INCREASED INFECTIVITY OF HEPATITIS-A VIRUS CDNA CLONES WITH ENGINEERED 5'-TERMINAL EXTRA-CISTRONIC SEQUENCES

Citation
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
Citations number
14
Categorie Soggetti
Microbiology
Journal title
The New microbiologica
ISSN journal
11217138 → ACNP
Volume
21
Issue
4
Year of publication
1998
Pages
321 - 327
Database
ISI
SICI code
1121-7138(1998)21:4<321:IIOHVC>2.0.ZU;2-H
Abstract
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.