Role of RNA structures present at the 3′UTR of dengue virus on translation, RNA synthesis, and viral replication

Abstract

We have developed a dengue virus replicon system that can be used to discriminate between translation and RNA replication. Using this system, we analyzed the functional role of well-defined RNA elements present at the 3'UTR of dengue virus in mammalian and mosquito cells. Our results show that deletion of individual domains of the 3'UTR did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. We demonstrated that complementarity between sequences present at the 5' and 3' ends of the genome is essential for dengue virus RNA synthesis, while deletion of domains A2 or A3 within the 3'UTR resulted in replicons with decreased RNA amplification. We also characterized the vaccine candidate rDEN2Delta30 in the replicon system and found that viral attenuation is caused by inefficient RNA synthesis. Furthermore, using both the replicon system and recombinant viruses, we identified an RNA region of the 3'UTR that enhances dengue virus replication in BHK cells while is dispensable in mosquito cells.

Dengue virus type 3 vaccine candidates generated by introduction of deletions in the 3′ untranslated region (3′-UTR) or by exchange of the DENV-3 3′-UTR with that of DENV-4

“…The DENV 3'-UTR is believed to play a key role in the regulation of viral replication by virtue of controlling RNA synthesis [35]. Both viral and cellular factors are known to bind to the 3'-UTR, and these interactions appear to regulate the efficiency of RNA replication [34].…”

“…Both viral and cellular factors are known to bind to the 3'-UTR, and these interactions appear to regulate the efficiency of RNA replication [34]. There is some evidence suggesting that the attenuating effects of deletion mutations in the 3'-UTR are mediated by a disruption in secondary structure that results in a decrease in the efficiency of RNA replication [35]. Studies by Men et al first characterized a panel of deletion mutations of varying length in the 3'-UTR of DENV-4 [33].…”

“…The only insight into the molecular mechanism by which the Δ30 mutation confers attenuation upon the DENV comes from a study of DENV replicons. Alvarez et al found that a replicon containing the 3'-UTR with the Δ30 deletion had 50-to 100-fold decreased RNA replication in BHK or C6/36 cells [35]. Further studies are needed to discern the structural basis for the divergent effects observed for deletion mutations in DENV-3 versus DENV-4.…”

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

“…As with the other positive strand RNA viruses, the 5′NCR and 3′NCR of DENV are predicted to form secondary RNA structures and cyclization which interact with cellular and viral proteins for viral protein translation (Ali and Siddiqui, 1995) and RNA genome replication (Guesdon et al, 2001;Kuhn et al, 2002). Sequences of 5′NCR together with capsid protein gene and 3′NCR are required to structurally interact during virus replication (Alvarez et al, 2005;You et al, 2001). The functional significance of dengue 5′NCR is demonstrated by the restricted growth of DENV-4 deletion mutants (Cahour et al, 1995), attenuation effect of the DENV-2 PDK-53 vaccine candidate (Butrapet et al, 2000), and reduced virus replication of DENV-2 5′NCR mutants (Leardkamolkarn et al, 2010;Sirigulpanit et al, 2007).…”

“…DENV-2 New Guinea C replicons containing GFP or luciferase reporter stabilized in BHK-21 cells were established for antiviral screening and siRNA testing (Ng et al, 2007). DENV-2 16681 replicon containing luciferase reporter was created and used to study 3′NCR structure and an anti-viral compound targeting the 3′NCR (Alvarez et al, 2005;Holden et al, 2006). In this study, we reported various strategies in constructing DENV-2 replicons with GFP reporter that provided a convenient and fast system to investigate the effects of 5′NCR mutations in viral replication.…”