The nucleotide sequence of the RNA genome of the human hepatitis C virus (HCV) has been determined from overlapping cDNA clones. The sequence (9379 nucleotides) has a single large open reading frme that could encode a viral polyprotein precursor of 3011 amino acids. While there is little overall amino acid and nucleotide sequence homology with other viruses, the 5' HCV nucleotide sequence upstream ofthis large open reading frame has substantial simiarit to the 5' termini of pestiviral genomes. The polyprotein also has significant sequence similarity to helicases encoded by animal pestiviruses, plant potyviruses, and human flaviviruses, and it contains sequence motifs widely conserved among viral replicases and trypsin-like proteases. A basic, presumed nucleocapsid domain is located at the N terminus upstream ofa region containing numerous potential N-linked glycosylation sites. These HCV domains are located in the same relative position as observed in the pestiviruses and flaviviruses and the hydrophobic profiles of all three viral polyproteins are similar. These combined data indicate that HCV is an unusual virus that is most related to the pestiviruses. Significant genome diversity is apparent within the putative 5' structural gene region of different HCV isolates, suggesting the presence of closely related but distinct viral genotypes.A recombinant immunoscreening approach has recently been used to isolate a cDNA clone (5-1-1) from the genome of an infectious human hepatitis agent that is immunologically unrelated to the hepatitis A and B viruses (1). Clone 5-1-1 and overlapping clones hybridized to a single-stranded RNA molecule that was present in infectious plasma and that encodes immunological epitopes that cross-react in non-A, non-B hepatitis (NANBH) cases from around the world (1, 2).Termed the hepatitis C virus (HCV), this agent appears to be the major cause of posttransfusion and sporadic NANBH worldwide and plays a major role in the development of chronic liver disease including hepatocellular carcinoma (ref.2; for review, see ref.3). We now report the nucleotide sequence of the HCV genome and we discuss its genetic organization and diversity. § MATERIALS AND METHODSThe nucleotide sequence was deduced from a large series of overlapping cDNA clones (150-800 base pairs) derived from the same random-primed Agtll cDNA library used to isolate clone 5-1-1 (1). The source of virus was a plasma pool derived from a chimpanzee with a chronic NANBH infection. This animal represented the second passage of the agent contaminating a human factor VIII concentrate (4). Based initially on the sequence of clone 5-1-1, 32P-labeled synthetic oligonucleotides (30-mers) were used as hybridization probes (5) to isolate cDNA clones overlapping with both termini of clone 5-1-1. Each successive cycle of this repetitive "walking" process usually involved the sequencing of at least 6 different cDNAs from each end. Each Agtll cDNA clone was sequenced on both strands after subcloning the EcoRI cDNA insert into bacterioph...

Saccharomyces cerevisiae cells were transformed with plasmids containing hybrid genes in which the sequence encoding mature human epidermal growth factor was joined to sequences encoding the leader region (preprosegment) of the precursor of the yeast mating pheromone afactor. These cells accurately process the hybrid protein and efficiently secrete authentic biologically active human epidermal growth factor into the medium. Previous studies on the expression of B-lactamase-proinsulin gene fusions in Escherichia coli showed that the /3-lactamase signal sequence is cleaved and proinsulin is secreted into the periplasm (4). We reasoned, therefore, that the leader sequences of secreted yeast proteins may allow more efficient processing and secretion of heterologous proteins by yeast.Most studies on secretion of yeast proteins have used enzymes such as invertase and acid phosphatase (2, 5, 6), which are secreted into the periplasmic space or cell wall.We have used the peptide mating pheromone a-factor, which is efficiently secreted into the medium. a-Factor is a 13-residue peptide, secreted by cells of the a mating type, that acts on cells of the opposite a mating type to promote efficient conjugation between the two cell types leading to the formation of aa diploid cells (7). Studies on the sequence of the afactor structural gene (8) and on the synthesis and processing of the a-factor peptide (9-11) have shown that a-factor is synthesized as a precursor of 165 amino acids containing an 83-residue leader and four a-factor coding regions, each preceded by a short spacer peptide. The leader and spacer amino acids appear to contain the signals necessary for proteolytic processing and secretion. A recent study (12) shows that fusion of the leader region of the a-factor precursor to invertase, another secreted yeast protein, directs its export.To investigate whether the a-factor leader sequences are sufficient to allow efficient processing and secretion, we have constructed plasmids in which the genes coding for foreign secreted proteins have been fused to the yeast a-factor gene and expressed under the control of the a-factor promoter. Several different constructions were made in which the

The trkB family of transmembrane proteins serves as receptors for BDNF and NT-4/5. The family is composed of a tyrosine kinase-containing isoform as well as several alternatively spliced "truncated receptors" with identical extracellular ligandbinding domains but very small intracellular domains. The two best-characterized truncated trkB receptors, designated as trkB.T1 and trkB.T2, contain intracellular domains of only 23 and 21 amino acids, respectively. Although it is known that the tyrosine kinase isoform (trkB.FL) is capable of initiating BDNF and NT-4/5-induced signal transduction, the functional role or roles of the truncated receptors remain enigmatic. At the same time, the potential importance of the truncated receptors in the development, maintenance, and regeneration of the nervous system has been highlighted by recent developmental and injury paradigm investigations. Here we have used trkB cDNA transfected cell lines to demonstrate that both trkB.T1 and trkB.T2 are capable of mediating BDNF-induced signal transduction. More specifically, BDNF activation of either trkB.T1 or trkB.T2 increases the rate of acidic metabolite release from the cell, a common physiological consequence of many signaling pathways. Further, these trkB.T1-and trkB.T2-mediated changes occur with kinetics distinct from changes mediated by trkB.FL, suggesting the participation of at least some unique rate-limiting component or components. Mutational analysis demonstrates that the isoform-specific sequences within the intracellular domains of each receptor are essential for signaling capability. Finally, inhibitor studies suggest that kinases are likely to be involved in the trkB.T1 and trkB.T2 signaling pathways.

Macrophages secrete a wide variety of proteins that mediate many aspects of acute and chronic inflammation (for review see reference 1). While some of these factors have been well characterized (e.g., IL-1 [2] and cachectin/TNF [3]), others remain poorly defined . Recently, we described the purification and characterization ofa new monokine found in the culture medium of an LPS-stimulated mouse macrophage tumor cell line (RAW264 .7) (4). This protein, termed macrophage inflammatory protein or MIP, has several properties indicative ofan endogenous mediator ofinflammation (e.g ., neutrophil attraction and activation) . Since MIP represents an important new addition to the family of activated macrophage products, it is important to investigate its structure and regulation on the molecular level. Here we describe the cloning and sequencing of the cDNA for murine MIP. Materials and MethodsConstruction ofthe cDNA Library. RAW264.7 cells were obtained from American Type Culture Collection (Rockville, MD) and grown in RPMI 1640 (Gibco Laboratories, Grand Island, NY) supplemented with 20 mM Hepes and 10% FCS (HyClone Laboratories, Logan, UT) until they reached confluency. The cells were then washed five times in HBSS (Gibco Laboratories) and the medium was replaced with serum-free RPMI supplemented with 1 Rg/ml of LPS W (Escherichia coli 0127:B8, Difco Laboratories, Detroit, MI). The cells were incubated at 37°C for 2 h and total RNA was extracted by the addition of6 M guanidinium thiocyanate (5) . Poly(A)' RNA was then isolated by two cycles of oligo-dT-cellulose chromatography, essentially as described by Maniatis et al. (6) . Double-stranded cDNA was prepared from the poly(A)' selected RNA as described by Gubler and Hoffman (7) . After methylation of the internal Eco RI sites and addition of Eco RI linkers, the cDNA was inserted into the Eco RI sites of the bacteriophage %gtl0 (8) .Construction of the Probe Pools . Oligonucleotide probe pools were synthesized as described by Warner et al. (9) against amino acids 22-30 of a partial NH2-terminal sequence . This portion ofthe polypeptide was selected because ofits lower degeneracy in the codon dictionary when compared with the remainder of the sequence . The resulting probe pools are two 512-fold degenerate pools of 26 nucleotides in length.

A number of macrophage-derived mediators have been implicated in the vascular changes of inflammation. We recently reported the isolation of a novel monokine, macrophage inflammatory protein 1 (MIP-1), which causes local inflammatory responses in vivo, and induces superoxide production by neutrophils in vitro. Purified native MIP-1 comprises two peptides with very similar physical characteristics. We report here the resolution of MIP-1 into component peptides by SDS-hydroxylapatite chromatography, and compare the NH2-terminal sequences of the two peptides, now referred to as MIP-1 alpha and MIP-1 beta. A synthetic oligonucleotide probe pool corresponding to the NH2-terminal amino acid sequence of MIP-1 beta was used to isolate a cDNA clone containing its coding sequence. The sequence codes for a 109 amino acid-long polypeptide, of which 69 amino acids correspond to the mature product. Comparison of this MIP-1 beta cDNA with our previously cloned MIP-1 alpha sequence reveals that the MIP-1 peptides, members of a growing family of potential inflammatory mediators, are distinct but highly homologous (58.9% sequence identity) products of different genes.

On our initial discovery that prion protein (PrP)-derived peptides were capable of capturing the pathogenic prion protein (PrP Sc ), we have been interested in how these peptides interact with PrP Sc . After screening peptides from the entire human PrP sequence, we found two peptides (PrP 19 -30 and PrP100-111) capable of binding full-length PrP Sc in plasma, a medium containing a complex mixture of other proteins including a vast excess of the normal prion protein (PrP C ). The limit of detection for captured PrP Sc was calculated to be 8 amol from a Ϸ10 5 -fold dilution of 10% (wt/vol) human variant Creutzfeldt-Jakob disease brain homogenate, with >3,800-fold binding specificity to PrP Sc over PrP C . Through extensive analyses, we show that positively charged amino acids play an important, but not exclusive, role in the interaction between the peptides and PrP Sc . Neither hydrophobic nor polar interactions appear to correlate with binding activity. The peptide-PrP Sc interaction was not sequence-specific, but amino acid composition affected binding. Binding occurs through a conformational domain that is only present in PrP Sc , is species-independent, and is not affected by proteinase K digestion. These and other findings suggest a mechanism by which cationic domains of PrP C may play a role in the recruitment of PrP C to PrP Sc .plasma ͉ Creutzfeldt-Jakob disease ͉ detection ͉ cationic interaction ͉ diagnostic

Broad, multispecific CD4؉ and CD8 ؉ T-cell responses to the hepatitis C virus (HCV), as well as viruscross-neutralizing antibodies, are associated with recovery from acute infection and may also be associated in chronic HCV patients with a favorable response to antiviral treatment. and CD8؉ T-cell responses to E1E2 and NS345 was obtained by first priming with Th1-adjuvanted proteins and then boosting with chimeric, defective alphaviruses expressing these HCV genes. In addition, this prime/ boost regimen resulted in the induction of anti-E1E2 antibodies capable of cross-neutralizing heterologous HCV isolates in vitro. This vaccine formulation and regimen may therefore be optimal in humans for protection against this highly heterogeneous global pathogen.

Naturally occurring hepatitis C virus (HCV) infection has long been thought to induce a weak immunity which is insufficient to protect an individual from subsequent infections and has cast doubt on the ability to develop effective vaccines. A series of intrahepatic genetic inoculations (IHGI) with type 1a HCV RNA were performed in a chimpanzee to determine whether a form of genetic immunization might stimulate protective immunity. We demonstrate that the chimpanzee not only developed protective immunity to the homologous type 1a RNA after rechallenge by IHGI but was also protected from chronic HCV infection after sequential rechallenge with 100 50% chimpanzee infectious doses of a heterologous type 1a (H77) and 1b (HC-J4) whole-virus inoculum. These results offer encouragement to pursue the development of HCV vaccines.