Display of whole proteins on inner and outer surfaces of grapevine fanleaf virus-like particles.

Lorène Belval,Catherine Reinbold,Claude Sauter,Corinne Schmitt‐Keichinger,Christophe Ritzenthaler,Léa Ackerer,Olivier Lemaire,Gérard Demangeat,François Berthold,Jean‐Daniel Fauny,Caroline Hemmer

doi:10.1111/pbi.12582

Abstract

SummaryVirus‐like particles (VLPs) derived from nonenveloped viruses result from the self‐assembly of capsid proteins (CPs). They generally show similar structural features to viral particles but are noninfectious and their inner cavity and outer surface can potentially be adapted to serve as nanocarriers of great biotechnological interest. While a VLP outer surface is generally amenable to chemical or genetic modifications, encaging a cargo within particles can be more complex and is often limited to small molecules or peptides. Examples where both inner cavity and outer surface have been used to simultaneously encapsulate and expose entire proteins remain scarce. Here, we describe the production of spherical VLPs exposing fluorescent proteins at either their outer surface or inner cavity as a result of the self‐assembly of a single genetically modified viral structural protein, the CP of grapevine fanleaf virus (GFLV). We found that the N‐ and C‐terminal ends of the GFLV CP allow the genetic fusion of proteins as large as 27 kDa and the plant‐based production of nucleic acid‐free VLPs. Remarkably, expression of N‐ or C‐terminal CP fusions resulted in the production of VLPs with recombinant proteins exposed to either the inner cavity or the outer surface, respectively, while coexpression of both fusion proteins led to the formation hybrid VLP, although rather inefficiently. Such properties are rather unique for a single viral structural protein and open new potential avenues for the design of safe and versatile nanocarriers, particularly for the targeted delivery of bioactive molecules.

Highlights

From a structural and biotechnological standpoint, virions can be viewed as self-assembled nanometre-scale cages or viral nanoparticles (VNPs) containing the viral genome
To address the ability of grapevine fanleaf virus (GFLV) capsid proteins (CPs) to produce virus-like particles (VLPs) in planta, the sequence encoding the CP from GFLV isolate F13 was introduced into the pEAQ-HT-DEST1 binary vector (Sainsbury et al, 2009) and used for transient expression in Nicotiana benthamiana leaves by agro-infiltration (Figure 1)
To test the ability of transiently expressed CP to self-assemble into VLPs, the same leaf extracts were further analysed by transmission electron microscopy (TEM) after immunocapture with GFLV-specific polyclonal antibodies

Summary

Introduction

From a structural and biotechnological standpoint, virions can be viewed as self-assembled nanometre-scale cages or viral nanoparticles (VNPs) containing the viral genome. As self-assembled cages, the inner cavity of VNPs can be used to encapsulate or encage a variety of active molecules, including pharmaceuticals, image enhancers and nucleic acids (Arcangeli et al, 2014; Bruckman et al, 2013; Mueller et al, 2011; Shriver et al, 2013) By acting on both external surface and inner cavity, VNPs can be adapted theoretically at will and are regarded as extremely versatile tools with great potentials in medicine, as enzyme nanocarriers or even as novel biomaterials (for review, see Reference Alonso et al, 2013; Cardinale et al, 2012; Pokorski and Steinmetz, 2010)

Results

Discussion

Conclusion