Engineering of a Sagiyama alphavirus RNA-based transient expression vector

Area of Science:

• Virology
• Molecular Biology
• Biotechnology

Background:

• Sagiyama virus (SAGV), an alphavirus, has a wide host range but is not pathogenic to humans.
• Alphavirus vectors are valuable tools in molecular biology and biotechnology.
• Transient expression vectors offer advantages for protein production and research.

Purpose of the Study:

• To engineer the Sagiyama virus (SAGV) genome into an efficient transient expression vector.
• To evaluate the expression efficiency of a reporter gene (GFP) using the engineered SAGV vector.
• To assess the suitability of SAGV-based pseudovirions for large-scale protein expression.

Main Methods:

• Engineered the SAGV genome using a full-length infectious cDNA clone (pSAG2).
• Inserted a green fluorescent protein (GFP) gene as a reporter, expressed from a subgenomic mRNA.
• Utilized a nonviable mutant construct (pSAG2.3L) as a helper for pseudovirion formation.
• Inoculated BHK21 and C6/36 mosquito cells with pseudovirions for GFP expression analysis.

Main Results:

• Efficient autoproteolysis released GFP from fused capsid-protease domains.
• High-level GFP expression was achieved in both BHK21 and C6/36 cells.
• The SAGV vector demonstrated efficient expression without a 5'-terminal capsid protein region, indicating no translation enhancer sequence.
• Pseudovirion inoculation yielded up to 50 pg of GFP from 1 X 10(6) BHK21 cells.

Conclusions:

• The engineered SAGV is an efficient and safe transient expression vector.
• SAGV-based pseudovirions facilitate high-level protein expression in mammalian and insect cells.
• The safety profile of SAGV offers an advantage over other alphavirus expression vectors for biotechnological applications.