Structure-function analysis of UAR complementary sequences during dengue virus replication

DIEGO E. ALVAREZ; CLAUDIA V. FILOMATORI; ANDREA V. GAMARNIK

Oregon

Congreso; XXVI Annual Meeting of the American Society for Virology; 2007

Positive stranded RNA viruses have evolved different strategies to control the utilization of the viral genome in translation, RNA synthesis, and encapsidation. In the case of flaviviruses, cyclization of the viral genome is required during RNA amplification. Using dengue virus as a model, we recently reported a mechanism for minus strand RNA synthesis that involves a promoter element at the 5’ end of the viral RNA. This finding provided a molecular explanation for the need of communication between the 5’ and 3’ ends of the genome. Two pairs of complementary sequences, named 5’-3’CS and 5’-3’UAR, at the ends of the viral RNA, were found to be involved in RNA-RNA interactions and genome cyclization in vitro. Functional evidence supporting the role of 5’-3’ CS hybridization in the replication of West Nile, Kunjin, dengue, and yellow fever viruses were previously reported. Here, we analyzed the role of 5’-3’ UAR complementarity using a dengue virus replicon system encoding a luciferase fused to the viral non-structural proteins. A systematic mutational analysis of UAR sequences was performed. We designed series of mutants carrying substitutions either in 5’UAR or 3’UAR, or compensatory mutations in both 5’ and 3’ UAR sequences, without altering the predicted secondary structures of the RNA. The effect of these mutations on translation and RNA synthesis was assessed by transfection of replicon RNAs into BHK cells. Single mismatches within UAR complementary sequences decreased about 1000 fold viral RNA synthesis, without affecting translation of the input RNA. In most of the cases, compensatory mutations that restored 5’-3’ UAR hybridization rescued viral RNA replication. To further confirm these observations, we assayed the infectivity of recombinant DV RNAs carrying specific mutations within UAR sequences. Viruses recovered from the supernatant of transfected BHK cells were characterized by sequence analysis. Interestingly, RNAs carrying mutations at 5’ or 3’ UAR evolved in cell culture showing sequences that differ from the input RNA. Spontaneous reversions and pseudo-reversions occurred to partially restore UAR base pairings. Taken together, our results provide strong evidence of a functional role of 5’-3’ UAR interactions during DV RNA synthesis.