INVESTIGADORES
GAMARNIK Andrea Vanesa
artículos
Título:
RNA Sequences and Structures Required for the Recruitment and Activity of the Dengue Virus Polymerase
Autor/es:
CLAUDIA FILOMATORI; IGLESIAS GABRIEL; VILLORDO SERGIO; ALVAREZ DIEGO; GAMARNIK ANDREA
Revista:
JOURNAL OF BIOLOGICAL CHEMISTRY
Editorial:
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Referencias:
Lugar: Bethesda, Maryland; Año: 2011 p. 6929 - 6939
ISSN:
0021-9258
Resumen:
Dengue virus RNA-dependent RNA polymerase specifically
binds to the viral genome by interacting with a promoter element known as
stem-loop A (SLA). Although a great deal has been learnt in recent years about
the function of this promoter in dengue virus infected cells, the molecular
details that explain how the SLA interacts with the polymerase to promote viral
RNA synthesis remain poorly understood. Using RNA binding and polymerase
activity assays, we defined two elements of the SLA that are involved in
polymerase interaction and RNA synthesis. Mutations at the top of the SLA resulted in RNAs that retained the ability to bind
the polymerase but impaired promoter-dependent RNA synthesis. These results
indicate that protein binding to the SLA is not sufficient to induce polymerase
activity, and that specific nucleotides of the SLA are necessary to render an
active polymerase-promoter complex for RNA synthesis. We also report that
protein binding to the viral RNA induces conformational changes downstream of
the promoter element. Furthermore, we found that structured RNA elements at the
3 end of
the template repress dengue virus polymerase activity in the context of a fully
active SLA promoter. Using assays to evaluate initiation of RNA synthesis at
the viral 3UTR, we found that the RNA-RNA interaction mediated by 5-3UAR
hybridization was able to release the silencing effect of the 3SL structure.
We propose that the long-range RNA-RNA interactions in the viral genome play multiple
roles during RNA synthesis. Together, we provide new molecular details about
the promoter-dependent dengue virus RNA polymerase activity.