The nucleotide sequence of the hepatitis C virus (HCV) RNA genome was determined from overlapping cDNA clones. The sequence, 9379 nucleotides long, contains a single large open reading frame (ORF) that could encode a polyprotein of 3011 amino acids. While there is little overall homology with other viruses, the 5' region of HCV shows similarity to pestiviral genomes. The polyprotein has significant sequence similarity to helicases from pestiviruses, potyviruses, and flaviviruses, and contains conserved motifs in viral replicases and trypsin-like proteases. A basic nucleocapsid domain is located at the N terminus, upstream of regions with many potential N-linked glycosylation sites. These domains are in the same relative position as in pestiviruses and flaviviruses, and the hydrophobic profiles of all three viral polyproteins are similar. These data suggest HCV is most related to pestiviruses. Significant genome diversity is apparent in the 5' structural gene region of different HCV isolates, indicating the presence of closely related but distinct viral genotypes. A recombinant immunoscreening approach was used to isolate a cDNA clone (5-1-1) from the genome of an infectious human hepatitis agent. Clone 5-1-1 and overlapping clones hybridized to a single-stranded RNA molecule present in infectious plasma, which encodes immunological epitopes cross-reacting in non-A, non-B hepatitis (NANBH) cases. This agent, termed HCV, is the major cause of posttransfusion and sporadic NANBH worldwide and plays a major role in chronic liver disease, including hepatocellular carcinoma. The nucleotide sequence was deduced from overlapping cDNA clones derived from a λ gt11 cDNA library. The sequence contains one large ORF that could encode a polyprotein of 3011 amino acids. The next largest ORF is 1156 nucleotides long and is present in the antigenomic sequence. The 3' noncoding region is short and may be incomplete. HCV RNA binds to oligo(dT)-cellulose, possibly due to an A-rich tract or a 3'-terminal poly(rA) sequence. The HCV polyprotein shows some homology with other viruses in three regions. One region shares homology with NTP-binding helicases from pestiviruses, plant potyviruses, and human flaviviruses. Another region contains 6 residues conserved among all viral-encoded RNA-dependent RNA polymerases. A third region contains amino acids conserved among putative trypsin-like serine proteases. The relative location of these domains is similar in HCV, flaviviruses, and pestiviruses, but not in plant viruses. The sequenceThe nucleotide sequence of the hepatitis C virus (HCV) RNA genome was determined from overlapping cDNA clones. The sequence, 9379 nucleotides long, contains a single large open reading frame (ORF) that could encode a polyprotein of 3011 amino acids. While there is little overall homology with other viruses, the 5' region of HCV shows similarity to pestiviral genomes. The polyprotein has significant sequence similarity to helicases from pestiviruses, potyviruses, and flaviviruses, and contains conserved motifs in viral replicases and trypsin-like proteases. A basic nucleocapsid domain is located at the N terminus, upstream of regions with many potential N-linked glycosylation sites. These domains are in the same relative position as in pestiviruses and flaviviruses, and the hydrophobic profiles of all three viral polyproteins are similar. These data suggest HCV is most related to pestiviruses. Significant genome diversity is apparent in the 5' structural gene region of different HCV isolates, indicating the presence of closely related but distinct viral genotypes. A recombinant immunoscreening approach was used to isolate a cDNA clone (5-1-1) from the genome of an infectious human hepatitis agent. Clone 5-1-1 and overlapping clones hybridized to a single-stranded RNA molecule present in infectious plasma, which encodes immunological epitopes cross-reacting in non-A, non-B hepatitis (NANBH) cases. This agent, termed HCV, is the major cause of posttransfusion and sporadic NANBH worldwide and plays a major role in chronic liver disease, including hepatocellular carcinoma. The nucleotide sequence was deduced from overlapping cDNA clones derived from a λ gt11 cDNA library. The sequence contains one large ORF that could encode a polyprotein of 3011 amino acids. The next largest ORF is 1156 nucleotides long and is present in the antigenomic sequence. The 3' noncoding region is short and may be incomplete. HCV RNA binds to oligo(dT)-cellulose, possibly due to an A-rich tract or a 3'-terminal poly(rA) sequence. The HCV polyprotein shows some homology with other viruses in three regions. One region shares homology with NTP-binding helicases from pestiviruses, plant potyviruses, and human flaviviruses. Another region contains 6 residues conserved among all viral-encoded RNA-dependent RNA polymerases. A third region contains amino acids conserved among putative trypsin-like serine proteases. The relative location of these domains is similar in HCV, flaviviruses, and pestiviruses, but not in plant viruses. The sequence