5' cap-independent translation of dengue virus genomic RNA

Abstract

Objetives. To analyze the involvement of methyl guanosine triphosphate cap (5’cap) and the start site of the genomic RNA of Dengue virus serotype 2 (DENV-2) American genotype in translation, using a cell-free system prepared from human placenta. Materials and methods. The recombinant plasmid pTZ18R-D2 was prepared containing DNA encoding the 5’UTR and the first 201 nucleotides of the viral capsid. This plasmid was used to transcribe the corresponding RNA (RNA-D2) without the 5’ cap. The RNA-D2 was translated in a system consisting of the postmitochondrial fraction (S-30) from human placenta and the  incorporation of [14C] aminoacids in the presence of RNA-D2 and in its absence (control) was evaluated. Seven antisense oligonucleotides (OAs1-7) directed against sequences of the SLA, SLB and CHP structures of RNA-D2 were designed and the effect thereof on RNA-D2 translation was analyzed. Results.The RNA-D2 produced a significant increase (p<0.001)
in the incorporation of [14C] amino acids, with 75% stimulation of translational activity compared to the control. Analysis of the translation products showed peak incorporation corresponding to peptides with apparent molecular weight close to the expected (7.746 kDa).The OAs5, complementary to a sequence of SLB structure of RNA-D2, completely inhibited translation. Conclusions. The RNA-D2 was translated specifically and efficiently under conditions similar to human intracellular conditions, by an alternative 5’ cap-independent mechanism, which would involve the SLB structure. This mechanism might be seen as an aim in the development of antisense therapies to inhibit virus replication.