Identification of novel sequences at the 5′ terminus of the hepatitis C virus genome

To permit accurate identification of the 5' end of the HCV genome, we used RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) where an RNA molecule of known sequence (transcribed in vitro from a cDNA template) was ligated to RNA purified from a hepatitis C virus (HCV)-infected liver sample. After ligation, the product was amplified by nested polymerase chain reaction (PCR) and amplicons that were equal to or greater in size than those predicted from the recognized 5' terminus of the HCV genome were cloned into pBluescriptKS. Twelve clones were sequenced, including three that were identical and contained an additional eight nucleotides, namely CCCCCCCA. Thus, the HCV 5' untranslated region (UTR) is now recognized as comprising 349 nucleotides, although it is possible to speculate that these additional nucleotides are part of a second, as yet undetected, stem-loop at the extreme 5' terminus of the genome.