Engineering of a Sagiyama alphavirus RNA-based transient expression vector.

Abstract

Sagiyama 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 pg from 1 X 10(6) 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.