Assembly of tomato blistering mosaic virus-like particles using a baculovirus expression vector system

Abstract

The expression of several structural proteins from a wide variety of viruses in heterologous cell culture systems results in the formation of virus-like particles (VLPs). These VLPs structurally resemble the wild-type virus particles and have been used to study viral assembly process and as antigens for diagnosis and/or vaccine development. Tomato blistering mosaic virus (ToBMV) is a tymovirus that has a 6.3-kb positive-sense ssRNA genome. We have employed the baculovirus expression vector system (BEVS) for the production of tymovirus-like particles (tVLPs) in insect cells. Two recombinant baculoviruses containing the ToBMV wild-type coat protein (CP) gene or a modified short amino-terminal deletion (Δ 2-24 CP) variant were constructed and used to infect insect cells. Both recombinant viruses were able to express ToBMV CP and Δ 2-24 CP from infected insect cells that self-assembled into tVLPs. Therefore, the N-terminal residues (2-24) of the native ToBMV CP were shown not to be essential for self-assembly of tVLPs. We also constructed a third recombinant baculovirus containing a small sequence coding for the major epitope of the chikungunya virus (CHIKV) envelope protein 2 (E2) replacing the native CP N-terminal 2-24 amino acids. This recombinant virus also produced tVLPs. In summary, ToBMV VLPs can be produced in a baculovirus/insect cell heterologous expression system, and the N-terminal residues 2-24 of the CP are not essential for this assembly, allowing its potential use as a protein carrier that facilitates antigen purification and might be used for diagnosis. Handling Editor: Sead Sabanadzovic.

Bacterial expression systems based on Tymovirus-like particles for the presentation of vaccine antigens

“…Turnip yellow mosaic virus crystallization experiments by Canady et al (1996) demonstrated that the N-terminal 26 amino acids of the CP A subunit are disordered, forming flexible chains (Barnhill et al, 2007), and are not essential for VLP self-assembly even after removing the first 26 amino acids of the N-terminus of the CP, resulting in no interference in either particle formation or infectivity (Powell et al, 2012). The same phenomenon was observed for PhMV CP VLPs expressed in Escherichia coli (Sastri et al, 1997(Sastri et al, , 1999 and for Tomato blistering mosaic virus (ToBMV)-like particles in a baculovirus expression system (Vasques et al, 2019), suggesting the high potential of the N-terminal end position as a possible antigen presentation locus. Taking this into account, several authors have demonstrated successful epitope insertion in the N-terminal ends of PhMV CP expressed in E. coli cell cultures (Hema et al, 2007; Chandran et al, 2009;Shahana et al, 2015) or model sequence insertions in TYMV CPs using agroinfiltration in Nicotiana benthamiana leaves (Shin et al, 2013(Shin et al, , 2018, all resulting in the formation of Tymovirus-like particles.…”

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

“…Similar to that in E. coli , this compartment exhibits the lowest degree of proteolytic activity and the purification of the potentially soluble structurally accurate protein can be significantly improved due to the presence of a few contaminating bacterial proteins. Unfortunately, this technique is not feasible for the large-scale processing of heterologous proteins in E. coli [ 120 ]. Proteins that are supposed to be secreted into the extracellular medium may traverse two separate membrane boundaries, namely, the cytoplasmic and the outer membranes [ 121 ].…”