Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic viruslike particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody

Virus-derived vectors for the expression of multiple proteins in plants

This chapter constitutes a practical guide to using the “pEAQ” vector series for transient or stable expression of one or more protein(s) in Nicotiana benthamiana plants. The pEAQ vectors are a series of small binary vectors designed for controlled expression of multiple proteins in plants. To achieve high levels of expression, an expression system based on translational enhancement by the untranslated regions of RNA-2 from cowpea mosaic virus (CPMV), named CPMV- HT, is used. The expression vector pEAQ- HT combines the user-friendly pEAQ plasmid with CPMV- HT to provide a system for high-level expression of proteins in plants

Bluetongue Virus Particles as Nanoreactors for Enzyme Delivery and Cancer Therapy

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Self-assembly of filamentous virus-like particles (VLPs) through transient expression in N. benthamiana of the coat protein (CP) of different sweet potato viruses

Trabajo presentado al International Advances In Plant Virology, celebrado on-line del 20 al 22 de abril de 2021.Peer reviewe

Engineering recombinant virus-like nanoparticles from plants for cellular delivery

Understanding capsid assembly following recombinant expression of viral structural proteins is critical to the design and modification of virus-like nanoparticles for biomedical and nanotechnology applications. Here, we use plant-based transient expression of the Bluetongue virus (BTV) structural proteins, VP3 and VP7, to obtain high yields of empty and green fluorescent protein (GFP)-encapsidating core-like particles (CLPs) from leaves. Single-particle cryo-electron microscopy of both types of particles revealed considerable differences in CLP structure compared to the crystal structure of infection-derived CLPs; in contrast, the two recombinant CLPs have an identical external structure. Using this insight, we exploited the unencumbered pore at the 5-fold axis of symmetry and the absence of encapsidated RNA to label the interior of empty CLPs with a fluorescent bioconjugate. CLPs containing 120 GFP molecules and those containing approximately 150 dye molecules were both shown to bind human integrin via a naturally occurring Arg-Gly-Asp motif found on an exposed loop of the VP7 trimeric spike. Furthermore, fluorescently labeled CLPs were shown to interact with a cell line overexpressing the surface receptor. Thus, BTV CLPs present themselves as a useful tool in targeted cargo delivery. These results highlight the importance of detailed structural analysis of VNPs in validating their molecular organization and the value of such analyses in aiding their design and further modification

Lessons from icosahedral and flexuous viral structures of whitefly-transmitted members of the genera Torradovirus and Ipomovirus

Trabajo presentado en la III Reunión de la Red Nacional de Virología de Plantas, RENAVIPLANT, celebrada en Murcia entre el 25 y el 27 de mayo de 2022.Virus-like particles (VLPs) have been used to understand the requirements for virion formation and to solve the structure of many plant viruses. For this purpose, plant transient expression systems have been successfully adopted (e.g.: pEAQ- and pEff-system). We explored the possibility of producing VLPs of two whitefly-transmitted viruses: i) tomato apex necrosis virus (ToANV, genus Torradovirus, family Secoviridae), which has icosahedral virions and causes infection in tomato crops; and ii) cucumber vein yellowing virus (CVYV, genus Ipomovirus, family Potyviridae), which has flexuous virions and causes infection in cucurbit crops. The pEAQ-vector system was initially used to produce the capsid protein (CP) of CVYV and the three CPs of ToANV in Nicotiana benthamiana plants. Successful expressions were confirmed by Western Blot (WB) with the corresponding specific anti-CP antibodies. However, with this system we were only able to confirm VLP production for ToANV by transmission electron microscopy (TEM). Thus, for CVYV we explored the possibility of using the replicative vector, pEff, which allows assembly of the CP on replicating RNA. The system allowed accumulation of CVYV CP, confirmed by WB, and the production of VLPs, confirmed by TEM. In addition, a high-resolution structure of ToANV VLPs was determined using cryo-electron microscopy (Cryo-EM) and compared with that of another aphid-transmitted member of the same family, broad bean wilt virus 1 (BBWV-1, genus Fabavirus, family Secoviridae). The novel structural information will be used for biotechnological applications, and to explore the mechanisms of transmission of these three viruses.Peer reviewe

Tandem core technology.

<p>a) The structure of a monomeric HBc VLP with one HBcAg dimer shown in a surface representation coloured yellow and green. b) Two HBcAg sequences fused together via a flexible linker makes a tandem core construct, with either full-length (hetero-tandem) or truncated (homo-tandem) C-terminus, and two modifiable major insertion regions (MIRs). c) Structure of a tandem core protein: N-terminal core 1 (in green) is fused via a flexible linker (red) to C-terminal core 2 (yellow). The two views are related by a 90° rotation.</p

Cryo-EM analysis of plant-produced CoHe-GFPL VLPs.

<p>a) Particles were flash-frozen in vitreous ice, then subjected to cryo-electron microscopy. Class averages were obtained from 441 individual particles using EMAN software. The expanded view (lower right) is of an average of all images used. b) 3D reconstruction of the particles using icosahedral symmetry, superimposed on the He map as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120751#pone.0120751.g003" target="_blank">Fig. 3</a>. The CoHe-GFPL map is coloured red-to-blue from the centre of the volume towards its edge; the He map is shown in grey.</p

Cryo-EM of plant-produced τGFP bound with GFP.

<p>a) Class averages computed using Relion of the τGFP particles. b) A 3D reconstruction (resolution estimate 25Å using the “gold standard” cross-FSC at cutoff 0.143) coloured by distance from the centre of the particle (red to blue). The map is shown viewed down a 5-fold axis with the He reconstruction on which the construct was based fitted within (grey surface). The projecting spikes represent density arising from the bound nanobody and GFP but do not occupy every position expected, instead appearing as an average of the density present with the highest intensity at the 2-fold (pseudo- 6-fold) axes and also at the 5-fold axis. These spikes are to some extent artefacts of the icosahedral symmetry imposed on the maps, but are reflected in the spikes also shown in the unaveraged class averages shown in a).</p