PROJECT SUMMARY In the long-term we aim to understand the genetic, biochemical and structural basis for extreme virus function and stability. Sulfolobus Spindle-shaped Virus 1 (SSV1) is astonishingly stable to both high temperatures (>80°C) and low pH (3 and below). This stability is despite being composed of proteins and nucleic acids with similar compositions to viruses and other macromolecular complexes found in less extreme conditions. However, it is not clear how SSV1’s extreme capsid stability is determined. We have developed robust genetic tools for SSV1 and standardized techniques for working with these extreme viruses, and demonstrated their use in undergraduate research. Together, these tools and the recently-determined structure allow probing of the determinants of stability as follows. Determine the amino acids in the SSV1 major capsid protein VP1 that are important for thermal stability using site directed mutagenesis (Aim 1). We have determined that the spindle-shaped SSV1 virion is composed of seven intertwined helical strands of the major capsid protein, VP1. Using the predicted structure of VP1 and the virion structure, we will make site-directed mutations in 3 sets of amino acid side chains: those predicted to be in loops and glycosylated, those predicted to make interactions between strands and those predicted to form intramolecular interactions in VP1. These mutant viruses will be screened for activity at multiple temperatures. Use in vitro evolution to select SSV1 VP1 and VP3 mutants for increased thermal stability (Aim 2). In order to perform an unbiased screen of amino acid substitutions that could confer stability to SSV1 we will make a library of mutants that contain random substitutions in the major and minor capsid proteins VP1 and VP3. These mutants will be selected in vitro for their ability to maintain infectivity at 80°C and 90°C. Comparison of mutant libraries before and after selection with high throughput sequencing will be used to determine which substitutions are permitted and which, if any, stabilize the virus. Impact: Characterizing SSV1 mutants in vitro (Aim 1) and in vivo (Aim 2) will provide insight into fundamental aspects of protein and virus stability including glycosylation. The identification of amino acid substitutions that stabilize or destabilize SSV will help refine the SSV1 structural model. The results obtained in this project could facilitate the creation of highly stable SSV-based nanoparticles, methods to destabilize pathogenic viruses and produce stabilized vaccines. Finally, this project will allow Portland State University (PSU) students to engage in cutting edge biomedical research and strengthen the PSU research environment.