Engineering of a Sagiyama Alphavirus RNA‐Based Transient Expression Vector

• DOI: 10.1111/j.1348-0421.2002.tb02668.x
• Publication Date: 2013-11-14T15:35:10Z
• Authors: Yuka Yamaguchi; Yukio Shirako
• Keywords: 0301 basic medicine; Luminescent Proteins; 0303 health sciences; 03 medical and health sciences; Insecta; Genetic Vectors; Green Fluorescent Proteins; Ross River virus; Animals; Cloning, Molecular; Cell Line; 3. Good health

AbstractSagiyama virus (SAGV), a strain of Getah virus in the genus Alphavirus in the family Togaviridae, has a broad host range in vertebrates and invertebrates but is not pathogenic for humans. We engineered the SAGV genome as an efficient transient expression vector using the full‐length infectious cDNA clone pSAG2 as the background. A green fluorescent protein (GFP) gene was used as a reporter gene and expressed from a subgenomic mRNA. When the GFP gene was placed downstream of the intact capsid protein gene or an internally deleted capsid protein gene encoding the N‐terminal 9 amino acids and C‐terminal 149 amino acids, autoproteolysis occurred efficiently at the boundary site to release GFP from the N‐terminally‐fused capsid‐protease domain. GFP was also expressed efficiently without the 5′‐terminal region of the capsid protein gene, suggesting that SAGV capsid protein gene does not have a translation enhancer sequence. To provide structural proteins for pseudovirion formation, a nonviable mutant construct, pSAG2.3L, which contains a Gly‐to‐Leu substitution at the − 2 position of the nsP3/4 cleavage site, was used as a helper. GFP was expressed up to 50 μg from 1 × 106 BHK21 cells after inoculation of pseudovirions. The C6/36 mosquito cell was also a suitable host for a large scale expression of GFP using pseudovirions. In addition to high‐level transient expression, safeness of SAGV should give an advantage over other alphavirus expression vectors.