The genomic RNA of Hepatitis A virus (HAV), a picornavirus of the hepatovirus group, is a single-stranded molecule, ca. 7.5 kb in length of positive polarity. Translation of this uncapped RNA starts at the 10th (or 11th) AUG triplet (position 734-36), by a mechanism of internal initiation of translation. The long sequences extending between the uncapped 5'-end and the translation initiation site contain two (instead of just one) pyrimidine-rich tracts (PRTs) spanning nucleotides 94-140 and 711-724, respectively. The latter lies only 11 nucleotides upstream from the initiation site of translation, and the question arose as to whether the notoriously poor replication ability of HAV was a consequence of a down regulation of translation due to the too short "spacer" sequence intervening between the 3'-PRT and the initiation of the main open reading frame. To address this issue, a series of full-length HAV cDNA clones were constructed in which the "spacer" sequence (normally 11 nts) was brought to 45 nts. Following transfection of COS-1 cells with these constructs, the amount of HAV (+)-strand RNA was determined by dot hybridization using a strand-specific RNA probe. HAV cDNA clones carrying a 45-nt "spacer" increased two-fold the rate of (+)-strand viral RNA synthesis, suggesting that the poor translation ability of HAV RNA may be one of the mechanisms responsible for the lengthy replication cycle of HAV.