Js. Carneiro et al., INCREASED INFECTIVITY OF HEPATITIS-A VIRUS CDNA CLONES WITH ENGINEERED 5'-TERMINAL EXTRA-CISTRONIC SEQUENCES, The New microbiologica, 21(4), 1998, pp. 321-327
The genomic RNA of Hepatitis A virus (HAV), a picornavirus of the hepa
tovirus 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 i
nitiation of translation. The long sequences extending between the unc
apped 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 upstre
am from the initiation site of translation, and the question arose as
to whether the notoriously poor replication ability of HAV was a conse
quence of a down regulation of translation due to the too short ''spac
er'' sequence intervening between the 3'-PRT and the initiation of the
main open reading frame. To address this issue, a series of full-leng
th 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 de
termined by dot hybridization using a strand- specific RNA probe. HAV
cDNA clones carrying a 45-nt ''spacer'' increased two- fold the rate o
f (+)-strand Viral RNA synthesis, suggesting that the poor translation
ability of HAV RNA may be one of the mechanisms responsible for the l
engthy replication cycle of HAV.