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 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.

Detection of rice grassy stunt tenuivirus nonstructural proteins p2, p5 and p6 from infected rice plants and from viruliferous brown planthoppers

“…The interaction was further examined in vitro by farWestern blots. As a probe for these blots, p5.H (p5 with six His residues at the C terminus) was prepared by using the (18), which is a helper construct providing all of the structural proteins for pseudovirion formation, were electroporated into BHK21 cells as described previously (18). After incubation of electroporated cells at 30°C for 2 days, two types of pseudovirions-one containing the p5-expressing replicon RNA and another containing structural protein-expressing helper RNA-were passaged again in BHK21 cells at 30°C for 5 days to increase the titer of replicon RNA-containing pseudovirions.…”

Rice Grassy Stunt Tenuivirus Nonstructural Protein p5 Interacts with Itself To Form Oligomeric Complexes In Vitro and In Vivo

“…The interaction was further examined in vitro by far-Western blots. As a probe for these blots, p5.H (p5 with six His residues at the C terminus) was prepared by using the Sagiyama Alphavirus transient expression vector (18) in cultured BHK21 cells as follows. The p5 gene followed by six-His codons and a TGA termination codon was inserted in-frame downstream of the N-terminally-deleted capsid protein gene in the place of the GFP.H gene in pSAG2.⌬C:GFP.H (18), designated pSAG2.⌬C:p5.H.…”

“…The p5 gene followed by six-His codons and a TGA termination codon was inserted in-frame downstream of the N-terminally-deleted capsid protein gene in the place of the GFP.H gene in pSAG2.⌬C:GFP.H (18), designated pSAG2.⌬C:p5.H. In vitro transcripts from pSAG2.⌬C: p5.H and pSAG2.3L (18), which is a helper construct providing all of the structural proteins for pseudovirion formation, were electroporated into BHK21 cells as described previously (18). After incubation of electroporated cells at 30°C for 2 days, two types of pseudovirions-one containing the p5-expressing replicon RNA and another containing structural protein-expressing helper RNA-were passaged again in BHK21 cells at 30°C for 5 days to increase the titer of replicon RNA-containing pseudovirions.…”

Barley Yellow Mosaic Virus VPg Is the Determinant Protein for Breaking eIF4E-Mediated Recessive Resistance in Barley Plants

“…Western blot analysis was performed as described previously ( Yamaguchi and Shirako, 2002 ). Polyclonal antibodies against BaYMV CP (coat protein), P2, and barley eIF4E were used as the primary antibodies.…”