999. siRNA-Mediated Interference of Double-Stranded RNA Binding Protein [E3L] Is an Effective Strategy to Inhibit Vaccinia Virus

Abstract

Top of pageAbstract Background: The Vaccinia virus E3L Interferon resistance gene encodes a 25kDa polypeptide which is synthesized early during the viral infection cycle. The polypeptide binds dsRNA and thus competes with the N-terminal dsRNA binding domain of protein kinase. This prevents PKR activation. However, siRNA-mediated down-regulation of E3L is hypothesized to eliminate the competition for dsRNA in favor of protein kinase leading to its activation and the subsequent cascade that establishes the anti-viral state. Methods: RNA interference (RNAi), a natural biological phenomenon mediated by small interfering RNA (siRNA) molecules, has been demonstrated to be an effective anti-viral strategy. However, no study thus far has utilized this emerging technology for Vaccinia virus, which is a useful surrogate for the Smallpox virus. In this study, we have used 21bp chemically synthesized siRNA duplexes whose sequences are derived from the E3L region of the Vaccinia genome and are conserved in smallpox and related poxviruses. Viral replication was assessed by measuring the |[beta]|-galactosidase activity in extracts prepared from infected HeLa cells. A recombinant strain of Vaccinia WR virus carrying the indicator gene was used. Production of infectious virus particles was measured by a plaque assay. Sonicated extracts from infected HeLa cells were used to quantify the amount of plaques generated on BSC-1 cells. Results: E3L-specific siRNAs exerted a potent antiviral effect on Vaccinia virus [WR] replication. Viral levels were reduced to below detection limits [MOI=0.01pfu/cell] and 7.6% of control infection [MOI=0.1pfu/cell]. Strong inhibition was observed even at extreme viral loads [15-30% of control infection at MOI=0.1-0.4pfu/cell]. Of the four E3L-specific siRNAs, E3L-C siRNA had the strongest effect [3.5% of control infection at MOI=0.1pfu/cell] followed by E3L-A siRNA [17.2% of control at MOI=0.1pfu/cell]. Silencing of E3L leads to a reduced production of infectious viral particles as well. When all the four E3L-specific siRNAs were multiplexed, infectious viral production was reduced to 4.5% of control infection [MOI=0.1]. Conclusions: The immediate potential use of the E3L-specific siRNA is as a biodefense tool against smallpox. These molecules seem to have a therapeutic potential that might be superior to the existing vaccination alternative. The value of these molecules has to be assessed in light of the morbidity associated with the small pox vaccine and potential complications that might be of concern for the immunocompromised population. Since dsRNA plays a crucial role in the generation of the antiviral state, the above described siRNA will be useful for dissecting the molecular mechanisms associated with this phenomenon.