INVESTIGADORES
ALVAREZ Diego Ezequiel
congresos y reuniones científicas
Título:
Structure-Function Analysis of UAR Complementary Sequences During Dengue Virus Replication
Autor/es:
DIEGO E. ALVAREZ; CLAUDIA V. FILOMATORI; ANDREA V. GAMARNIK
Lugar:
Corvallis, Oregon
Reunión:
Congreso; American Society for Virology 26th Annual Meeting; 2007
Institución organizadora:
American Society for Virology
Resumen:
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 5-3CS and 5-3UAR, 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 5UAR or
3UAR, 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.