Abstract
The 579-nucleotide 5′ untranslated region (5′UTR) of the Rhopalosiphum padi virus (RhPV) possesses a cross-kingdom internal ribosome entry site (IRES) activity that functions in insect, mammalian, and plant-derived in vitro translation systems, and six TAAG motifs within the DNA fragment encoding the RhPV 5′UTR were previously found to confer the RhPV 5′UTR with late promoter activity in baculovirus. In the present study, various truncated RhPV 5′UTR sequences were produced, and among them, a fragment of 110 bp ranging from nucleotides 309 to 418 was identified to be the shortest fragment responsible for the late promoter activity in baculovirus infected Sf21 cells. This 110 bp fragment contains a TAAG tandem repeat that retains more than 60% of the late promoter activity of the full length RhPV 5′UTR sequence. Further, IRES activity remained unchanged in all truncated RhPV 5′UTR constructs. Taken together, this novel 110 bp fragment having late promoter activity in baculovirus as well as IRES activity in mammalian cell, renders it a useful tool for the development of a “shuttle” bi-cistronic baculovirus gene expression and/or delivery vector.
Highlights
Translation in eukaryotes may be achieved in a cap-dependent or a cap-independent manner.An unusual cap-independent translation initiation mechanism is mediated by an RNA element termed an internal ribosomal entry site (IRES)
Tandem Repeated TAAG Motifs Are Responsible for the Promoter Activity of Rhopalosiphum padi virus (RhPV) internal ribosome entry site (IRES) in Baculovirus Infected Sf21 Cells
In order to characterize which of the six TAAG motifs mediated the promoter activity, truncated RhPV 5′ untranslated region (5′UTR) sequences were produced by making stepwise deletions from either ends; these truncated sequences were subject to functional promoter assays
Summary
Translation in eukaryotes may be achieved in a cap-dependent or a cap-independent manner. Interesting, we have shown that the cDNA of the RhPV 5′UTR IRES possesses six TAAG motifs and can act as a cryptic promoter activity in baculovirus-infected Sf21 cells [19]. We demonstrate that a small fragment in the RhPV 5′UTR IRES cDNA, corresponding to nt 309–418, retains significant IRES activity in CHO cells, and possesses baculovirus-dependent promoter activity in Sf21 insect cells. This novel transcription and translation coupled activity may be attributed to (1) tandem repeat TAAG motifs that can act as a baculovirus late or very late promoter transcription initiation and (2) the low level of. Secondary structure within this region, with only 40%–60% nucleotides being base-paired [16], which may permit access to ribosomes and protein factors for internal translation initiation
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