Friday, May 9, 2025
5/9/2025
Negative regulation of human antiviral RNAi by PACT - PROJECT SUMMARY Small interfering RNAs (siRNAs) processed from virus-produced double-stranded RNA (dsRNA) mediate potent antiviral immunity, often termed as antiviral RNA interference (RNAi), in fungi, plants and invertebrates. Antiviral RNAi is often initiated by dicer proteins which chop viral dsRNAs into siRNAs. Argonaut (AGO) proteins recruit dicer-produced siRNAs and use them as sequence guide to identify and destroy viral RNA transcripts with matching sequence. The destruction of target viral transcripts is mediated by the slicer activity of AGO proteins. dsRNA binding proteins (DRBPs) also contribute to antiviral RNAi by facilitating siRNA production or loading into AGO proteins. Currently, whether RNAi mediates antiviral immunity in mammals under physiological conditions is still under hot debate. Although the antiviral activity of mammalian RNAi is active in non-differentiated stem cells and newborn mice it becomes undetectable in differentiated cells and fully developed mice. The fact that both dicer and the AGO protein required for siRNA production remain functionally active in differentiated cells suggest that there is a regulatory mechanism that actively suppresses the antiviral activity of mammalian RNAi in differentiated cells. Currently, how this regulatory mechanism works remains largely unknown. Since key mammalian RNAi genes, such as those encoding dicer and Ago2, are required for the biogenesis or function of miRNAs, which regulate essential cell differentiation and division, it is impossible to knock out these genes to study the antiviral function of mammalian RNAi without causing cell growth arrest or lethality. By co-delivery of human dicer and Ago2, one of the human Ago proteins with slicer activity, the PI’s lab successfully reconstituted human antiviral RNAi in C. elegans. Importantly, the antiviral activity is further enhanced in the presence of a human TRBP transgene but appears to be suppressed in the presence of a human PACT transgene. Both TRBP and PACT are dsRNA-binding proteins sharing similar domain structure. Previous in vitro studies have demonstrated that whereas TRBP facilitates the processing of dsRNA by dicer PACT seems to inhibit dicer processing of dsRNA. These observations together with our finding suggest that TRBP and PACT conversely regulate the antiviral activity of mammalian RNAi and PACT may dominate the regulation, leading to suppressed antiviral activity under physiological conditions. Here we propose to study the negative regulation of human antiviral RNAi by PACT in C. elegans. Findings from the proposed research may not only allow us to gain insight into the mechanism by which mammalian antiviral RNAi is regulated in differentiated cells but also facilitate the development of novel therapeutic strategies for viral infection prevention.
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