Establishing intein-based tools to study filovirus replication

The overall goals of this application are to harness the power of intein chemistry to invent useful technologies to study Ebola virus (EBOV), a deadly reemerging pathogen with case fatality rates of 40-90%. Inteins (intervening proteins) are translated within the context of other proteins and are removed through protein splicing. In this intein-mediated reaction, the two peptide bonds surrounding the intein are rearranged to release the intein from the flanking protein sequences, which are concurrently joined by a peptide bond. Because of this unique functionality, inteins have proven exceptionally useful in protein engineering. In one application, a heterologous protein can be inserted within an intein, with both the intein and the inserted protein retaining function. Leveraging this property, we recently developed an intein-based system for adding reporter genes to EBOV. In this self-removing reporter, a fluorescent protein is inserted within an intein, with the hybrid protein maintaining fluorescence and splicing capability. Importantly, the reporter does not remain fused to any viral protein or require an additional transcriptional unit, both which can disrupt viral growth. Recombinant EBOV containing this intein-based reporter displayed fluorescence and remained infectious, demonstrating in principle the validity of our reporter design. While a growth defect was observed for EBOV expressing this reporter, we found that serially passaged virus grew significantly better than the original recombinant virus. Excitingly, passaged virus had mutations that enhanced the catalytic properties of the intein itself. This proposal will both build upon recent discoveries as well as establish new lines of investigation into the use of inteins as tools to study viruses. Aim 1 will systematically evaluate the sequence and structural features of natural intein insertion sites, information that will be used to develop improved intein-based technologies for use in EBOV and other systems. Aim 1 will also develop additional intein-based reporters using our strategy of passaging recombinant EBOV. Intein-reporter fusions will be assessed by evaluating splicing efficiency, reporter activity, and viral protein function using E. coli and mammalian systems. Importantly, Aim 1 will directly compare our optimized intein-based reporters to traditional reporter virus approaches. As splicing must occur for intein-containing proteins to become active, Aim 2 will establish an intein-based "off-switch" that can be used to inactivate newly translated protein via inhibition of splicing. Using this strategy, a recombinant EBOV containing an off-switch within the transcriptional activator VP30 will be developed and used to investigate the role of VP30 during early viral replication. In principle, our innovative off-switch design could be extended to reversibly inactivate any newly translated protein. This work will establish new, widely applicable intein-based technologies that will be of immediate use for the study of EBOV mechanisms of pathogenesis. Further, as our strategy of passaging virus allows for the identification of intein variants with enhanced catalytic properties, our work will impact the broad community of researchers employing intein-based applications.