Graduation date: 2007
In this thesis, I studied the translation of dengue virus RNA using a luciferase reporter gene system in Vero cells. The dengue reporter mRNA construct, which harbors 5´-terminal viral nts and 3´-terminal nts, could be translated efficiently compared to an alpha globin reporter construct. The 5´-cap structure and 3´-untranslated region (UTR) of dengue virus RNA were found to contribute to strong enhancement of translation. The 5´-cap and 3´-UTR act synergistically to enhance translation like the 5´-cap and 3´-polyA tail of a cellular mRNA. Deletion analysis of each individual structural element in the dengue 3´-UTR indicated a cumulative effect in enhancing dengue translation. The cyclization sequences, which are present in the 5´ and 3´ portion of the genome to facilitate the end-to-end interactio, were tested for their importance in translation. The finding that a mutation of the sequence in the 3´-UTR, but not in the 5´, exhibited a deteriorating effect, indicates that cyclization of the genome via the sequences is not required for efficient dengue translation. The importance of a 5´-cap in dengue RNA translation was supported by using an inhibitor of cap-dependent translation, LY294002, whose presence significantly inhibited dengue reporter RNA translation in vivo. Dengue viral replication was also found to be sensitive to the presence of inhibitor, indicating that dengue viral translation does not take advantage of inhibition of cap-dependent cellular translation, unlike mRNA with an internal ribosomal entry site. Consistent with our finding of 5´-cap dependency of dengue RNA translation, introduction of a stable stem loop structure in front of the dengue reporter construct significantly inhibited translation, strongly suggesting that dengue RNA does not harbor an internal ribosome entry site.
To examine whether ribosomes scan through the 5´-UTR of dengue RNA, various upstream AUGs (uAUG) were introduced in the 5´-UTR of a dengue construct with a green fluorescent reporter protein. uAUG containing reporter constructs showed reduced translation from the authentic AUG in vivo and in rabbit reticulocyte lysate, suggesting that dengue translation is utilizing the conventional scanning mode of ribosome for AUG recognition. All together, dengue virus utilizes the cap-dependent scanning mode for translation initiation.