**5.1. Introduction**

the Tn7‐mediated site‐specific transposition reaction to direct integration of expression cassettes contained in the transfer vector into a baculovirus backbone vector (bacmid) preex‐ isting in the *E. coli* DH10Bac strain. In this case, the bacmid is a mini‐F replicon with the baculovirus genome and has a kanamycin resistance marker. In addition, the *E. coli* strain contains a helper plasmid that expresses the Tn7 transposase gene. The system was designed in such a way that the recombinant Tn7 transposon from the transfer vector will be integrated into a mini‐attTn7 in the *lacZα* gene fragment contained within the recombinant viral genome, causing inactivation of the α‐complementation of *lacZ*. The desired recombinant transformants will be resistant to tetracycline, kanamycin, and gentamicin and can be easily distinguished

The BacMam transfer vectors described here are derivatives of pFastBac1 of the Bac‐to‐Bac system (InvitrogenTM). Originally, the AcMNPV *polh* promoter of pFastBac1 was deleted for the introduction of cassettes containing a mammalian promoter. Later, the CMV immediate early promoter was inserted to allow expression of the cloned cDNA sequences in mammalian cells. The vector pFastBacMam‐1, in addition, contains a neomycin resistance gene driven by the SV40 promoter. The neomycin resistance marker allows selection of stable cell lines following BacMam transduction. Using this vector a new version was constructed (pFast‐

Heterologous peptides can be inserted between the signal peptide and the mature domain of the envelope fusion protein GP64, and this feature has been exploited for surface display of peptides to improve the virus transduction [103, 104], for ligand‐directed targeting if an appropriate ligand is chosen [105, 106]. When a short peptide motif from gp350/220 of Epstein– Barr virus (EBV, which naturally infects B cells) was displayed as GP64 fusion peptide on the baculovirus envelope [107], the efficiency of transduction to B lymphocytes was increased. Another paradigm is the display of the immunoglobulin Fc region on the baculovirus surface [108]. Fc receptors (FcRs) are membrane proteins that bind to the Fc region of antibody and mediate the phagocytosis and antigen presentation. The Fc display allows for specific bacu‐ lovirus targeting to cell lines and antigen presenting cells (APCs) expressing FcRs, hence augmenting the vaccine effect. The display system also allows for the surface presentation of

Aside from the gp64‐aided display, expression of vesicular stomatitis virus G protein (VSVG) [109], influenza virus neuraminidase [110], *Spodoptera exigua* multiple nucleopolyhedrovirus F protein, single chain antibody fragments and human endogenous retrovirus envelope protein [111] in insect cells also leads to incorporation of the protein into baculovirus envelope. Among these strategies, display of VSVG or heterologous peptide/protein via the VSVG anchor is the most widely adopted and can tremendously enhance baculovirus transduction in vitro

Backmam‐NA) to accommodate ORFs cloned in GatewayTM vectors (Invitrogen).

**4.3.1. Surface display via gp64 fusion or expression of heterologous protein**

from nonrecombinants by blue/white selection on X‐gal plates.

96 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

**4.3 Strategies to improve baculovirus transduction**

functional membrane proteins to simplify subsequent isolation.

and in vivo.

Recently, a novel molecular biology tool was established by the development of baculovirus surface display [117‐123], using different strategies for presentation of foreign peptides and proteins on the surface of budded virions. This eukaryotic display system enables presentation of large complex proteins on the surface of baculovirus particles and has thereby become a versatile system in molecular biology.

The baculovirus system offers great potential as an eukaryotic surface display system, since the post‐translational modification of the recombinant proteins is efficient and high transfec‐ tion rates can be reached. These features are important for the generation of efficient surface display libraries. The principal applications of such strategies are ligand screening of surface expression libraries, for example epitope mapping, antigen display for induction of specific antibodies and presentation of proteins that increase binding to mammalian host cells. Moreover, display strategies play an important role, as they may be used to enhance the efficiency and specificity of viral binding and entry to mammalian cells. In addition, baculo‐ virus surface display vectors have been engineered to contain mammalian promoter elements designed for gene delivery both in vitro and in vivo. Moreover, baculovirus capsid display has recently been developed; this holds promise for intracellular targeting of the viral capsid and subsequent cytosolic delivery of desired protein moieties. Finally, the viruses can accommo‐ date large insertions of foreign DNA and replicate only in insect cells. Together, these are attributes that are very likely to make them important tools in functional genomics and proteomics.

duced mammalian cells. Ideally, capsid display should thus enable transfer of functional

Genetic Engineering of Baculoviruses http://dx.doi.org/10.5772/56976 99

In the first constructions used to display peptides on the surface of the budded virions the foreign open reading frames were fused to the complete GP64 coding sequence, with the parental baculovirus retaining a wild type GP64 copy [126]. The foreign genes were cloned between the gp64 signal peptide and the mature gp64 peptide. The mechanism of incorpora‐ tion into the viral particle probably involves the oligomerisation of the fusion construct with wild‐type GP64. Until now, only small peptides have been inserted into the protein gp64 [127, 128]. When entire proteins were inserted the virus budding efficiency decreased drastically, and titres similar to those of gp64‐deletion mutants were obtained [129]. By comparison of different positions within the gp64 sequence using specific antibody epitopes, it was found that the surface probability of the inserted peptide strongly depends on the position, structural framework and the adjacent amino acids [128]. Incorporation of the fusion protein onto the viral surface usually represents only a small proportion of the total fusion protein and the levels of incorporation into the budded virus are variable and cannot be predicted. The position not only affects the viral titres obtained, but also influences the presentation of the epitope. In addition to the oligomerization domain and fusion domain, the N‐terminal part of the protein also contains essential structural or sequential motifs that are more sensitive to changes than

Different promoters for the GP64 fusion protein have been evaluated to increase incorporation rates and presentation of the displayed peptide [130]. It was noticed that the use of early promoters resulted in more complete post‐translational processing of glycoproteins; but the level of fusion protein detected on the surface of cells and budded virus particles was signif‐ icantly enhanced when strong, very late polyhedrin promoter was used. High concentrations of the target protein are required on the cell surface in order to reach a signal‐to‐noise ratio that allows cell sorting to be performed by fluorescence‐activated cell sorting, which, at the moment, is the only practical technique for selecting specific clones from baculovirus surface

As an alternative to using either the entire GP64 or portions of GP64 protein as the scaffold for protein presentation, the coat protein of a different virus, vesicular stomatitis virus (VSV), or its membrane anchor domain, has also been evaluated. It was shown that by using this strategy, incorporation eGFP was extremely high [131]. The avidity of the display virus increased significantly, without putting a direct limit on the size of the target gene. In the latter cases,

The study of baculoviruses is a traditional field in virology. In particular, genetic engineering of AcMNPV emerged in the 1980s, and several systems for various purposes have been

wild‐type gp64 was still expressed in order to maintain efficient infectivity.

molecules into the cytoplasm and/or the nucleus of the target cells.

**5.2. Baculoviral display cloning**

the rest of the protein.

display libraries.

**6. Conclusions and perspectives**

Display of foreign proteins or peptides on the surface of various virus particles has been valuable in a number of areas within life sciences, ranging from basic research such as protein structure–function studies to diagnostics and gene therapy. One of the most successful examples of display technology is the isolation of antibodies from large combinatorial libraries displayed on the surface of the bacteriophages [124]. The versatile principle of phage display is based on the direct physical linkage between genotype and phenotype. This linkage enables the selection of basically any protein with the desired characteristics, such as increased binding affinity or improved catalytic properties from a suitable display library [125]. Phage display comprises some severe limitations imposed by expression in the bacterial host, however, for example when large complex eukaryotic proteins that require glycosylation or particular protein folding are under study.

Over the past few years, the ability to present large complex glycoproteins on the surface of AcMNPV, has been developed into a versatile system in molecular biology. Expression of proteins or peptides on the baculoviral surface, or more recently also on the viral capsid, without compromising replication in insect cells, has shown to be useful for important applications, both *in vivo* and *in vitro*. The major envelope glycoprotein of AcMNPV is generally known as gp64. The corresponding gene encodes a type I integral membrane glycoprotein with an amino‐terminal signal sequence and a carboxy‐proximal transmembrane domain. The GP64 protein occurs on the viral particle as a disulphide‐linked oligomer, most likely a trimer, and is responsible for viral cell entry mediated by acid‐triggered membrane fusion. Structural studies on the GP64 protein have identified separate domains responsible for oligomer formation and membrane fusion. These structural characteristics of gp64 make it a good candidate as a presentation platform for the development of a eukaryotic‐based viral surface display system. Modification of viral surface structures by display techniques has enabled the use of baculovirus for enhanced targeting to mammalian cells in vitro. Based on the fact that surface display may interfere with baculovirus infectivity, and that molecules which are displayed on the baculovirus envelope end up in the lysosomes of the mammalian cell and subsequent acid‐induced fusion of the viral envelope in the endosomes, an approach for display of foreign protein moieties on the capsid of AcMNPV was recently developed. This system allows for presentation of desired proteins as fusions with the baculovirus major capsid protein VP39. By contrast, molecules displayed on the baculovirus capsid should escape endosomes and thereby follow the capsid through the cytoplasm into the nucleus of trans‐ duced mammalian cells. Ideally, capsid display should thus enable transfer of functional molecules into the cytoplasm and/or the nucleus of the target cells.

#### **5.2. Baculoviral display cloning**

Moreover, display strategies play an important role, as they may be used to enhance the efficiency and specificity of viral binding and entry to mammalian cells. In addition, baculo‐ virus surface display vectors have been engineered to contain mammalian promoter elements designed for gene delivery both in vitro and in vivo. Moreover, baculovirus capsid display has recently been developed; this holds promise for intracellular targeting of the viral capsid and subsequent cytosolic delivery of desired protein moieties. Finally, the viruses can accommo‐ date large insertions of foreign DNA and replicate only in insect cells. Together, these are attributes that are very likely to make them important tools in functional genomics and

98 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

Display of foreign proteins or peptides on the surface of various virus particles has been valuable in a number of areas within life sciences, ranging from basic research such as protein structure–function studies to diagnostics and gene therapy. One of the most successful examples of display technology is the isolation of antibodies from large combinatorial libraries displayed on the surface of the bacteriophages [124]. The versatile principle of phage display is based on the direct physical linkage between genotype and phenotype. This linkage enables the selection of basically any protein with the desired characteristics, such as increased binding affinity or improved catalytic properties from a suitable display library [125]. Phage display comprises some severe limitations imposed by expression in the bacterial host, however, for example when large complex eukaryotic proteins that require glycosylation or particular

Over the past few years, the ability to present large complex glycoproteins on the surface of AcMNPV, has been developed into a versatile system in molecular biology. Expression of proteins or peptides on the baculoviral surface, or more recently also on the viral capsid, without compromising replication in insect cells, has shown to be useful for important applications, both *in vivo* and *in vitro*. The major envelope glycoprotein of AcMNPV is generally known as gp64. The corresponding gene encodes a type I integral membrane glycoprotein with an amino‐terminal signal sequence and a carboxy‐proximal transmembrane domain. The GP64 protein occurs on the viral particle as a disulphide‐linked oligomer, most likely a trimer, and is responsible for viral cell entry mediated by acid‐triggered membrane fusion. Structural studies on the GP64 protein have identified separate domains responsible for oligomer formation and membrane fusion. These structural characteristics of gp64 make it a good candidate as a presentation platform for the development of a eukaryotic‐based viral surface display system. Modification of viral surface structures by display techniques has enabled the use of baculovirus for enhanced targeting to mammalian cells in vitro. Based on the fact that surface display may interfere with baculovirus infectivity, and that molecules which are displayed on the baculovirus envelope end up in the lysosomes of the mammalian cell and subsequent acid‐induced fusion of the viral envelope in the endosomes, an approach for display of foreign protein moieties on the capsid of AcMNPV was recently developed. This system allows for presentation of desired proteins as fusions with the baculovirus major capsid protein VP39. By contrast, molecules displayed on the baculovirus capsid should escape endosomes and thereby follow the capsid through the cytoplasm into the nucleus of trans‐

proteomics.

protein folding are under study.

In the first constructions used to display peptides on the surface of the budded virions the foreign open reading frames were fused to the complete GP64 coding sequence, with the parental baculovirus retaining a wild type GP64 copy [126]. The foreign genes were cloned between the gp64 signal peptide and the mature gp64 peptide. The mechanism of incorpora‐ tion into the viral particle probably involves the oligomerisation of the fusion construct with wild‐type GP64. Until now, only small peptides have been inserted into the protein gp64 [127, 128]. When entire proteins were inserted the virus budding efficiency decreased drastically, and titres similar to those of gp64‐deletion mutants were obtained [129]. By comparison of different positions within the gp64 sequence using specific antibody epitopes, it was found that the surface probability of the inserted peptide strongly depends on the position, structural framework and the adjacent amino acids [128]. Incorporation of the fusion protein onto the viral surface usually represents only a small proportion of the total fusion protein and the levels of incorporation into the budded virus are variable and cannot be predicted. The position not only affects the viral titres obtained, but also influences the presentation of the epitope. In addition to the oligomerization domain and fusion domain, the N‐terminal part of the protein also contains essential structural or sequential motifs that are more sensitive to changes than the rest of the protein.

Different promoters for the GP64 fusion protein have been evaluated to increase incorporation rates and presentation of the displayed peptide [130]. It was noticed that the use of early promoters resulted in more complete post‐translational processing of glycoproteins; but the level of fusion protein detected on the surface of cells and budded virus particles was signif‐ icantly enhanced when strong, very late polyhedrin promoter was used. High concentrations of the target protein are required on the cell surface in order to reach a signal‐to‐noise ratio that allows cell sorting to be performed by fluorescence‐activated cell sorting, which, at the moment, is the only practical technique for selecting specific clones from baculovirus surface display libraries.

As an alternative to using either the entire GP64 or portions of GP64 protein as the scaffold for protein presentation, the coat protein of a different virus, vesicular stomatitis virus (VSV), or its membrane anchor domain, has also been evaluated. It was shown that by using this strategy, incorporation eGFP was extremely high [131]. The avidity of the display virus increased significantly, without putting a direct limit on the size of the target gene. In the latter cases, wild‐type gp64 was still expressed in order to maintain efficient infectivity.
