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...

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We have determined the nucleotide sequence at the extreme 5' and 3' termini of the hepatitis C virus (HCV) genome. Our analyses ofthese sequences show (t) the nucleotide sequence in the 5' untranslated region is highly conserved among HCV isolates of widely varying geographical orign, (i) within this region, there are blocks of nucleotide sequence homology with pestiviruses but not with other viruses, (ii) the relative position of short open reading frames present in the same region of the HCV genome is similar to that of the pestiviral genome, (iv) RNAs truncated at the 5' and 3' ends are found, but the origin and functions of these RNAs are unknown, and (v) poly(A) tails appear to be present on 3' subgenomic RNAs. (ORFs). The HCV genome, however, displays singular characteristics at each terminus. We detect a hairpin structure at the 5' end of the genome as well as 5' and 3' subgenomic RNAs, the latter of which are polyadenylylated. These are consistent with a polyadenylylated 3' terminus of the viral genome and perhaps of functional subgenomic RNAs. Our data provide insights into the organization of the HCV genome, which may have important ramifications regarding the replication strategy and evolution of the virus. MATERIALS AND METHODSRNA was extracted from a high-titer plasma of an experimentally infected chimpanzee (6) and plasma (or serum) from HCV-positive or negative blood donors by a low-temperature guanidinium thiocyanate method (7). Poly(A)+ RNA was isolated from the liver ofthe same infected chimpanzee by the guanidinium thiocyanate/urea method (8). cDNA was synthesized from RNA according to Han and Rutter (9) and amplified by polymerase chain reaction (PCR) according to Saiki et al. (10). Briefly, RNA isolated from about 500 Al of plasma or S Ag of poly(A)+ liver RNA was converted into single-stranded cDNA by reverse transcriptase (BRL) using 150 pmol of the appropriate cDNA primer. For 5' end characterization by primer extension (7), first-strand cDNA was precipitated by spermine (11) and tailed with dA (9). Tailed or untailed cDNA was converted into double-stranded cDNA using a second-strand cDNA primer (9). This doublestranded cDNA was amplified using the indicated HCVspecific PCR primers for 35 cycles (940C, 1.5 min; 60'C, 2 min; 720C, 3 min). PCR without cDNA template was routinely performed to check for possible contamination during PCR. The PCR product was analyzed by Southern blot hybridization using a 32P-labeled oligonucleotide probe. The sequences and locations in the HCV genome of various cDNA and PCR primers are shown in Figs. 2 and 5. Most primers were designed to contain a Not I site for subsequent cloning of the PCR products into pUC18S, which contains a polylinker derived from Agt22 (9). DNA sequence was obtained by the supercoil sequencing (12) or the direct PCR sequencing method (13). RESULTSBased on the sequence of the major part of the HCV genome determined from overlapping A clones (2), we devised a directed strategy for obtaining clones representing the remaini...