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The Dengue Virus Unplugged

al from cellular RNA, to replicate the right molecule?

Last year, Gamarnik got her first hint when her group identified two RNA sequences located at the ends of the dengue virus genome. These short sequences interact during RNA replication, shaping the viral RNA genome into a circle. Gamarnik's team published those findings in the June 2005 issue of the Journal of Virology.

Further studies of the dengue virus life cycle revealed another piece of the virus's RNA that recruits the enzyme RdRp. Found at one tip of the genome, that sequence adopts a characteristic stem-loop structure that the scientists suspected might be important to its function.

To test whether RdRp was relying on that stem-loop shape to recognize the viral RNA, the scientists created copies of the dengue genome with minor changes that would alter its structures. The mutated RNAs were then inserted into mosquito cells or hamster cells to see if the viral RNA would be copied.

To their surprise, the scientists found that the stem-loop or SLA sequence is essential for viral replication. Changes in even one or two building blocks in this structure were enough to halt the replication process. 'That told us that RdRp probably discriminates the viral RNA by recognizing SLA,'Gamarnik said.

To confirm the vital link between RdRp and SLA, the researchers allowed virus particles that couldn't replicate to evolve in cells grown in lab dishes. Spontaneous mutations that occurred in the SLA often restored RdRp's activity and full viral replication capacity.

The scientists didn't expect to find that RdRp activity relies on a sequence at the far end of the genome, thousands of nucleotides away from the end where the enzyme begins copying the RNA.

The new discovery makes sense, Gamarnik said, because the circular shape adopted by the virus brings the distant ends of its genome together. At first we were puzzled by the cyclizati
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