RNA structures play key roles in the replication of RNA viruses. Sequence alignment software, thermodynamic RNA folding programs, and classical comparative phylogenetic analysis were used to build models of six RNA elements in the coding region of the hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B. The importance of five of these elements was evaluated by site-directed mutagenesis of a subgenomic HCV replicon. Mutations disrupting one of the predicted stem-loop structures, designated 5BSL3.2, blocked RNA replication, implicating it as an essential cis-acting replication element (CRE). 5BSL3.2 is about 50 bases in length and is part of a larger predicted cruciform structure (5BSL3). As confirmed by RNA structure probing, 5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop, and an 8-base internal loop. Mutational analysis and structure probing were used to explore the importance of these features. Primary sequences in the loops were shown to be important for HCV RNA replication, and the upper helix appears to serve as an essential scaffold that helps maintain the overall RNA structure. Unlike certain picornavirus CREs, whose function is position independent, 5BSL3.2 function appears to be context dependent. Understanding the role of 5BSL3.2 and determining how this new CRE functions in the context of previously identified elements at the 5 and 3 ends of the RNA genome should provide new insights into HCV RNA replication.The first molecular clones of hepatitis C virus (HCV) were reported in 1989 (11). Comparative sequence analysis revealed that HCV is related to flavi-and pestiviruses (12), and HCV was subsequently placed in the family Flaviviridae. The genomic RNA of viruses in this family has a long open reading frame (ORF) flanked by nontranslated regions (NTRs) at the 5Ј and 3Ј termini (48). HCV initiates translation of the ORF via an internal ribosome entry site (IRES) (25, 51). Translation of the ORF yields a polyprotein that is cleaved co-and posttranslationally by host and viral proteases. The HCV polyprotein gives rise to 10 viral proteins: core, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (3). NS5B, the viral RNAdependent RNA polymerase, comprises the C-terminal portion of the polyprotein. The 5Ј and 3Ј NTRs of HCV have cis-acting replication elements (CREs) which are essential for the viral life cycle (14,32,33,36,70) and are known to bind cellular proteins (15,21,26,46,57,67).Coding regions of viral RNAs often have embedded nucleic acid signals and overlapping reading frames that encode proteins, as in X174 (52), hepatitis B virus (16), and Rous sarcoma virus (27). Embedded RNA signals include promoters (29), nucleation sites for encapsidation (2,30,35,49), and other types of CREs (5,18,40,43,44,53). Among the best characterized is the picornavirus CRE (17,18,40,43,45,71). The picornavirus CRE acts as a template for the uridylylation of VPg, the protein primer for genome replication, and thus plays a direct role in initiation of RNA replication. The exac...

Many viruses have overlapping genes and/or regions in which a nucleic acid signal is embedded in a coding sequence. To search for dual-use regions in the hepatitis C virus (HCV), we developed a facile computer-based sequence analysis method to map dual-use regions in coding sequences. Eight diverse full-length HCV RNA and polyprotein sequences were aligned and analyzed. A cluster of unusually conserved synonymous codons was found in the core-encoding region, indicating a potential overlapping open reading frame (ORF). Four peptides (A1, A2, A3, and A4) representing this alternate reading frame protein (ARFP), two others from the HCV core protein, and one from bovine serum albumin (BSA) were conjugated to BSA and used in western blots to test sera for specific antibodies from 100 chronic HCV patients, 44 healthy controls, and 60 patients with non-HCV liver disease. At a 1:20,000 dilution, specific IgGs to three of the four ARFP peptides were detected in chronic HCV sera. Reactivity to either the A1 or A3 peptides (both ARFP derived) was significantly associated with chronic HCV infection, when compared to non-HCV liver disease serum samples (10/100 versus 1/60; p , 0.025). Antibodies to A4 were not detected in any serum sample. Our western blot assays confirmed the presence of specific antibodies to a new HCV antigen encoded, at least in part, in an alternate reading frame (ARF) overlapping the core-encoding region. Because this novel HCV protein stimulates specific immune responses, it has potential value in diagnostic tests and as a component of vaccines. This protein is predicted to be highly basic and may play a role in HCV replication, pathogenesis, and carcinogenesis.

To examine whether fatty acid transport is abnormal in obesity, the kinetics of [ 3 H]oleate uptake by hepatocytes, cardiac myocytes, and adipocytes from adult male Wistar (؉/؉), Zucker lean (fa/؉) and fatty (fa/fa), and Zucker diabetic fatty (ZDF) rats were studied. A tissuespecific increase in oleate uptake was found in fa/fa and ZDF adipocytes, in which the V max was increased 9-fold (p < 0.005) and 13-fold (p < 0.001), respectively. This increase greatly exceeded the 2-fold increase in the surface area of adipocytes from obese animals, and did not result from trans-stimulation secondary to increased lipolysis. Adipocyte tumor necrosis factor-␣ mRNA levels, assayed by Northern hybridization, increased in the order ؉/؉ < fa/fa < ZDF. Oleate uptake was also studied in adipocytes from 20 -24-day-old male ؉/؉, fa/؉, and fa/fa weanlings. These animals were not obese, and had equivalent plasma fatty acid and glucose levels. Tumor necrosis factor-␣ mRNA levels in ؉/؉ and fa/fa cells also were similar. Nevertheless, V max was increased 2.9-fold (p < 0.005) in fa/fa compared ؉/؉ cells. These studies indicate 1) that regulation of fatty acid uptake is tissuespecific and 2) that up-regulation of adipocyte fatty acid uptake is an early event in Zucker fa/fa rats. These findings are independent of the role of any particular fatty acid transporter. Adipocyte mRNA levels of three putative transporters, mitochondrial aspartate aminotransferase, fatty acid translocase, and fatty acid transporting protein (FATP) were also determined; mitochondrial aspartate aminotransferase and FATP mRNAs correlated strongly with fatty acid uptake.

June 19, 1987) ABSTRACT A portion of the hepatocellular uptake of nonesterified long-chain fatty acids is mediated by a specific 40-kDa plasma membrane fatty acid binding protein, which has also been isolated from the gut. To investigate whether a similar transport process exists in other tissues with high transmembrane fatty acid fluxes, initial rates (Vd) of [3H]-