New clues to flavivirus replication
Scientists find first 5' promoter element in dengue genome, perhaps explaining why virus loops before being copied
For the first time, researchers have found
a promoter element in a flavivirus -- specifically, the dengue virus
genome. The 5' location of the promoter, revealed in this month's Genes & Development
, may explain why the genome of this mosquito-transmitted pathogen must form a loop before it can be copied, opening up new avenues for researchers to explore in developing antiviral therapeutics.
"There's this emerging concept in RNA virus replication in which the promoter for RNA synthesis sits on the 5' end of the genome and the genome sort of loops back into the 3' end of the genome to deliver [replication machinery]," said Raul Andino
at the University of California, San Francisco. The paper employs a "very diverse set of approaches to very solidly demonstrate this elegant gymnastics" in a new class of viruses, he said.
RNA-dependent RNA polymerases (RdRps) encoded in the genomes of certain RNA viruses help catalyze their rapid replication, enabling viruses to make up to 10,000 copies within a few hours. Researchers were in the dark, however, about how RdRps can distinguish viral RNA from the cellular mRNA of the virus's host.
For this study, Andrea Gamarnik
and her team at the Fundacion Instituto Leloir in Buenos Aires compared RNA synthesis of whole and truncated dengue genomes. In radio-labeled replication assays, the researchers found the 5' untranslated region (UTR) of the genome template was far more critical for replication than the 3' UTR. Using a series of mutations and deletions, they narrowed down a putative promoter element to a large stem loop in the 5' UTR.
This is an unusual location, according to Gamarnik, since the RdRp must begin RNA synthesis at the 3' end of the genome, "thousands of nucleotides apart from the [putative] promoter." But last year, her group found
that the dengue genome forms a circle when it replicates, similar to polio, rotavirus, and other viruses. In the case of dengue, the virus forms the loop by matching up complementary sequences on its 5' and 3' UTRs. These ends might be coming together to bring the RdRp from its recruitment site to where it can begin doing its job, they reasoned.
Atomic force microscopy revealed the interaction between the RdRp and looped RNA in detail. "One picture is very powerful to provide a sense of what is going on at a molecular level," said Andino, not a co-author. When the researchers transfected cells with the virus, they found that those without the intact promoter could not replicate, even if their genomes could form the proper loop -- suggesting the 5' promoter is likely playing a key role in the process. A series of other biochemical and genetic tests reaffirmed these results.
In RNA viruses like dengue with plus-stranded genomes, genomic RNA is a template for translation as well as for RNA replication. Based on the researchers' findings, RNA polymerases likely compete with ribosomes to start their work at the 5' end, said John Patton
at the National Institute of Allergy and Infectious Diseases, who did not participate in this study. The dengue virus genome may use this competition to control when it is replicated or translated, Patton explained, by allowing polymerases to edge out ribosomes or vice versa; Gamarnik and Andino have observed
this pattern in poliovirus.
What's more, the loop requirement "ensures that the only [genomes] that are replicated are full length," explained Eva Harris
at the University of California, Berkeley, also not a co-author. "That's another check mechanism to make sure that what's actually being packaged is capable of going through the complete infectious process."
"Because the essential RNA signals found for dengue virus replication are also present in other flavivirus genomes, such as yellow fever, West Nile, and Japanese encephalitis virus, it is likely that our findings can be extrapolated to other members of this family," Gamarnik wrote in an E-mail.
Gamarnik added that her team is now trying to understand exactly how the polymerase binds to the promoter and passes from the 5' to 3' end, as well as how it is regulated. Researchers may eventually build on these findings to develop antiviral therapeutics that interfere with RNA synthesis by, for instance, blocking the 5' promoter, Harris added.
Links within this article
C.V. Filomatori, "A 5' RNA element promotes dengue virus RNA Synthesis on a Circular Genome," Genes Dev
, 20:2238-49, 2006.
T. Toma, "Original sin and dengue fever," The Scientist
, June 16, 2003.
D.E. Alvarez, et al., "Long-range RNA-RNA interactions circularize the dengue virus genome," J. Virol.
, 79:6631-43, 2005.
A.V. Gamarnik and R. Andino, "Switch from translation to RNA replication in a positive-stranded RNA virus," Genes Dev.
, 12:2293-304, 1998.