Yeasts are widely used model microorganism for virus research. In addition, some yeasts own double-stranded RNA viruses that are considered as non-infectious since no naturally occurring extracellular route of transmission has been determined [1]. Yeasts are also an attractive platform to synthesize virus-like particles (VLPs) due to numerous advantages such as capability for post-translational modifications, high yield of recombinant protein and simple cultivation procedure. The majority of virus-like particles were biosynthesized in S. cerevisiae expression systems followed by P. pastoris and H. polymorpha. In the past several years, significant changes occurred in virus-like particle biosynthesis strategies including multilayered VLPs or VLPs produced of more than one type of structural protein [2].
In previous work, the dsRNA virus TdV-1 of the Partitiviridae virus family was identified in nonconventional yeast Torulaspora delbrueckii. The partitiviruses are typically isolated from filamentous fungi, protozoan and plant hosts [3]. The Partitiviridae virus forms non-enveloped particles in the range of 25 nm to 43 nm in diameter. Therefore, TdV-1 is the first virus to be discovered in non-filamentous fungi, belonging to the Partitiviridae family. This study aimed to characterize the properties of the capsid protein encoded by TdV-1 virus.
To produce TdV-1 capsid protein, yeast expression plasmids encoding recombinant TdV-1 capsid protein were constructed and protein biosynthesis in S. cerevisiae has been performed. Protein analysis confirmed that TdV-1 recombinant capsid protein biosynthesis occurs in S. cerevisiae. According to transmission electron microscopy (TEM) analysis, the recombinant TdV-1 capsid protein is capable to form virus-like particles of 30 nm in diameter. To evaluate TdV-1 virus-like particle assembly capabilities, capsid protein was fused with the fluorescent protein mCherry and biosynthesis of modified recombinant capsid protein was performed in S. cerevisiae. Microscopy analysis of TEM demonstrated that modified TdV-1 capsid protein is able to assemble into VLPs, thus mCherry fluorophore does not interfere with VLPs assembly. To identify TdV-1 recombinant capsid protein C-terminus localization in VLP, it was labeled with 6xHis affinity tag and immobilized metal affinity chromatography purification was carried out. Protein fraction profile analysis suggested that C-terminus of TdV-1 capsid protein is located on the inner surface of VLP.