Improved protein splicing through viral passaging

Intervening proteins (inteins) are translated as subdomains within host proteins and removed through an intein-driven splicing reaction where the flankingsequences (exteins) are joined with a peptide bond. Previously, we developed a self-removing translation reporter for labeling Ebola virus (EBOV). In this reporter, an intein (RadA) containing the fluorescentprotein ZsGreen (ZsG) is inserted within the EBOV protein VP30. Upon VP30-RadA-ZsG expression from the viral genome, RadA-ZsG is removed from VP30 through the protein splicing activity of RadA, generating functional, non-tagged VP30 and functional ZsGreen. While incorporation of our VP30-RadA-ZsG fusion reporter into recombinant EBOV (rEBOV-RadA-ZsG) resulted in an infectious virus that expresses ZsG upon infection of cells, this virus displayed a replication defect compared to wild-type EBOV, which might be the result of insufficientRadA splicing. Here, we demonstrate that the serial passaging of rEBOV-RadA-ZsG in human cells led to an increase in replication efficiencycompared to unpassaged rEBOV-RadA-ZsG. Sequencing of passaged viruses revealed intein-specificmutations. These mutations improve intein activity in both prokaryotic and eukaryotic systems, as well as in multiple extein contexts. Taken together, our findingsoffera novel means to select for inteins with enhanced catalytic properties that appear independent of extein context and expression system.