IIBBA   05544
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE BUENOS AIRES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Location and Function of Dengue Virus Cyclization Sequences
Autor/es:
SERGIO VILLORDO, CLAUDIA FILOMATORI, DIEGO ALVAREZ, AND ANDREA GAMARNIK
Lugar:
Vancouver, CANADA
Reunión:
Congreso; 28th Anual Meeting American Society of Virology; 2009
Resumen:
Location and Function of Dengue Virus Cyclization Sequences
Sergio Villordo, Claudia Filomatori, Diego Alvarez, and Andrea Gamarnik
Fundación Instituto Leloir, Av. Patricias Argentinas 435, Buenos Aires, Argentina
Dengue virus (DENV) belongs to the Flaviviridae family and represents an important human pathogen. It is estimated that more than 50 million infections occur annually worldwide. Dengue is an enveloped virus with a positive single stranded RNA genome of about 11 kb that encodes a single open reading frame (ORF). The viral genome is flanked by 5 and 3 untranslated regions (UTRs) of 100 and 450 nucleotides, respectively. Two pairs of complementary sequences, named 5-3CS and 5-3UAR, were previously found to be involved in long range RNA-RNA interactions, genome cyclization, and viral RNA amplification. 5UAR is located upstream of the translation initiator AUG and is complementary to a 17 nucleotide-long sequence located within the viral 3SL. In contrast, 5CS is located inside the capsid coding region and is complementary to an 11 nucleotide-long sequence located upstream of the 3SL. Because the location of these two pairs of complementary sequences is conserved in mosquito-borne flaviviruses, we investigated the functionality of these signals in different places of the viral genome.
First, to analyze the role of the location of 5 and 3 UAR in viral replication, we designed reporter DENVs encoding a luciferase gene in which the 5UAR was introduced inside of the ORF. In these constructs, the translation initiation context was maintained. Translation of these RNAs was as efficient as the parental RNA, while RNA synthesis was reduced 20-30 fold. In contrast, when the 3UAR was located outside of the 3SL, viral RNA synthesis was impaired. These results indicate that while the 5UAR was functional inside the ORF, the location of the 3UAR within the 3SL was crucial. In addition, viruses that contained a unique cyclization sequence, named DENVCS+, which was sufficient to mediate long range RNA-RNA interaction and RNA polymerase activity in vitro, were not competent for viral replication in transfected cells, suggesting a requirement of a bipartite cyclization sequence for DENV replication. These studies indicate that 5-3 RNA-RNA hybridization plays an additional role in DENV replication besides cyclization of the viral RNA, presumably modulating local secondary and tertiary structure of the viral genome.