biostudies-literatureHume AJHHS | National Institutes of HealthHHS | National Institutes of Health (NIH)NIAID NIH HHSNIGMS NIH HHSe0098424https://www.ebi.ac.uk/biostudies/studies/S-EPMC11237716biostudies-literatureUnknown15(6)Intervening proteins (inteins) are translated as subdomains within host proteins and removed through an intein-driven splicing reaction where the flanking sequences (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 fluorescent protein 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 mBioImproved protein splicing through viral passaging.PMC11237716R01AI133486, R21AI169646R15GM143662, P20GM103436P20 GM103436R15 GM143662R21 AI169646R01 AI133486Connor JHHume AJSmetana JSMuhlberger ELennon CWTurcinovic JDeeney DJBelfort MfalseImproved 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 flanking sequences (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 fluorescent protein 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 2024-01-01T00:00:00Z2024 Jun2026-04-29T12:08:28.648Z2025-04-07T12:04:12.752ZS-EPMC112377163878026610.1128/mbio.00984-24