Abstract
A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae) dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs) as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8+ memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i) a heterologous model protein (GFP), (ii) a per se toxic protein (K28 α-subunit), and (iii) a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A). Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.
Highlights
Viral expression systems can be classified into three types based on the regulatory and/or structural viral component that drives protein expression: (i) plasmid-based vectors containing promoter elements from either pro- or eukaryotic viruses; (ii) infectious viral vectors in which the gene of interest is integrated into the viral genome and expressed from a viral promoter in an appropriate host; (iii) virus-like particles (VLPs), called pseudovirions, representing subunit structures composed of multiple copies of a viral capsid and/or envelope protein capable to self-assemble into VLPs of defined spherical symmetry in vivo [1,2,3]
Chimeric Gag assembles into yeast VLPs
Electron micrograph of recombinant Gag/EstA particles prepared from yeast were purified by sucrose gradient centrifugation, negatively stained with uranyl acetate/methyl cellulose and subsequently used for electron microscopy. (B) Linear correlation between the 4nitrophenol concentration of up to 1 mM and its absorption at 405 nm. (C) Kinetics of Gag/EstA-driven hydrolysis of 4-nitrophenylacetate (280 mM) to 4-nitrophenol and acetate at 25uC in PBS50 buffer. (D) Coomassie-Blue staining and western analysis of Gag/EstA particles before and after cata-lysis and recycling by ultracentrifugation
Summary
Viral expression systems can be classified into three types based on the regulatory and/or structural viral component that drives protein expression: (i) plasmid-based vectors containing promoter elements from either pro- or eukaryotic viruses; (ii) infectious viral vectors in which the gene of interest is integrated into the viral genome and expressed from a viral promoter in an appropriate host; (iii) virus-like particles (VLPs), called pseudovirions, representing subunit structures composed of multiple copies of a viral capsid and/or envelope protein capable to self-assemble into VLPs of defined spherical symmetry in vivo [1,2,3]. Most VLPs can be produced in large quantity in a heterologous host. Due to their particle structure and high molecular weight, VLPs can be purified in a preparative scale. A number of particle forming proteins tolerate insertion of foreign amino acid sequences without affecting in vivo self-assembly competence. Such chimeric or hybrid VLPs, exploited as platform for the display of antigenic determinants in a polyvalent manner, have already been shown to be promising candidates in the development of various subunit vaccines [5]
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