viruses
Review
MultiBac: Baculovirus-Mediated Multigene DNA
Cargo Delivery in Insect and Mammalian Cells
Kapil Gupta 1,2 , Christine Tölzer 1,2 , Duygu Sari-Ak 3 , Daniel J. Fitzgerald 4,
Christiane Schaffitzel 1,2 and Imre Berger 1,2,*
1 School of Biochemistry, Biomedical Sciences, University of Bristol, 1 Tankard’s Close, Bristol BS8 1TD, UK;
kapil.gupta@bristol.ac.uk (K.G.); ct17870@bristol.ac.uk (C.T.);
christiane.berger-schaffitzel@bristol.ac.uk (C.S.)
2 Bristol Synthetic Biology Centre BrisSynBio, University of Bristol, 4 Tyndall Ave, Bristol BS8 1TQ, UK
3 European Molecular Biology Laboratory EMBL, 71 Avenue des Martyrs, 38000 Grenoble, France;
duygusari05@gmail.com
4 Geneva Biotech SARL, 64 Avenue de la Roseraie, 1205 Genève, Switzerland; df@geneva-biotech.com
* Correspondence: imre.berger@bristol.ac.uk; Tel.: +44-117-394-1857
Received: 30 January 2019; Accepted: 21 February 2019; Published: 26 February 2019
Abstract: The baculovirus/insect cell system (BICS) is widely used in academia and industry to
produce eukaryotic proteins for many applications, ranging from structure analysis to drug screening
and the provision of protein biologics and therapeutics. Multi-protein complexes have emerged
as vital catalysts of cellular function. In order to unlock the structure and mechanism of these
essential molecular machines and decipher their function, we developed MultiBac, a BICS particularly
tailored for heterologous multigene transfer and multi-protein complex production. Baculovirus
is unique among common viral vectors in its capacity to accommodate very large quantities of
heterologous DNA and to faithfully deliver this cargo to a host cell of choice. We exploited this
beneficial feature to outfit insect cells with synthetic DNA circuitry conferring new functionality
during heterologous protein expression, and developing customized MultiBac baculovirus variants
in the process. By altering its tropism, recombinant baculovirions can be used for the highly efficient
delivery of a customized DNA cargo in mammalian cells and tissues. Current advances in synthetic
biology greatly facilitate the construction or recombinant baculoviral genomes for gene editing and
genome engineering, mediated by a MultiBac baculovirus tailored to this purpose. Here, recent
developments and exploits of the MultiBac system are presented and discussed.
Keywords: baculovirus; heterologous expression; multiprotein complex; human TFIID; GPCR;
virus-like particle VLP; transduction; drug screening; genome engineering; synthetic biology
1. Introduction
In 1983, Max Summers and his team reported the successful production of a heterologous protein,
human IFN-β, in insect cells by using a recombinant baculovirus [1]. They had modified the genome of
Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and transfected insect cell cultures
derived from the fall armyworm with the recombinant AcMNPV genome. Previously, they had
realized that a viral protein, polyhedrin, is expressed at very high levels very late in the viral life
cycle but was dispensable for maintaining an infectious virus in laboratory culture. By replacing the
polyhedrin gene with their heterologous gene of choice, Summers and his team could exploit the
machinery of the virus to drive the high-level expression of IFN-β which they could then purify [2].
This remarkable feat demonstrated the utility of baculovirus for heterologous protein production, and
the baculovirus/insect cell system (BICS) has since been used to produce many proteins, accelerating
research and development in laboratories world-wide, in academia and industry.
Viruses 2019, 11, 198; doi:10.3390/v11030198 www.mdpi.com/journal/viruses
Viruses 2019, 11, 198 2 of 14
Originally used for recombinant protein production, the scope of the technology experienced
significant expansion with the discovery that the tropism of the virus could be altered to transduce
mammalian cells, acting as a DNA delivery tool [3–5]. If genes were now placed under mammalian
active promoters, baculovirus could be manufactured in insect cells and then administered to
mammalian cells and even tissues or organisms to realize this particular genetic information,
introducing baculovirus as a gene therapy vector. These developments have been authoritatively
reviewed in numerous publications including this Special Issue in Viruses [2,6–11]. We will thus focus
here on our own contributions to baculovirus technology, the MultiBac system, which we introduced
some fifteen years ago [12–14]. We had the privilege over the years to contribute periodical reviews
about MultiBac developments and—to our delight—its increasingly widespread adoption in the
research community [13,15–24]. To forestall boring the audience, we will therefore restrict ourselves
to just briefly summarizing the essentials of MultiBac, and then proceed to highlight in the present
review the most recent exploits and developments, by us and others, of this baculoviral system we
have conceived.
2. The MultiBac BICS: Enabling Multiprotein Complex Structure Analysis
Our incentive to develop MultiBac originated from the emerging realization that proteins in
cells rarely function in isolation, but often accommodate in supramolecular assemblies with several
to many other proteins and other biomolecules to carry out their chores [25]. Elucidating how
these ensembles work at a molecular resolution necessitates methods to produce and purify them
in the quality and quantity required for structural and mechanistic studies. Some assemblies such
as ribosomes or proteasomes are prevalent in cells and can be purified from native source material.
Many others however are characterized by a low abundance requiring recombinant overexpression.
Recombinant baculovirus can express heterologous proteins at high levels. Further, it uses eukaryotic
insect cell culture as a host and can thus provide authentic post-translational modification which may
be important for the integrity and activity of a specimen studied. Furthermore, the narrow host range
of baculovirus requires only standard laboratory safety provisions. All these were good reasons to
choose baculovirus as the system to produce those essential molecular machines.
Baculovirus/insect cell systems were already available back then including tool-kits which
conveniently utilized a baculoviral genome in the form of a bacterial artificial chromosome (BAC),
originally developed by Luckow and colleagues [26]. This BAC is propagated in E. coli cells and relies
on Tn7 transposase mediated integration of the foreign gene of interest from a transfer plasmid into
the BAC [27]. This setup allowed us to reengineer the baculoviral genome in a rather straight forward
fashion in E. coli, eliminating undesired functions such as viral proteolytic and apoptotic activities and
adding advantageous modalities such as a site-specific recombination sequence (LoxP) to integrate
additional foreign genes distal from the Tn7 attachment site (Figure 1) [14,21].
Viruses 2019, 11, 198 3 of 14
Viruses 2019, 11, x FOR PEER REVIEW 3 of 14
Figure 1. The MultiBac system. MultiBac consists of an engineered baculoviral genome optimized for
multigene delivery and protein complex expression (left). The genome is present as a bacterial
artificial chromosome (BAC) in E. coli cells supplying the Tn7 transposase. Expression cassettes
comprising genes of interest and gene regulatory elements are assembled into the multi-gene
expression constructs and inserted into the MultiBac genome by Tn7 transposition. The Tn7
attachment site is embedded in a LacZα gene enabling blue-white screening of recombinants. A
second entry option into the viral backbone is provided distal from the Tn7 site, relying on Cre
recombinase catalysed site-specific integration into a LoxP sequence on the viral backbone (circle
filled in red). This site can be used to customize MultiBac by providing additional functionalities.
Composite MultiBac baculoviral DNA containing all DNA elements of choice is extracted from E.coli
cultures, followed by transfection into insect cell cultures to manufacture functional MultiBac virions.
These are then used for a wide range of applications (right), by the infection of insect cell cultures or
transduction of mammalian cells, tissues and organisms. Cryo-EM, electron cryo-microscopy; X-ray,
crystallography; BV, baculovirus; VLP, virus-like particle; SBDD, structure-based drug design; HCS,
high-content screening. Amp, ampicillin; Kan, kanamycin; F, bacterial F replicon; LacZα, gene
encoding B-galactosidase α fragment; mini-attTn7, minimal Tn7 attachment site. Virion image is
adapted from drawing kindly provided by Kari Airenne[28].
To facilitate the assembly of multi-gene expression cassettes, we developed recombineeringbased,
parallelized, automatable approaches relying on synthetic DNA plasmid modules that could
be conjoined into elaborate transfer plasmids for integration into the Tn7 and LoxP sites, respectively
[29–32]. We followed a ‘reduce to the max’ approach in designing these reagents, eliminating from
our plasmid modules DNA elements with unclear or, for our purposes unnecessary, functions
present in common plasmids, keeping only the bare minimum of DNA elements conferring defined
functions (origin, resistance marker, promoters and terminators), adding the specific integration
sequences we needed (LoxP, Tn7) and short DNA sequences for multiplying the expression cassettes.
This approach yielded a significantly streamlined, functional tool-box of DNA substantially
simplifying the construction of multifunctional recombinant baculovirus specimens which could be
customized with ease [16,24,33]. More recently we have extended this reductionist approach also to
the baculoviral genome itself, a considerable fraction of which appears to be dispensable for, or even
detrimental to, maintaining and deploying it in the laboratory [19,23]. Of note, all these developments
greatly profit from the remarkable ongoing reduction in cost of custom DNA synthesis. We anticipate
that in the near future, the cross-over may be reached to render commercial DNA synthesis a bona
Figure 1. The MultiBac system. MultiBac consists of an engineered baculoviral genome optimized for
multigene delivery and protein complex expression (left). The genome is present as a bacterial artificial
chromosome (BAC) in E. coli cells supplying the Tn7 transposase. Expression cassettes comprising
genes of interest and gene regulatory elements are assembled into the multi-gene expression constructs
and inserted into the MultiBac genome by Tn7 transposition. The Tn7 attachment site is embedded
in a LacZα gene enabling blue-white screening of recombinants. A second entry option into the
viral backbone is provided distal from the Tn7 site, relying on Cre recombinase catalysed site-specific
integration into a LoxP sequence on the viral backbone (circle filled in red). This site can be used to
customize MultiBac by providing additional functionalities. Composite MultiBac baculoviral DNA
containing all DNA elements of choice is extracted from E. coli cultures, followed by transfection
into insect cell cultures to manufacture functional MultiBac virions. These are then used for a wide
range of applications (right), by the infection of insect cell cultures or transduction of mammalian cells,
tissues and organisms. Cryo-EM, electron cryo-microscopy; X-ray, crystallography; BV, baculovirus;
VLP, virus-like particle; SBDD, structure-based drug design; HCS, high-content screening. Amp,
ampicillin; Kan, kanamycin; F, bacterial F replicon; LacZα, gene encoding B-galactosidase α fragment;
mini-attTn7, minimal Tn7 attachment site. Virion image is adapted from drawing kindly provided by
Kari Airenne [28].
To facilitate the assembly of multi-gene expression cassettes, we developed recombineering-based,
parallelized, automatable approaches relying on synthetic DNA plasmid modules that could
be conjoined into elaborate transfer plasmids for integration into the Tn7 and LoxP sites,
respectively [29–32]. We followed a ‘reduce to the max’ approach in designing these reagents,
eliminating from our plasmid modules DNA elements with unclear or, for our purposes unnecessary,
functions present in common plasmids, keeping only the bare minimum of DNA elements conferring
defined functions (origin, resistance marker, promoters and terminators), adding the specific
integration sequences we needed (LoxP, Tn7) and short DNA sequences for multiplying the expression
cassettes. This approach yielded a significantly streamlined, functional tool-box of DNA substantially
simplifying the construction of multifunctional recombinant baculovirus specimens which could be
customized with ease [16,24,33]. More recently we have extended this reductionist approach also to
the baculoviral genome itself, a considerable fraction of which appears to be dispensable for, or even
detrimental to, maintaining and deploying it in the laboratory [19,23]. Of note, all these developments
greatly profit from the remarkable ongoing reduction in cost of custom DNA synthesis. We anticipate
that in the near future, the cross-over may be reached to render commercial DNA synthesis a bona fide
economic alternative even to the streamlined current assembly approaches for constructing multigene
expression cassettes, which we and others have put in place [31,32,34,35].
Viruses 2019, 11, 198 4 of 14
In addition to developing the reagents, we worked out detailed, simple and user-friendly protocols
for the multi-gene expression cassette assembly, composite baculovirus generation, insect cell culture
maintenance as well as virus amplification and protein complex production. A particularly useful asset
was the incorporation of a gene encoding yellow fluorescent protein (YFP) into the backbone of our
engineered MultiBac baculovirus. This enables users to follow virus performance and heterologous
protein complex production simply by tracking the YFP signal, providing a quantitative means to
assess virus quality and protein product quantity, as well as variability from batch to batch. We
validated our system and communicated the MultiBac reagents and protocols we developed which
were immediately well received by the community, underscoring the existing need for heterologous
expression systems that could enable the high quality production of large multi-subunit protein
assemblies. The superior performance of our MultiBac system, compared to other existing baculoviral
systems, was compellingly demonstrated recently in an elaborate study undertaken by the network of
protein production core facilities in Europe (P4EU) [36]. In the meantime, a large and rapidly growing
number of multiprotein complexes have now been produced, by us and many others, unlocking
their exciting structures and mechanisms at high resolution by X-ray crystallography and electron
cryo-microscopy (cryo-EM), resulting in a host of high profile publications. A by no means exhaustive
selection of impressive structures reported only in the short time span since our last MultiBac review
in 2017 [19] is depicted in Figure 2, exemplifying the fast pace of the field, enabled in part by the
baculoviral multiprotein complex production tool-kit we contributed.
Viruses 2019, 11, x FOR PEER REVIEW 4 of 14
fide economic alternative even to the streamlined current assembly approaches for constructing
multigene expression cassettes, which we and others have put in place [31,32,34,35].
In addition to developing the reagents, we worked out detailed, simple and user-friendly
protocols for the multi-gene expression cassette assembly, composite baculovirus generation, insect
cell culture maintenance as well as virus amplification and protein complex production. A
particularly useful asset was the incorporation of a gene encoding yellow fluorescent protein (YFP)
into the backbone of our engineered MultiBac baculovirus. This enables users to follow virus
performance and heterologous protein complex production simply by tracking the YFP signal,
providing a quantitative means to assess virus quality and protein product quantity, as well as
variability from batch to batch. We validated our system and communicated the MultiBac reagents
and protocols we developed which were immediately well received by the community, underscoring
the existing need for heterologous expression systems that could enable the high quality production
of large multi-subunit protein assemblies. The superior performance of our MultiBac system,
compared to other existing baculoviral systems, was compellingly demonstrated recently in an
elaborate study undertaken by the network of protein production core facilities in Europe (P4EU)
[36]. In the meantime, a large and rapidly growing number of multiprotein complexes have now been
produced, by us and many others, unlocking their exciting structures and mechanisms at high
resolution by X-ray crystallography and electron cryo-microscopy (cryo-EM), resulting in a host of
high profile publications. A by no means exhaustive selection of impressive structures reported only
in the short time span since our last MultiBac review in 2017 [19] is depicted in Figure 2, exemplifying
the fast pace of the field, enabled in part by the baculoviral multiprotein complex production tool-kit
we contributed.
Figure 2. Multiprotein complex structures. Chromatin remodeling enzymes SWR1 (14 subunits) and
INO80 (11 subunits) were produced by using MultiBac, bound to nucleosomes and the molecular
structures determined by electron cryo-microscopy [37–42]. The yeast polII DSIF-PAF-Spt6 cryo-EM
structure utilized RNA polymerase II from native source reconstituted with recombinantly expressed
associated proteins [43]. Further recent structures of sample produced with the MultiBac BICS include
the CENPN-CENPA-nucleosome complex [44], the human CPSF-160–WDR33–CPSF-30–PAS RNA
Figure 2. Multiprotein complex structures. Chromatin remodeling enzymes SWR1 (14 subunits) and
INO80 (11 subunits) were produced by using MultiBac, bound to nucleosomes and the molecular
structures determined by electron cryo-microscopy [37–42]. The yeast polII DSIF-PAF-Spt6 cryo-EM
structure utilized RNA polymerase II from native source reconstituted with recombinantly expressed
associated proteins [43]. Further recent structures of sample produced with the MultiBac BICS include
the CENPN-CENPA-nucleosome complex [44], the human CPSF-160–WDR33–CPSF-30–PAS RNA
quaternary complex [45], the E9 polymerase [46], the USP18-ISG15 complex [47], the Separase-Securin
complex [48] and the cohesion loader Scc [49].
Viruses 2019, 11, 198 5 of 14
3. Mechanisms of Transcription Factor Complex Assembly
The impetus for our own team in putting the MultiBac system in place was derived from our desire
to unlock the structure, mechanism and cellular assembly of key multiprotein complexes in human gene
expression, with specific focus on human general transcription factors (GTFs) and the pre-initiation
complex (PIC) [50]. A central cornerstone of this elaborate machinery of more than a hundred proteins
is GTF TFIID, a megadalton-sized multiprotein complex comprising about 20 subunits made up of
the TATA-box binding protein (TBP) and its associated factors (TAFs) [50]. TFIID is the first factor to
bind the core promoter, thereby nucleating PIC formation. Notably the discovery of distinct TFIID
complexes, some of them confined to specific tissues, with different subunit composition, and the
observation that several TFIID subunits are shared with other regulatory complexes, imply a dedicated
assembly mechanism at work in cells [50]. The underlying mechanisms, however, remain poorly
understood. We are addressing these questions by producing partial and holo-TFIID complexes by
using MultiBac, determining structures by hybrid methods and scrutinizing cellular compartments
in vivo for functional subassemblies and their specific mechanisms which may shed light on TFIID
assembly pathways and the cellular factors involved (Figure 3) [50–54].Viruses 2019, 11, x FOR PEER REVIEW 6 of 14
Figure 3. Partial TFIID complexes and mechanisms of TFIID assembly. (a) The architecture of
TAF11/TAF13/TBP revealed an unexpected binding mode with the TAFs invading the DNA binding
groove of TBP [53]. A MultiBac baculovirus customized for genetic code expansion, MultiBac-TAG,
was utilized to probe the interaction interfaces by inserting UV-activatable amino acids for crosslinking
[55]. (b) A nuclear import particle (NIP) formed by TAF8, TAF10, TAF2 and importin is
shown, reconstituted from recombinant TAF proteins co-expressed by MultiBac and Importin
produced in bacteria [54]. The structures of TAF8/TAF10 and TAF8/Importin were determined by Xray
crystallography, a homology model of TAF2 is shown [50]. (c) X-ray crystallography combined
with mutational studies, proteomics and cell-based experiments revealed a crucial checkpoint
function of the adenylate-dependent chaperonin CCT in the formation of a TAF5/TAF6/TAF9
complex in early stages of human TFIID assembly. CCT captures nascent TAF5 as it emerges from the
ribosome, and hands-over folded TAF5 to a preformed TAF6/TAF9 complex in the cytoplasm [51].
4. G-Protein Coupled Receptor (GPCR) Structure and Mechanism
We observed early on that the alterations we had engineered into the MultiBac baculoviral
genome resulted in delayed lysis of cells upon infection with our recombinant virus. Baculovirus
takes over the host cell machinery upon infection and either shuts down or reprograms cellular
functions to ramp up virus production. Given that many cellular functions terminate, cells upon
infection can be looked at as enveloped micro-bioreactor devices rather than ‘living’ cells. Typically,
in BICS, heterologous protein expression is driven by viral promoters which are maximally active in
Figure 3. Partial TFIID complexes and mechanisms of TFIID assembly. (a) The architecture of
TAF11/TAF13/TBP revealed an unexpected binding mode with the TAFs invading the DNA binding
groove of TBP [53]. A MultiBac baculovirus customized for genetic code expansion, MultiBac-TAG, was
utilized to probe the interaction interfaces by inserting UV-activatable amino acids for cross-linking [55].
(b) A nuclear import particle (NIP) formed by TAF8, TAF10, TAF2 and importin is shown, reconstituted
from recombinant TAF proteins co-expressed by MultiBac and Importin produced in bacteria [54]. The
structures of TAF8/TAF10 and TAF8/Importin were determined by X-ray crystallography, a homology
model of TAF2 is shown [50]. (c) X-ray crystallography combined with mutational studies, proteomics
and cell-based experiments revealed a crucial checkpoint function of the adenylate-dependent
chaperonin CCT in the formation of a TAF5/TAF6/TAF9 complex in early stages of human TFIID
assembly. CCT captures nascent TAF5 as it emerges from the ribosome, and hands-over folded TAF5 to
a preformed TAF6/TAF9 complex in the cytoplasm [51].
We already determined by hybrid methods the architecture of a core-TFIID complex present as a
distinct, stable entity, possibly with own, specific gene regulatory functions, in the nuclei of cells [52].
Viruses 2019, 11, 198 6 of 14
Moreover, we identified hitherto unexpected modalities of TBP binding by two TFIID subunits, TAF11
and TAF13, in vitro and in vivo, hinting at substantial conformational dynamics within TFIID to
initiate PIC formation and ultimately transcription, possibly depending on the promoter context
(Figure 3a) [53]. In this study, we implemented genetic code expansion by engineering MultiBac-TAG,
our customized BICS to probe molecular interfaces by incorporating UV-activatable amino acids at
specific positions in the primary sequence of the proteins studied [53,55].
Probing the cytoplasm of HeLa cells, we discovered a nuclear import particle (NIP) formed
by TFIID subunits TAF2, TAF8, TAF10 and importin, implicating nuclear transport as a regulatory
mechanism for holo-TFIID assembly (Figure 3b) [54]. To balance the stoichiometry of the TAFs
produced, we implemented a polyprotein strategy [56]. Here, subunits of a complex are produced from
a single open reading frame including the gene for a highly specific protease, which efficiently cleaves at
specific sites in between the individual subunits within the polyprotein produced [56,57]. Interestingly,
our findings triggered our first collaborative study with clinicians investigating genomic mutations
present at the interaction interfaces we had identified within the NIP, that lead to severe mental
retardation [58]. This study demonstrated the essential nature of these interactions for holo-TFIID
integrity and activity. At the same time, it challenged the prevailing concept that complete holo-TFIID
is absolutely required for life. In immortalized fibroblasts from the patient, who clearly is alive,
practically no holo-TFIID complex could be identified, it had seemingly fallen apart [58].
Recently, we implemented a state of the art proteomics approach to scrutinize in unprecedented
detail the presence of TAFs and TBP in cellular compartments. We found a different partial TFIID
complex, formed by TAF5, TAF6 and TAF9, existing as a prevalent, distinct entity in the cytoplasm.
In the process, we discovered a critical checkpoint function of a chaperonin, CCT, in catalyzing the
assembly of this complex (Figure 3c) [51,59]. This intriguing finding sheds first light on the involvement
of a cellular factor in early stages of TFIID assembly and may be paradigmatic for the formation of
other multiprotein complexes regulating transcription and gene expression, and other vital activities,
in cells.
4. G-Protein Coupled Receptor (GPCR) Structure and Mechanism
We observed early on that the alterations we had engineered into the MultiBac baculoviral genome
resulted in delayed lysis of cells upon infection with our recombinant virus. Baculovirus takes over the
host cell machinery upon infection and either shuts down or reprograms cellular functions to ramp up
virus production. Given that many cellular functions terminate, cells upon infection can be looked at as
enveloped micro-bioreactor devices rather than ‘living’ cells. Typically, in BICS, heterologous protein
expression is driven by viral promoters which are maximally active in the late or very late phase of
the viral life cycle in the infected cells, most prominent are the p10 and polh promoters. In wild-type
virus, this stage coincides with host liquefaction to release the virus into the environment for infecting
new hosts [2,60]. In cell culture, the late and very late phase is characterized by a wide-spread lysis
of cells (i.e., the mirco-bioreactors), release of cellular components into the media and accumulation
of cell debris (Figure 4a). However, this is also the stage where recombinant protein production is
maximal, implying a trade-off between the integrity of the micro-bioreactor device and its capacity to
accumulate a heterologous product. This could be particularly relevant for membrane proteins that
occupy the cell plasma membrane—it is conceivable that micro-bioreactors with an intact membrane
envelope would be advantageous for their production as compared to bio-reactors that become leaky
and disintegrate when production of the membrane protein of choice is at its peak.
Among membrane proteins, G-protein coupled receptors (GPCRs) have gained especial
prominence. GPCRs are key components of a multitude of signaling cascades. Moreover, they
are present on cell plasma membranes and thus more easily ‘reachable’ for modulators as compared to
intracellular proteins where one or several membrane bilayer barriers must be navigated before the
target is bound. Thus, for many reasons, GPCRs are highly attractive drug targets and their structure
determination, bound to agonists, antagonists, small molecules and protein ligands is intensely
Viruses 2019, 11, 198 7 of 14
pursued in the life sciences and pharmaceutical drug development [61–68]. Insect cells appear to
be well suited for producing heterologous G-protein coupled receptors, consequently, many GPCRs
are being produced by BICS. The MultiBac system we have developed is contributing to this trend,
and a selection of recent high-impact ligand-bound GPCR structures, enabled by MultiBac [69–71], is
depicted below (Figure 4b–d). As hetero-oligomeric GPCRs and GPCRs complexed with accessory
proteins come increasingly in focus, we expect the impact of MultiBac to significantly increase in this
vibrant research field [72,73].
Viruses 2019, 11, x FOR PEER REVIEW 7 of 14
present on cell plasma membranes and thus more easily ‘reachable’ for modulators as compared to
intracellular proteins where one or several membrane bilayer barriers must be navigated before the
target is bound. Thus, for many reasons, GPCRs are highly attractive drug targets and their structure
determination, bound to agonists, antagonists, small molecules and protein ligands is intensely
pursued in the life sciences and pharmaceutical drug development [61–68]. Insect cells appear to be
well suited for producing heterologous G-protein coupled receptors, consequently, many GPCRs are
being produced by BICS. The MultiBac system we have developed is contributing to this trend, and
a selection of recent high-impact ligand-bound GPCR structures, enabled by MultiBac [69–71], is
depicted below (Figure 4b–d). As hetero-oligomeric GPCRs and GPCRs complexed with accessory
proteins come increasingly in focus, we expect the impact of MultiBac to significantly increase in this
vibrant research field [72,73].
Figure 4. GPCRs made by MultiBac. (a) Micrographs show cells 72 h after infection with MultiBac
and, for comparison, a commercial competitor baculovirus (Bac-to-Bac, Invitrogen), respectively
[12,14]. MultiBac-infected insect cells (I.) are uniformly round and appear intact in contrast to the cell
lysis prevalent for Bac-to-Bac infected cells (II.). The elimination of proteolytic and apoptotic factors
from the MultiBac backbone significantly improved the integrity of membrane bilayers of infected
insect cells at very late stages of the viral life cycle. Scale bars, 20 μm. (b–d) GPCRs were produced by
using the MultiBac BICS and crystallized. X-ray structures of ligand-bound free fatty acid receptor
GPR40 (violet) bound to a proprietary ligand (compound 1, red) [70] (b), neurokinin 1 receptor NK1R
(green) bound to ligand aprepitant (white) [71] (c) and parathyroid hormone 1 receptor PTH1R (blue
and red) bound to peptide ligand ePTH (yellow) [69] (d) are shown. The membrane bilayer is depicted
schematically (grey bar).
5. VLP-factoryTM: Customized MultiBac Baculovirus for Virus-Like Particle Production
Infectious diseases continue to plague populations and economies. Influenza, for example, while
rarely deadly, still causes a substantial global economic shortfall every year. In addition, the
significant threat of pandemics remains highly acute [74]. Vaccines constitute a premier defense
against infectious diseases including influenza [75]. Recombinant virus-like particles (VLPs) which
simulate live viruses but lack genetic content and thus are safe, constitute attractive, cost-effective
alternatives to inactivated viruses which have dominated the selection of influenza vaccinations to
Figure 4. GPCRs made by MultiBac. (a) Micrographs show cells 72 h after infection with MultiBac and,
for comparison, a commercial competitor baculovirus (Bac-to-Bac, Invitrogen), respectively [12,14].
MultiBac-infected insect cells (I.) are uniformly round and appear intact in contrast to the cell lysis
prevalent for Bac-to-Bac infected cells (II.). The elimination of proteolytic and apoptotic factors from the
MultiBac backbone significantly improved the integrity of membrane bilayers of infected insect cells
at very late stages of the viral life cycle. Scale bars, 20 µm. (b–d) GPCRs were produced by using the
MultiBac BICS and crystallized. X-ray structures of ligand-bound free fatty acid receptor GPR40 (violet)
bound to a proprietary ligand (compound 1, red) [70] (b), neurokinin 1 receptor NK1R (green) bound
to ligand aprepitant (white) [71] (c) and parathyroid hormone 1 receptor PTH1R (blue and red) bound
to peptide ligand ePTH (yellow) [69] (d) are shown. The membrane bilayer is depicted schematically
(grey bar).
5. VLP-FactoryTM: Customized MultiBac Baculovirus for Virus-Like Particle Production
Infectious diseases continue to plague populations and economies. Influenza, for example, while
rarely deadly, still causes a substantial global economic shortfall every year. In addition, the significant
threat of pandemics remains highly acute [74]. Vaccines constitute a premier defense against infectious
diseases including influenza [75]. Recombinant virus-like particles (VLPs) which simulate live viruses
but lack genetic content and thus are safe, constitute attractive, cost-effective alternatives to inactivated
viruses which have dominated the selection of influenza vaccinations to date [76,77]. We recently
engineered a version of MultiBac for efficient expression of VLPs based on the influenza M1 capsid
protein [78]. We chose M1 from the influenza strain H1N1 as, already by itself, H1N1 M1 produced
stable enveloped capsids budding off the infected insect cells in superior quantities as compared
Viruses 2019, 11, 198 8 of 14
to M1 originating from other strains. The gene encoding for H1N1 M1 was inserted into the viral
backbone at the LoxP site (Figure 5). A gene encoding a fluorescent protein (mCherry) was supplied
along with H1N1 M1 to monitor virus performance and VLP production (Figure 5). This customized
MultiBac variant, called VLP-factoryTM, enabled production of an array of influenza VLPs presenting
hemagglutinin (HA) and neuraminidase (NA) proteins from various influenza strains, including
HA mutants thought to modulate the host immune system [79]. These influenza VLP variants hold
promise to develop into VLP-based hyper-immunogenic antigens that could lead to broadly protecting
influenza vaccines by eliciting a strong antibody titre upon immunization in contrast to wild-type HA
which by itself is not particularly immunogenic.
Viruses 2019, 11, x FOR PEER REVIEW 8 of 14
date [76,77]. We recently engineered a version of MultiBac for efficient expression of VLPs based on
the influenza M1 capsid protein [78]. We chose M1 from the influenza strain H1N1 as, already by
itself, H1N1 M1 produced stable enveloped capsids budding off the infected insect cells in superior
quantities as compared to M1 originating from other strains. The gene encoding for H1N1 M1 was
inserted into the viral backbone at the LoxP site (Figure 5). A gene encoding a fluorescent protein
(mCherry) was supplied along with H1N1 M1 to monitor virus performance and VLP production
(Figure 5). This customized MultiBac variant, called VLP-factoryTM, enabled production of an array
of influenza VLPs presenting hemagglutinin (HA) and neuraminidase (NA) proteins from various
influenza strains, including HA mutants thought to modulate the host immune system [79]. These
influenza VLP variants hold promise to develop into VLP-based hyper-immunogenic antigens that
could lead to broadly protecting influenza vaccines by eliciting a strong antibody titre upon
immunization in contrast to wild-type HA which by itself is not particularly immunogenic.
Figure 5. MultiBac-based VLP-factoryTM. A plasmid module comprising expression cassettes for the
capsid-forming influenza H1N1 M1 protein (colored in grey) and a fluorescent protein marker,
mCherry (colored in red), was introduced into the MultiBac baculoviral genome by Cre recombinase
enzyme mediated plasmid fusion into the LoxP site (circle filled in red, gradient) present on the viral
backbone (top right). Genes encoding for hemagglutinin (HA) and neuraminidase (NA) from one or
several influenza strains can be integrated into the Tn7 attachment site distal from LoxP. Coexpression
of HA, NA and M1 yields synthetic influenza virus-like particles (VLPs) resembling live
influenza virus as shown in electron micrographs (boxed) [78]. Scale bars, 100nm. Abbreviations as
in Figure 1; oriR6Kγ
, conditional origin of replication; Chl, chloramphenicol.
Our VLP-factoryTM is not restricted to producing influenza VLPs only. Most viral envelope
proteins that can be expressed efficiently in insect cells will be incorporated in M1-based VLPs during
the budding process. Thus, many other enveloped VLPs, displaying antigenic proteins from a wide
range of viruses, can conceivably be manufactured using our approach, to produce potent VLP-based
vaccines to combat infectious disease.
6. MultiBacMam-BiFC: Cell-Based Screening by Bimolecular Fluorescence Complementation
The scope of baculovirus-based DNA delivery was substantially expanded by the finding that
mammalian cells can be transduced by a recombinant baculovirus and used for heterologous
expression if appropriate, mammalian-cell active promoters were provided [3–5]. We showed
recently that appropriately modified MultiBac baculovirus, MultiBacMam, can be efficiently
deployed to deliver complex multifunctional DNA circuitry in mammalian cells and tissues including
Figure 5. MultiBac-based VLP-factoryTM. A plasmid module comprising expression cassettes for
the capsid-forming influenza H1N1 M1 protein (colored in grey) and a fluorescent protein marker,
mCherry (colored in red), was introduced into the MultiBac baculoviral genome by Cre recombinase
enzyme mediated plasmid fusion into the LoxP site (circle filled in red, gradient) present on the viral
backbone (top right). Genes encoding for hemagglutinin (HA) and neuraminidase (NA) from one or
several influenza strains can be integrated into the Tn7 attachment site distal from LoxP. Co-expression
of HA, NA and M1 yields synthetic influenza virus-like particles (VLPs) resembling live influenza
virus as shown in electron micrographs (boxed) [78]. Scale bars, 100nm. Abbreviations as in Figure 1;
oriR6Kγ, conditional origin of replication; Chl, chloramphenicol.
Our VLP-factoryTM is not restricted to producing influenza VLPs only. Most viral envelope
proteins that can be expressed efficiently in insect cells will be incorporated in M1-based VLPs during
the budding process. Thus, many other enveloped VLPs, displaying antigenic proteins from a wide
range of viruses, can conceivably be manufactured using our approach, to produce potent VLP-based
vaccines to combat infectious disease.
6. MultiBacMam-BiFC: Cell-Based Screening by Bimolecular Fluorescence Complementation
The scope of baculovirus-based DNA delivery was substantially expanded by the finding that
mammalian cells can be transduced by a recombinant baculovirus and used for heterologous expression
if appropriate, mammalian-cell active promoters were provided [3–5]. We showed recently that
appropriately modified MultiBac baculovirus, MultiBacMam, can be efficiently deployed to deliver
complex multifunctional DNA circuitry in mammalian cells and tissues including cell types known
to be recalcitrant to transfection by conventional, plasmid-based approaches [19,80]. We used our
Viruses 2019, 11, 198 9 of 14
approach successfully to facilitate gene editing by CRISPR-Cas9 in mammalian cells including primary
neurons [80].
Building on these results, we have constructed a MultiBacMam variant to assay protein-protein
interactions (PPIs) by florescence complementation in living mammalian cells [81]. PPIs are central
to the interplay of cellular factors in health and disease, and targeting PPIs, for example by small
molecules, is at the forefront of drug discovery. Instrumental to understanding how small molecules
can enhance or disrupt PPIs are cellular assays which closely recapitulate native conditions in vivo.
These depend on non-invasive, highly sensitive and faithful readout of the PPI and the impact of
additives under scrutiny. Bimolecular fluorescence complementation (BiFC) can provide such a
readout [82–84]. Therefore, we have integrated modalities for BiFC into our engineered MultiBacMam
baculovirus (Figure 6). We interfaced our reagents with high-content screening (HCS), resulting in
a powerful system for assay development, identification and characterization of small molecule PPI
modulators [81]. We implemented our assay to scrutinize chemical compounds modulating the PPI
formed by cyclin-dependent kinase 5 (CDK5) and p25, a fragment of the p35 activator. The CDK5-p25
PPI is implicated in many diseases including Alzheimer’s [85–87]. With our MultiBacMam-BiFC
tool-kit, we implemented cell-based screening to analyse small molecules in a dose-dependent manner
and discovered novel compounds which effectively abolished the CDK5-p25 PPI [86].
Viruses 2019, 11, x FOR PEER REVIEW 9 of 14
cell types known to be recalcitrant to transfection by conventional, plasmid-based approaches [19,80].
We used our approach successfully to facilitate gene editing by CRISPR-Cas9 in mammalian cells
including primary neurons [80].
Building on these results, we have constructed a MultiBacMam variant to assay protein-protein
interactions (PPIs) by florescence complementation in living mammalian cells [81]. PPIs are central
to the interplay of cellular factors in health and disease, and targeting PPIs, for example by small
molecules, is at the forefront of drug discovery. Instrumental to understanding how small molecules
can enhance or disrupt PPIs are cellular assays which closely recapitulate native conditions in vivo.
These depend on non-invasive, highly sensitive and faithful readout of the PPI and the impact of
additives under scrutiny. Bimolecular fluorescence complementation (BiFC) can provide such a
readout [82–84]. Therefore, we have integrated modalities for BiFC into our engineered
MultiBacMam baculovirus (Figure 6). We interfaced our reagents with high-content screening (HCS),
resulting in a powerful system for assay development, identification and characterization of small
molecule PPI modulators [81]. We implemented our assay to scrutinize chemical compounds
modulating the PPI formed by cyclin-dependent kinase 5 (CDK5) and p25, a fragment of the p35
activator. The CDK5-p25 PPI is implicated in many diseases including Alzheimer’s [85–87]. With our
MultiBacMam-BiFC tool-kit, we implemented cell-based screening to analyse small molecules in a
dose-dependent manner and discovered novel compounds which effectively abolished the CDK5p25
PPI [86].
Figure 6. MultiBacMam-BiFC for compound screening. The MultiBacMam baculovirus is shown (top
left). Genes encoding for vesicular stomatitis virus glycoprotein (VSV-G) and mCherry to track virus
performance during manufacturing have been integrated into the baculoviral backbone, each
controlled by baculoviral late promoters. MultiBacMam was outfitted in the Tn7 site with genes
encoding for CDK5 and p25, each fused to complementary fragments of a split fluorescent protein,
which, upon CDK5-p25 complex formation reconstitute complete, active fluorophore (bottom left).
Our setup can be adapted to any PPI. Composite MultiBacMam baculovirus is produced in insect cells
and then used to transduce mammalian cells (here U2OS) with superior efficacy as compared to
plasmid transfection (top right). BiFC signal is shown in green and Hoechst 33342 nuclear staining in
blue. Scale bar, 50 μm. A selection of chemical compounds inhibiting the CDK5-p25 PPI is depicted
(bottom right), identified by using our MultiBac-BiFC cell-based screening assay. Abbreviations as in
Figure 1.
Figure 6. MultiBacMam-BiFC for compound screening. The MultiBacMam baculovirus is shown
(top left). Genes encoding for vesicular stomatitis virus glycoprotein (VSV-G) and mCherry to track
virus performance during manufacturing have been integrated into the baculoviral backbone, each
controlled by baculoviral late promoters. MultiBacMam was outfitted in the Tn7 site with genes
encoding for CDK5 and p25, each fused to complementary fragments of a split fluorescent protein,
which, upon CDK5-p25 complex formation reconstitute complete, active fluorophore (bottom left). Our
setup can be adapted to any PPI. Composite MultiBacMam baculovirus is produced in insect cells and
then used to transduce mammalian cells (here U2OS) with superior efficacy as compared to plasmid
transfection (top right). BiFC signal is shown in green and Hoechst 33342 nuclear staining in blue. Scale
bar, 50 µm. A selection of chemical compounds inhibiting the CDK5-p25 PPI is depicted (bottom right),
identified by using our MultiBac-BiFC cell-based screening assay. Abbreviations as in Figure 1.
Viruses 2019, 11, 198 10 of 14
7. Conclusions and Outlook
In 2004, we first communicated our original MultiBac BICS, comprising an engineered baculovirus
and a set of reagents specifically tailored to produce multiprotein complexes in the quality and quantity
required for high-resolution structural and mechanistic studies. Today, MultiBac is put to excellent use
in many laboratories world-wide, in academia and industry, accelerating research and development.
Many multiprotein complexes, vital catalysts of cellular function, have been produced and their
molecular structures determined, shedding light on their function. The inherent, large heterologous
DNA cargo capacity of the baculovirion allowed us and others to insert many genes encoding for
the subunits of multiprotein complexes together with synthetic promoters, terminators and diverse
gene regulatory elements into recombinant MultiBac baculoviral genomes. In addition to this DNA
cargo, a variety of other functions can be provided if needed, including markers to monitor virus
performance, insect cell infection levels and the time of harvest when maximal protein production is
achieved. Simultaneously, DNA encoded functions can be added including kinases, phosphatases,
chaperones and other factors to activate or inactivate a given complex or to assist in proper folding.
All this foreign DNA is delivered to insect cell cultures upon infection and translated into elaborate
programs yielding high quality complex biological specimens.
This unprecedented, large DNA cargo capacity also renders baculovirus highly attractive as a tool
for DNA delivery in mammalian cell types, for a range of applications including drug discovery, cellular
reprogramming and genome engineering, some of which we have touched upon here. We anticipate
that the emergence of innovative methods in synthetic biology will enable cost-effective crafting of
new and powerful designer baculoviral genomes from scratch, tailored to deploy multifunctional DNA
circuitry faithfully in cells, tissues and even organisms, translating encoded messages into elaborate
programs to carry out a multitude of tasks, accelerating discovery.
Author Contributions: I.B. conceived the contribution. K.G., C.T., D.S.-A., D.J.F., C.S. and I.B. prepared figures
and data. I.B. wrote the contribution with input from all authors.
Funding: The work in our laboratory reviewed here was funded by the European Commission (EC) Framework
Programme (FP) 7 grants ComplexINC (contract number 279039) and SynSignal (contract number 613879) and the
Agence National de Recherche (ANR) (grant ID ANR-13-BSV8-0021). I.B. is Senior Investigator of the Wellcome
Trust (106115/Z/14/Z). The MultiBac platform at the School of Biochemistry, University of Bristol, receives
support from BrisSynBio, a BBSRC/EPSRC Research Centre for synthetic biology at the University of Bristol
(BB/L01386X/1).
Acknowledgments: We thank all past and present members of the Berger laboratories and our collaborators for
their contributions and Dale Wigley (Imperial College, London) for sharing data. We thank Timothy J. Richmond
(ETH Zürich, Switzerland) for his support early on in the MultiBac project, as well as Polly Roy (The London
School of Hygiene and Tropical Medicine, UK) and Jane Endicott (Newcastle University, UK) for invaluable advice.
Conflicts of Interest: The authors declare conflict of interest: I.B. and D.J.F. are inventors on patents covering
various aspects and applications of the MultiBac technology, and are shareholders of Geneva Biotech SARL
(Geneva, Switzerland) which commercializes MultiBac. The funders of the work presented had no role in study
design, in the collection, analyses, or interpretation of any data presented; in the writing of the manuscript, or in
the decision to publish the results.
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