Multi-Gene Recombinant Baculovirus Expression Systems: From Inception to Contemporary Applications

Abstract

Many protein expression systems are primarily utilised to produce a single, specific recombinant protein. In contrast, most biological processes such as virus assembly rely upon a complex of several interacting proteins rather than the activity of a sole protein. The high complexity of the baculovirus genome, coupled with a multiphase replication cycle incorporating distinct transcriptional steps, made it the ideal system to manipulate for high-level expression of a single, or co-expression of multiple, foreign proteins within a single cell. We have developed and utilised a series of recombinant baculovirus systems to unravel the sequential assembly process of a complex non-enveloped model virus, bluetongue virus (BTV). The high protein yields expressed by the baculovirus system not only facilitated structure-function analysis of each viral protein but were also advantageous to crystallography studies and supported the first atomic-level resolution of a recombinant viral protein, the major BTV capsid protein. Further, the formation of recombinant double-shelled virus-like particles (VLPs) provided insights into the structure-function relationships among the four major structural proteins of the BTV whilst also representing a potential candidate for a viral vaccine. The baculovirus multi-gene expression system facilitated the study of structurally complex viruses (both non-enveloped and enveloped viruses) and heralded a new generation of viral vaccines.

Keywords: baculovirus expression system; bluetongue virus; multi-gene transfer vectors; virus assembly; virus-like particle viral vaccines.

Figure 1

Baculovirus Dual Gene Transfer Vector for CLP Expression. General organisation of vectors pAcVC2 and pAcVC3. Red arrows represent polyhedron promoter (Pph) and indicate the direction of transcription. Viral genes are represented by coloured blocks. (A) pAvCV2 contains genes expressing the LCMV-N and polyhedrin proteins. (Adapted from [8]). (B) In pAvCV3, the genes expressing LCMV-N and polyhedrin have been replaced with genes expressing the VP7 and VP3 of BTV (adapted from [6]). (C) EM of empty BTV CLPs comprised of the two major core proteins VP3 and VP7, expressed from a dual recombinant baculovirus (Adapted from [7]).

Figure 2

Baculovirus Quadruple and Quintuple Gene Transfer Vector. General organisation of vectors pAcAB4 and pAcBT. Arrows represent promoters and indicate the direction of transcription. Blue arrows represent polyhedron promoter (Pph) and red arrows represent p10 promoter (Pp10). Boxes represent transcriptional termination signals with the polyhedron (ph) termination signal in blue, the SV40 termination signal in green and the p10 termination signal in yellow. (A) In pAcAB4, the restriction sites relative to the promoter and termination signals are indicated along with the four BTV genes inserted into the pAcAB4 vector (Adapted from [18]). (B) In pAcBT, the five BTV genes inserted into the pAcBT vector are indicated, adjacent to their relevant promoters and termination signals (Adapted from [19]).

Figure 3

Modification of Genomic Bacmid DNA for Multi-gene Expression. (A) Diagram of AcMNPV genome showing additional genetic loci identified within the baculovirus genome which could support high-level expression of foreign genes. (B) Recombinant baculoviruses were selected using a bipartite selection cassette comprising LoxP sites (purple boxes) flanking a Zeocin resistance gene (Zeo^R) and LacZα marker (blue boxes). Foreign genes (orange box) were inserted between the p35 promoter (P_p35) and the polyhedrin polyadenylation sequences (T_ph) (white boxes) (adapted from [21]).

Figure 4

Comparison of 3D Cryo-EM Structures. Comparison of authentic virus and core structures with recombinant double-shelled VLPs and single-shelled CLPs. Recombinant proteins form structures which mimic the BTV core and virion particles. The BTV VP2 trimers are represented on the virion (A) and VLP (B) structures in red or cyan, respectively, and the VP5 trimers are yellow in both images. The VP7 trimers are represented on the core (C) and CLP (D) in yellow or blue, respectively, and the VP3 layer is coloured blue or green, respectively (Adapted from [24,25]).

Figure 5

Diagram of Proposed VP7 Assembly Pathway. VP7 trimers are labelled P, Q, R, S and T. In the proposed model of core assembly, multiple sheets of VP7 form at different nucleation sites. A process which is initiated by a single VP7 trimer (‘T’) occupying the ‘preferred’ site for assembly on the VP3. The ‘T’ trimer becomes the most tightly attached trimer, which is subsequently followed by the progressively weaker attachment of four more VP7 trimers (R-Q-S-P) which ‘fill the gaps’ (Adapted from [27]).

Figure 6

Serum Neutralising Antibody Responses Following Immunisation with Recombinant VP2 or Different Doses of Double-Shelled VLPs. (A) Plaque reduction serum antibody titres against BTV10 following immunisation with recombinant VP2 alone or doubled-shelled VLPs, demonstrating the difference in 50% titres. (Adapted from [7]) (B) Average serum neutralising antibody titres from sheep (n = 4) immunised with two doses of VLPs at 10, 50, 100 or 200 µg. At day 117, sheep were challenged with virulent BTV. No signs of BT disease or viremia were detected in any of the VLP-immunised animals (Adapted from [29]).

Figure 7

SARS-CoV-1 VLP Characterisation and Neutralising Antibody Responses. (A) Expression of S, M and E SARS-CoV-1 proteins in insect cells infected with a triple recombinant baculovirus, analysed by SDS-PAGE followed by Coomassie blue staining. (B) EM of immunogold-labelled SARS-CoV-1 VLPs probed with anti-S monoclonal antibody (adapted from [39]). (C) IC90 neutralising antibody titre against SARS-CoV-1 pseudotyped lentivirus, using sera from 3 mice immunised with SARS-CoV-1 VLPs, 1 mouse immunised with Rotavirus VLP and one serum obtained from a SARS-CoV-1 convalescent patient (adapted from [40]).

Figure 8

Design of Multi-gene Transfer Vector for SARS-CoV-2 VLP Expression and Efficacy of SARS-CoV-2 VLPs against Disease Associated Weight Loss following Heterologous Challenge. (A) Diagram of gene arrangement in the transfer vector used to generate the recombinant baculovirus expressing SARS-CoV-2 VLPs. The M protein was expressed from the p10 locus whilst the S and E proteins were expressed by back-to-back polyhedrin promoters (ph) within the ph locus. (B) Average weight loss of Syrian hamster groups following live virus challenge. Closed green circles represent VLP-immunised animals. Closed red circles represent the control animals (adapted from [41]).