2006 Ohio Student Research Forum

Abstract

HSV-1 Works to Trigger and Abrogate an Interfering RNA Response in Cells
Suzanna Jones
Ohio State University
Department of Molecular Genetics
Mentor: Dr. Deborah Parris

RNA interference (RNAi) is an evolutionary-conserved process that regulates gene expression. RNAi pathways are involved in sequence-specific gene silencing and are triggered by double-stranded RNA precursor molecules. These duplex RNAs are processed by the type III endoribonuclease Dicer into 21-bp to 24-bp RNA duplexes known as short-interfering RNAs (siRNAs). Specific siRNAs are incorporated into a ribonucleoprotein complex known as RNA-induced silencing complex (RISC) that guides a strand of the siRNA to its complementary target mRNA for degradation. In plants, gene silencing by RNAi is an innate defense against viruses and retrotransposons. In fact, all pathogenic plant viruses have developed mechanisms of evading the RNAi pathway by encoding suppressor proteins that enable them to interfere with various steps in the RNAi response pathway. Previous studies with mammalian cells have shown that introducing synthetic 21-bp duplex siRNAs into cells can trigger an RNAi response by targeting complementary sequences for degradation, indicating that RNAi pathways are active in these cells. However, it remains unknown whether mammalian cells utilize RNAi as an antiviral defense. If so, then successful mammalian viruses would be expected to encode suppressors of RNAi. Previous work in our laboratory has shown that HSV-1 encodes a protein, US11, that blocks the ability of plants to silence via the anti-viral RNAi pathway, suggesting that HSV-1 can trigger and abrogate an interfering RNAi response in mammalian cells. In this study, we sought to determine the importance of the RNAi pathway in limiting virus replication. Specifically, we examined whether knock-down of the RNAi pathway would result in an increased yield of progeny virus produced in mammalian cells. To knock-down an important component of the RNAi pathway, we utilized synthetic siRNAs directed to Dicer. Vero cells were transfected with these inhibitory Dicer-siRNAs or control short duplex RNAs and then infected with wild-type HSV-1 or a mutant virus deleted for US11 at a multiplicity of 0.1 plaque forming units per cell. After one-cycle of infection (18 hours), the amount of progeny virus was determined with plaque assays. The results suggest that there was no significant difference for the yields of progeny produced in control vs Dicer siRNA-transfected cells for either the wild-type or mutant virus.