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Viral siRNA Carriers Key to Powerful New Gene Silencing Technique

By BiotechDaily International staff writers
Posted on 25 Sep 2013
Image: A computer-generated model of the surface of an alphavirus derived by cryoelectron microscopy. Alphaviruses are RNA-containing viruses that cause a wide variety of mosquito-transmitted diseases (Photo courtesy of the [US] Centers for Disease Control and Prevention (CDC)).
Image: A computer-generated model of the surface of an alphavirus derived by cryoelectron microscopy. Alphaviruses are RNA-containing viruses that cause a wide variety of mosquito-transmitted diseases (Photo courtesy of the [US] Centers for Disease Control and Prevention (CDC)).
A screening technique based on viruses modified to carry discrete gene-silencing small interfering RNA (siRNA) molecules is being employed to follow the interaction between viruses and their natural host cells.

RNA interference (RNAi) has been extensively used to identify host factors affecting virus infection but requires exogenous delivery of siRNAs and the use of cell types, such as cancer cells or fibroblasts, which are easy to grow in culture. Results of such studies may not explain what is happening in cells that most viruses actually attack.

An improved RNAi technique was recently described by investigators at the Mount Sinai Medical Center (New York, NY, USA). They modified an alphavirus, a class of viruses with more than 10,000 family members, so that each family carried a distinct siRNA, which inhibited a single host gene and additionally acted as a molecular "barcode.” The viruses were used to infect mice by mimicking the natural route of infection. A week after virus infection, the investigators were able to pinpoint which viruses grew faster than others did, and could read the "barcode" that indicated which genes were silenced.

Results published in the September 11, 2013, online edition of the journal Cell Host & Microbe revealed that natural selection, defined by siRNA production, permitted the identification of host restriction factors through virus enrichment during physiological infection. Monitoring virus evolution across four independent screens identified two categories of enriched siRNAs: specific effectors of the direct antiviral arsenal and host factors that indirectly dampened the overall antiviral response.

“We have a powerful system [RNAi] in place today to investigate ways in which viruses interact with cells, which has yielded fundamental insights. But has significant limitations such as cost, difficulty of use, and the problem that the cells we have to use are not in any way physiologically relevant to the virus we want to study,” said senior author Dr. Benjamin tenOever, professor of medicine at Mount Sinai Medical Center. “The new system that we developed is much less costly, can be transferable from the study of one virus to another and, best of all, allows us to use the real virus in the real environment it infects.”

“We created a virus family identical in all respects, except that each member of the family carries a different siRNA,” said Dr. tenOever. “So in this swarm of viral soldiers, each one has a very small trick up its sleeve—it can silence one thing in the host cell and because of this, we can use the cells that viruses actually infect, such as lung cells. It could be used to generate cell cultures that allow enhanced vaccine production. You could improve the capacity of a therapeutic virus to get into a particular tissue, to kill tumor cells, or to chase after metastatic cancer cells. There is potentially no end to uses of this technology.”

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Mount Sinai Medical Center



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