Increased infectivity of hepatitis A virus cDNA clones with engineered 5'-terminal extra-cistronic sequences.
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.
Silveira Carneiro
J
J
Dept. Cellular & Developmental Biology, University of Rome, Italy.
Bucci
M
M
Pierangeli
A
A
Equestre
M
M
Pagnotti
P
P
Pérez Bercoff
R
R
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D018076:DNA, Complementary / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry*; D005814:Genes, Viral / Q000502:physiology*; D006506:Hepatitis A / Q000821:virology*; D006507:Hepatovirus / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009693:Nucleic Acid Hybridization; D016366:Open Reading Frames / Q000502:physiology; D016133:Polymerase Chain Reaction; D015347:RNA Probes / Q000737:chemistry; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D014162:Transfection; D015678:Viral Structural Proteins / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics
D018076:DNA, Complementary; D004279:DNA, Viral; D015347:RNA Probes; D012367:RNA, Viral; D015678:Viral Structural Proteins