Elucidating the antiviral mechanisms of OAS family innate immune sensors.

About This Grant

Project Summary/Abstract Viral pandemics remain a major threat to human health due to their capacity to transmit and cause disease globally. There is an urgent need to define the mechanisms by which viruses interface with our immune system and how our cells initiate antiviral responses. The OAS-RNase L innate immune pathway is an essential component of the antiviral response that restricts viral replication by sensing double-stranded RNA (dsRNA) and initiating RNA degradation. Oligoadenylate synthetase (OAS) proteins sense dsRNA and produce oligoadenylate molecules that activate RNase L to initiate robust RNA decay. Each catalytic OAS (OAS1, 2, and 3) is canonically thought to bind dsRNA and activate RNase L, although emerging evidence suggests each OAS protein may have non-redundant antiviral functions. Recent studies have revealed that OAS3 acts as the primary activator of RNase L and mediates this activation via condensation on dsRNA. In contrast, OAS1 and OAS2 do not often condense on dsRNA but rather bind and aggregate viral single-stranded RNA (ssRNA), potentially eliciting antiviral activity independent of RNase L. Additionally, OASL, a non-catalytic OAS, localizes to ribonucleoprotein granules, suggesting a potential role in RNA metabolism or translational control. Thus, there is an urgent need to define how each OAS elicits antiviral activity. This F31 fellowship proposal seeks to uncover the molecular mechanisms and RNA substrates recognized by each OAS protein in activating innate immune responses. The overarching hypothesis is that OAS3 recognizes dsRNA through multivalent interactions with dsRNA, while OAS1, OAS2, and OASL recognize ssRNA substrates and elicit antiviral activity independent of RNase L through unique protein-protein interactions. This hypothesis will be tested through three complimentary aims: 1) solve conformation of full-length OAS3 bound to dsRNA via structural studies and identify subcellular location of sensing via TEM, 2) identify the ssRNA sequence motifs recognized by OAS1, OAS2, and OASL during viral infection by using CLIPseq, and 3) define the OAS interactomes during infection using TurboID-based proximity labeling and mass spectrometry. These studies will characterize the molecular mechanisms of condensation, distinct RNA substrates recognized by OAS proteins, and the interactome of each OAS. The proposed experiments will be performed at the UF Scripps Research Institute which houses state-of-the-art facilities and a collaborative research environment. Access to advanced instruments and guidance from an experienced multidisciplinary teams of mentors will ensure the successful execution of the proposed training plan. The strong mentorship and collaborative setting will provide the ideal foundation for developing the technical and professional skills needed for a career as an independent researcher.