Sunday, May 18, 2025
5/18/2025
Exploiting pseudogenetic screens to unravel small DNA virus persistence - PROJECT SUMMARY Polyomavirus (PyV)-associated diseases arise in immunocompromised hosts and involve reactivation of viral replication from a persistent reservoir. However, little is known about the factors that control reactivation. There is an urgent need to fill in these gaps because developing a more thorough understanding of how viruses establish, maintain, and reactivate from persistent infection may yield information on effective treatment options for PyV disease and allow more patients to benefit from immunosuppressive therapies without the threat of developing PyV-associated diseases. Our long-term goal is to screen PyVs armed with genome-wide shRNA libraries. Our objective here is to optimize PyV shRNA expression and develop methods to conduct screens of shRNA-expressing PyVs. We hypothesize that PyV genomes can tolerate shRNA insertions and that these will be active during infection. The rationale for this proposed research is that the mechanism of PyV persistence/reactivation has been unresolved for decades, and that deciphering this is key to any true understanding of PyV biology. Further, such understanding may lead to new approaches for the prevention and treatment of PyV-associated disease as well as be informative more broadly to small DNA virus persistence. We plan to test our central hypothesis and complete the objectives outlined in this proposal via the following two specific aims: 1) Optimize PyV shRNA expression, and 2) Conduct proof-of-principle pilot screen on shRNA- armed PyVs. This application utilizes two recent advances including non-invasive assays of virus shedding over time and a proven combined shRNA molecular barcode strategy that monitors changes in the composition of a population of viruses. In Aim 1, varied shRNA backbone elements and genomic orientations are probed to generate the most active shRNA inserts with minimal impact on the PyV lifecycle. For the second aim, a pilot shRNA-armed PyV library targeting a subset of components of the type I interferon (IFN) pathway will be screened to assess altered acute and persistent shedding and persistent organ infection. Our contribution here is expected to be a proof-of-principle for shRNA library technologies for small DNA viruses, which will allow broader understanding of small DNA virus persistence and may lead to new approaches for preventing and treating PyV-associated diseases. This is expected to be significant because it will provide a foundation for future knowledge of translational importance regarding PyV-associated diseases while also helping to solve a long- standing mystery: How do small DNA viruses undergo long-term and in some cases life-long infections? The research proposed in this application is innovative because it focuses on arming individual viruses with shRNAs capable of dissecting the functional contribution of numerous host genes to acute and persistent infection in a single experiment format – a potentially powerful approach that has so far been absent in the study of small DNA viruses.
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