Dengue virus genome plasticity and host-specific functions of viral RNA structures

VILLORDO S *; FILOMATORI C * EQUAL CONTRIBUTION TO THIS WORK; GAMARNIK A

Boston

Simposio; Positive strand RNA-viruses. Keystone Symposia; 2013

Keystone Symposia

Dengue virus genome is a single stranded RNA molecule of positive polarity. We have previously defined conserved RNA structures that serve as promoters, enhancers and silencers of viral replication. These elements are mainly located at the viral 5´ and 3´ UTRs and they communicate by long range RNA-RNA interactions. Also, an absolute requirement for RNA replication of two mutually exclusive RNA structures was found, indicating that the viral RNA is a dynamic molecule, which acquires alternative conformations during RNA synthesis. More recently, studying replication of recombinant dengue viruses in mosquito and human cells, we identified a sequence essential for viral replication in mosquito cells but dispensable in human cells. Based on these observations and taking into account that dengue virus cycles in nature between mosquitoes and humans, we performed a systematic study to define functions and mechanisms of RNA structures in the two hosts. Unexpectedly , successive passages of dengue virus in mosquito cells resulted in possitive selection of genomes with large deletions and mutations. These changes yielded viruses with replication capacities about 50-fold higher than the parental virus. In contrast, the same viruses replicate in human cells with lower fitness than the parental virus. Deep sequencing analysis from different viral populations, together with SHAPE analysis, indicated that viral adaptation to mosquito cells is associated to drastic changes of defined RNA structures. Interestingly, the parental RNA structures were quickly recovered in culture after switching to human cells. We are currently investigating the mechanism by which these RNA elements provide a host specific advantage. Our results highlight the plasticity of the dengue virus genome during host adaptation and provide novel information about functions of dengue virus RNA structures in mosquito and human cells.