The lipid droplet (LD) is an organelle that is used for the storage of neutral lipids. It dynamically moves through the cytoplasm, interacting with other organelles, including the endoplasmic reticulum (ER). These interactions are thought to facilitate the transport of lipids and proteins to other organelles. The hepatitis C virus (HCV) is a causative agent of chronic liver diseases. HCV capsid protein (Core) associates with the LD, envelope proteins E1 and E2 reside in the ER lumen, and the viral replicase is assumed to localize on ER-derived membranes. How and where HCV particles are assembled, however, is poorly understood. Here, we show that the LD is involved in the production of infectious virus particles. We demonstrate that Core recruits nonstructural (NS) proteins and replication complexes to LD-associated membranes, and that this recruitment is critical for producing infectious viruses. Furthermore, virus particles were observed in close proximity to LDs, indicating that some steps of virus assembly take place around LDs. This study reveals a novel function of LDs in the assembly of infectious HCV and provides a new perspective on how viruses usurp cellular functions.

MicroRNAs (miRNAs) are a non-coding family of genes involved in post-transcriptional gene regulation. These transcripts are associated with cell proliferation, cell differentiation, cell death and carcinogenesis. We analysed the miRNA expression profiles in 25 pairs of hepatocellular carcinoma (HCC) and adjacent nontumorous tissue (NT) and nine additional chronic hepatitis (CH) specimens using a human miRNA microarray. Targets and references samples were co-hybridized to a microarray containing whole human mature and precursor miRNA sequences. Whereas three miRNAs exhibited higher expression in the HCC samples than that in the NT samples, five miRNAs demonstrated lower expression in the HCC samples than in the NT samples (Po0.0001). Classification of samples as HCC or NT by using support vector machine algorithms based on these data provided an overall prediction accuracy of 97.8% (45/46). In addition, the expression levels of four miRNAs were inversely correlated with the degree of HCC differentiation (Po0.01). A comparison of CH and liver cirrhosis samples revealed significantly different pattern of miRNA expression (Po0.01). There were no differences, however, between hepatitis B-positive and hepatitis C-positive samples. This information may help clarify the molecular mechanisms involved in the progression of liver disease, potentially serving as a diagnostic tool of HCC.

The nucleotide sequence of the Japanese type of hepatitis C virus (HCV-J) genome, consisting of 9413 nucleotides, was determined by analyses of cDNA clones from plasma specimens from Japanese patients with chronic hepatitis. HCV-J genome contains a long open reading frame that can encode a sequence of 3010 amino acid residues. Comparison ofHCV-J with the American isolate of HCV showed 22.6% difference in nucleotide sequence and 15.1% difference in amino acid sequence. Thus HCV-J and the American isolate of HCV are probably different subtypes of HCV. The relationship of HCV-J with other animal RNA virus families and the putative organization of the HCV-J genome are discussed.

DNA methyltransferases

Processing of the putative structural proteins of hepatitis C virus was examined by using an in vitro expression system. An RNA transcript for cell-free translation was prepared from a cDNA construct that encompasses the region encoding the 980 amino-terminal residues of the viral polyprotein precursor. Processing of the in vitro translation product proceeded cotranslationally in the presence of microsomal membranes and generated four major membrane-associated products. Two of these four major products, named gp35 and gp7O, were shown to be transported into microsomes and heavily glycosylated, suggesting that the processing events are partly mediated by the signal peptidase of the endoplasmic reticulum. The other two products, p19 and p21, were probably associated with the outer surface of the microsomal membrane. Analysis of processed proteins translated from a series of truncated forms of the cDNA construct as well as determination of amino-terminal amino acid sequences of gp35 and gp7O indicated that these four products are arranged from the amino-terminal end of the polyprotein precursor in the order: NH-p22-gp35-gp7O-pl9. Both gp35 and gp7O could be candidates of initially processed forms of envelope proteins of the hepatitis C virus.Hepatitis C virus (HCV) is considered to be a causative agent of post-transfusion non-A, non-B hepatitis (1, 2). The enveloped virions consist of unknown species of structural proteins encoded by positive-stranded RNA genomes. This virus is probably related to pestiviruses and to flaviviruses judging from the similarities of the deduced amino acid sequences of the putative viral proteins (refs. 3 and 4 and unpublished results). Our recent study showed that genomic RNA ofHCV from Japanese patients with non-A, non-B hepatitis is more than 9413 nucleotides long and includes a single open reading frame (ORF) encoding a precursor polyprotein of 3010 amino acids (4). This precursor polyprotein has many basic amino acid residues clustered in its 120 amino-terminal residues deduced from the sequence of the ORF, as in the aminoterminal regions of nucleocapsid (C) proteins of flaviviruses (4,5). It also has 15 potential aspargine-linked glycosylation (N-glycosylation) sites clustered in the region of amino acid residues 196-645 like those found in putative envelope (E) glycoproteins of pestiviruses (4, 6). These data suggest that the genetic organization of HCV is almost identical to those of pestiviruses or flaviviruses and that the 5' portion of the genome encodes viral structural proteins.The gene order in the genome of flaviviruses has been determined to be C-premembrane (preM)-