Labeling Ebola Virus with a Self-Splicing Fluorescent Reporter.

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Inteins are protein segments that excise themselves from precursor proteins via protein splicing.
• This process joins flanking protein sequences (exteins) and has applications in protein engineering.
• Heterologous proteins can be inserted into inteins, allowing for co-expression of additional coding sequences.

Purpose of the Study:

• To demonstrate the insertion of a fluorescent protein (ZsGreen) into an intein for creating a novel reporter system.
• To develop a method for generating recombinant fluorescent viruses without additional gene expression.
• To investigate the feasibility of using intein-mediated protein splicing for reporter gene insertion in viral systems.

Main Methods:

• Engineered the *Pyrococcus horikoshii* RadA intein with a ZsGreen insertion (ZsG-Int).
• Identified an optimal insertion site within the Ebola virus VP30 gene for ZsG-Int.
• Generated a recombinant Ebola virus expressing the ZsG-Int-VP30 fusion protein and assessed its fluorescence and splicing efficiency in mammalian cells.

Main Results:

• The ZsG-Int hybrid protein retained both fluorescence and protein splicing capabilities.
• Efficient intein splicing and VP30 protein function were maintained at the identified insertion site within the Ebola virus VP30 gene.
• Recombinant Ebola virus expressing the ZsG-Int-VP30 fusion protein displayed fluorescence in infected cells.

Conclusions:

• Intein-based insertion of reporter proteins offers a novel strategy for generating fluorescent viruses without requiring separate reporter genes.
• This approach facilitates reporter expression only upon successful translation of the target protein.
• The developed system demonstrates a new application of inteins in protein engineering and viral reporter development.