| Summary: | 碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 100 === Abstract
Dengue virus is a positive stranded RNA virus. The 5’ and 3’ untranslated regions (5’ and 3’ UTRs) flanking viral RNA genome contain highly conserved RNA elements essential for viral replication. Stem-loop A (SLA) and stem-loop B (SLB) reside in 5’UTR. The small hairpin loop (sHP) and 3’ stem-loop (3’SL) near the 3’ terminus of viral genome are two of the stem-loop structures of 3’UTR. The terminal regions of viral genome contain three pairs of complementary sequences named as UAR (upstream AUG region), DAR (downstream AUG region), and CS (conserved region). The hybridization of these complementary sequences results in viral genome circularization, which could disrupt the base pairing interaction of the bottom region of 3’SL and bring the SLA (the promoter for viral RNA dependent RNA polymerase) and the 3’ terminus of viral genome in close proximity for viral negative stranded RNA synthesis. Interesting, 5’UAR overlaps with SLB and sHP is made from sequences of 3’UAR and 3’DAR. Previous study shows that the relative stability of the competing, mutually exclusive UAR and sHP structures modulates the efficiency of viral replication. Hence, the conversion between the circular and linear forms of viral genome appeared to be crucial for virus genome multiplication. In this thesis, mutagenesis analysis of the SLB and 5’UAR was performed in the context of a selectable dengue virus subgenome replicon. The effect of substitution mutation on viral RNA replication was evaluated by the ability of the mutant replicon to form stable cell colonies in the presence of selecting antibiotics. Suppressors of defective substitution mutation were screened and the function of selected suppressors in restoring the replication ability of the mutant replicon was evaluated. The alteration in SLB formation caused from the original mutation, the suppressor, or together was detected by the in-line probing of a dengue 5’ RNA fragment of 180 nt long. The findings of this study show that SLB is an essential RNA element although it can be replaced by an alternative stem-loop formed by 5’UAR sequence, and that UAR allows no more than one extra mismatched basepair to maintain the viral replication ability. Hence, both SLB and UAR are required RNA elements.
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