It is well established that RNA molecules in the cell interact with a myriad of factors. Our overarching hypothesis is that viral frameshifting RNAs can be also regulated both directly and indirectly by cellular factors such as RNA binding proteins or non-coding RNAs during an infection. Our research questions focus on what regulatory proteins or other factors interact with frameshifting complexes, how they control translation pathways during viral infections and how these processes can be directly modulated. Ultimately, we aim to use this knowledge for designing antivirals targeting frameshifting RNAs during translation of viral RNAs. In the first funding period, we have established a frameshift interactome capture assay and discovered host encoded RNA-binding factors that interact with the frameshift RNAs in HIV-1 and SARS-CoV-2. Through these efforts, we identified several host-encoded candidate proteins that interact with the frameshift element. In one of the studies, we identified a human interferon-induced protein called the short isoform of the zinc-finger antiviral protein ZAP-S, as the direct regulator of frameshifting on SARS-CoV-2. ZAP-S is an RNA binding protein that specifically interact with the frameshift stimulating RNA pseudoknot of the SARS coronaviruses. During translation of the viral RNA, the interaction of ZAP-S with the frameshift RNA alters the stability of the secondary structure, which cannot fold into the pseudoknot essential for frameshifting. Ultimately, at high levels of ZAP-S, frameshift rates decrease, which lead to a drop in the viral polymerase level and consequently impede viral replication (Zimmer and Kibe et al., Nat. Comm., 2021).
Our mechanistic studies of the RNA interactions relies on using a combination of structural, biochemical and single molecule analysis tools. Using this toolset we have dissected interaction principles of frameshift RNA elements in unprecedented detail. We also integrated infection assays to study the functions of the trans-acting factors and the interplay between frameshift RNAs and the host and viral factors. Our interdisciplinary approach represents a benchmark to study the emerging concept of protein-mediated frameshifting events, which can open doors for new immune modulatory and antiviral intervention strategies. Highlights of our work are summarized below.
In the upcoming period, we will further develop our assays to directly visualize how cis and trans-acting elements interact with the translation apparatus in real-time.
