSV-1)-infected cells with an apparent Mr of ca. 62,000 (6). This DNA-binding protein and 65KTIF have the same electrophoretic mobilities in sodium dodecyl sulfate-5 to 12.5 polyacrylamide gels, and so we designated the DNA-binding protein 65KDBP. It seemed important to establish whether 65KDBp and 65KTIF were one and the same protein and whether 65KTIF can bind to DNA. This study addresses these questions. We characterized the two proteins and show that 65KDBp and 65KTIF are distinct and that 65KTIF does not detectably bind to DNA under the conditions of our assay. MATERIALS AND METHODS Cells. BHK21 clone 13 cells (34) were used throughout. Viruses. HSV-1 strain 17syn+ (9) and HSV-2 strain HG52 (51) were used in this study. The isolation of HSV-1/HSV-2 intertypic recombinants and the determination of their genome structures have been previously reported (12, 15, 37, 46, 54). Radioactive labeling. Confluent monolayers in 50-mmdiameter dishes or roller bottles were infected at a multiplicity of infection of 5 to 20 PFU of HSV-1 per cell. After 1 h, * Corresponding author.