Sequences in the 5 ' nontranslated region of hepatitis C virus required for RNA replication

Sequences in the 5' and 3' termini of plus-strand RNA viruses harbor cis-ac ting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flavi viridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5' end of the genome. This sequence contains an internal ribosome entry si te (IRES) that is located downstream of an about 40-nucleotide-long sequenc e of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5' NTR required for RNA r eplication. We show that deletions introduced into the 5' terminal 40 nucle otides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5' NT Rs, we could show that the first 125 nucleotides of the HCV genome are esse ntial and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5' NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5' NTR are sufficie nt for RNA replication, but such replicon molecules are severely impaired f or multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of sig nals in the 5' NTR of HCV RNA essential for replication of this virus.