Cis acting RNA elements present at the 5'UTR of dengue virus genome

LODEIRO, FERNANDA AND GAMARNIK, ANDREA

Madison USA

Congreso; 25th Annual Meeting of the American Society; 2006

Cis acting RNA elements present at the 5’UTR of dengue virus genome Lodeiro, M. Fernanda and Gamarnik, Andrea. Fundación Instituto Leloir, Argentina. mflodeiro@leloir.org.ar Dengue virus belongs to the Flaviviridae family together with other important human pathogens. Dengue is an enveloped virus with a positive single stranded RNA genome of about 11 kb that encodes a single ORF. The viral genome is flanked by 5’ and 3’ untranslated regions (UTRs) of 100 and 450 nucleotides, respectively. The 5’ UTR has a cap structure at the 5’ end and the 3’ lacks a poly(A) tail, but contains a number of conserved RNA structures. In order to understand molecular details of viral replication, we analyzed the role of RNA sequences and structures present at the 5’UTR of the viral genome. The predicted secondary structure of the 5’UTR is highly conserved among the different DV isolates. This structure consists of a large stem-loop (SLA) that includes a side loop, and a second short stem-loop (SLB), which terminates in the translation initiator AUG. We performed mutations within these two RNA elements, SLA and SLB, in the context of a full-length cDNA clone of DV type 2. Recombinant infectious RNAs were generated and transfected into BHK cells. Infectivity of the RNAs was assessed by IFA and characterization of recovered infectious viruses. We found that mutations within SLA that changed the structure of the stem abolished viral replication. However, reconstitution of base pairing, regardless of the sequence, restored viral replication. In addition, mutations at the top loop of SLA yielded non-replicating RNAs that evolved in culture generating revertant viruses. Sequence analysis and characterization of the revertant viruses revealed an absolute requirement of specific sequences within this RNA element. In the case of SLB, mutations that disrupted the structure also abolished viral replication, but in contrast to SLA, reconstitution of the stem was not sufficient to rescue viral replication, indicating that the nucleotide sequence within SLB is also required. It is important to mention that within SLB there is one of the two complementary sequences previously identified to be required for flavivirus genome cyclization (5’UAR). To further investigate the role of each RNA element of the 5’UTR during the viral life cycle, we are currently analyzing each step of viral replication introducing mutations within SLA and SLB in the context of genomic and subgenomic replicons.