Record:   Prev Next
Author Reichert, Erin Donohue
Title Construction and characterization of a Dengue virus type 2 subgenomic replicon and analysis of the 3'untranslated region
book jacket
Descript 314 p
Note Source: Dissertation Abstracts International, Volume: 67-05, Section: B, page: 2359
Adviser: Radhakrishnan Padmanabhan
Thesis (Ph.D.)--Georgetown University Medical Center, 2006
To study the requirement of RNA elements in dengue virus translation and replication, a bicistronic subgenomic replicon was created by fusing a Renilla luciferase (Rluc) gene in-frame with the viral capsid (C) coding region followed by an Encephalomyocarditis virus (EMCV) Internal Ribosome Entry Site (IRES) to drive translation of the viral nonstructural (NS) proteins, NS1 to NS5. BHK-21 cells transfected with replicon RNA were positive for NS5 expression and viral RNA synthesis. To quantify translation and replication, BHK-21 cells were transfected with either replication-competent or replication-deficient replicon RNA. Rluc activity was observed over time with activity at 2 hours post-transfection correlating with translation of the input RNA and activity at 96 hours post-transfection correlating with replication of the input RNA. Using the replicon system, the requirement of conserved elements in the 3'-untranslated region (3'UTR) was evaluated. Deletion of TL2 with the associated stem region did not affect translation or replication. Deletion of TL1 with the associated stem region did not affect translation, but affected replication. Deletion of both TL1 and TL2 with the associated stem regions resulted in a complete loss of replication without affecting translation. Disruption of either base-pairing that might be involved in formation of predicted pseudoknot structures, PK1 and PK2, did not affect translation and an increase in Rluc activity was observed from 2 hours to 96 hours, implying that the mutations did not affect replication. However, disruption of both PK1 and PK2 simultaneously affected replication without affecting translation to an appreciable extent. To evaluate the requirement of 3'-terminal nucleotides for efficient translation and replication in vivo, point mutations were engineered into the four 3'-terminal nucleotides of the replicon. Mutating any of the four 3'-terminal nucleotides had an effect on replication without affecting translation to an appreciable extent. A cell line that persistently expressed replicon RNA was constructed using a replicon that expressed both Rluc and Neomycin (Neo). Transfection of BHK-21 cells with replicon RNA followed by exposure to G418 resulted in a cell line that persistently expressed replicon RNA. This cell line was positive for Rluc activity, replicon RNA synthesis, and NS5 expression
School code: 0544
Host Item Dissertation Abstracts International 67-05B
Subject Biology, Microbiology
Biology, Molecular
Alt Author Georgetown University Medical Center
Record:   Prev Next