**1. Introduction**

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

1997;15 275–283.

Baculoviruses are enveloped viruses that infect insect larvae mainly from the order Lepidop‐ tera. Their genomes are circular double-stranded DNA molecules of about 80 to 180 kbp and are packed in rod-shaped nucleocapsids with a typical size of 40-50 nm in diameter and 200-400 nm in length.

Among the numerous baculoviruses, *Autographa californica* multiplenucleopolyhedrovirus (AcMNPV) is the most widely studied and used in biotechnology.

During its infection cycle it produces two phenotypes. Occlusion derived viruses (ODV) initiate the infection at the larvae midgut. After this primary infection, the viral progeny consists of budded viruses (BV) that carry on the systemic infection in larvae. These types of virions differ in their efficiencies of infection for different cell types; ODV infect midgut epithelial cells up to 10,000 fold more efficiently than BV. In contrast, BV are up to 1,000-fold more efficient at infecting cultured cells than ODV. As the viral propagation in cell culture is mediated by BV phenotype (Rohrmann, 2011), most of the knowledge regarding baculovirus infection cycle is based on studies performed in insect cells infected by BV (Figure 1).

Cell entry is mediated by a class III viral glycoprotein located at the virion surface, Gp64, which interacts with an unknown cell receptor (Backovic & Jardetzky, 2009). This interaction triggers clathrin-dependent endosomal internalization. This internalized vesicle becomes subsequent‐ ly acidified. This causes a conformational change in Gp64 that result in the fusion of the viral envelope with the endosome membrane. Thus the nucleocapsid is released in the cytoplasm and migrates to the nucleus. Once in the nucleus, DNA is uncoated and the transcriptional cascade begins (Figure 2).

© 2013 Pidre et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

AcMNPV genome encodes about 150 genes which are transcribed in a temporal fashion. Firstly, immediate early genes are transcribed by the host RNA-polymerase II. These genes generally encode for transcription factors, like Ie1, that aid the subsequent transcription of genes. After this early phase DNA replication occurs. Immediately after DNA replication there may be a transient period when proteins are not bound to the DNA and this might expose late promoters and facilitate their activation (Rohrmann, 2011). Baculoviruses also encode a novel RNA polymerase that transcribes late and very late genes and that recognizes the unique baculoviral promoter consensus sequence DTAAG. During the systemic infection nucleocap‐ sids are assembled in the virogenic stroma. The envelope proteins are synthesized, translated in association with the endoplasmic reticulum, glycosylated and transported to and incorpo‐ rated into the cytoplasmic membrane via the Golgi apparatus. Nucleocapsids destined to become BV exit the nucleus. They move to the cytoplasmic membrane at the site where envelope proteins (Gp64 and F protein) concentrate, and bud through obtaining their enve‐ lopes. Early in the systemic infection more BV are produced which spread the infection throughout the insect. Finally, late in infection, occluded virions are produced, and the cell

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There are many biotechnological uses for baculoviruses. One of the most widespread is their use as insecticide agents. There have been much work on the development of baculoviruses to control insects but the acceptance and use of viruses for insect control has been limited. This can be attributed to their slow speed of kill and their limited host range. At present many research groups are working with the aim of overcoming these limitations developing novel strategies such as baculovirus-mediated expression of toxic proteins for insects. Moreover, recombinant baculoviruses have been extensively used as expression vectors in insect cell cultures. A variety of technological improvements have eliminated the tedious procedures to isolate the recombinant viruses turning the baculovirus-based expression system in a safe, easy

Another application of baculovirus is their use as expression vectors for eukariotic proteins. Their ability to include quite large DNA extra fragments in their genomes and the possibility to use their very strong polihedryn promoter, which activates upon infection, make baculo‐ viruses a very useful tool in biotechnology for the production of recombinant proteins in insect

In addition, protein expression in larvae or cell culture is not the only application of baculo‐ viruses. In fact, baculoviruses are widely used in the development of strategies for displaying foreign peptides and proteins on the virus surface as well as mammalian cell transduction using different mammalian expression cassettes. Baculovirus display consists of the expression of proteins or peptides in the surface of a baculovirus. This is achieved by fusing the protein of interest with the major baculoviral envelope glycoprotein Gp64, resulting in the localization of the chimeric protein on the viral envelope and the plasmatic membraneof infected cells. The surface displaying of antigenic epitopes make baculoviruses efficient vaccine vehicles capable

The aim of this chapter will be to describe the biotechnological utilities of baculovirus display. Particularly, it will describe this technique for vaccination and gene delivery. It will discuss

dies releasing the occlusion bodies.

to use and scale up system. (Kost et al., 2005).

of mounting a strong specific immune response.

cells.

**Figure 1.** Structure of the budded virus.

**Figure 2. Baculovirus replication cycle.** Infection cycle initiates when a budded virus (BV) interacts with the cell membrane and is endocytosed. When the endocytic vesicle is acidified, GP64 fusion protein, located at the BV mem‐ brane, trigger the fusion of the plasma membrane and the BV envelope releasing the nucleocapsid in the cytoplasm. The nucleocapsid is then transported to the nuclus where it transcribes its genes, replicates its DNA in the virogenic stroma where new nucleocapsids are assembled. Nucleocapsids then egress from the nucleus, travel to the cytoplas‐ mic membrane and bud through aquiring an envelope containing the surface protein GP64.

AcMNPV genome encodes about 150 genes which are transcribed in a temporal fashion. Firstly, immediate early genes are transcribed by the host RNA-polymerase II. These genes generally encode for transcription factors, like Ie1, that aid the subsequent transcription of genes. After this early phase DNA replication occurs. Immediately after DNA replication there may be a transient period when proteins are not bound to the DNA and this might expose late promoters and facilitate their activation (Rohrmann, 2011). Baculoviruses also encode a novel RNA polymerase that transcribes late and very late genes and that recognizes the unique baculoviral promoter consensus sequence DTAAG. During the systemic infection nucleocap‐ sids are assembled in the virogenic stroma. The envelope proteins are synthesized, translated in association with the endoplasmic reticulum, glycosylated and transported to and incorpo‐ rated into the cytoplasmic membrane via the Golgi apparatus. Nucleocapsids destined to become BV exit the nucleus. They move to the cytoplasmic membrane at the site where envelope proteins (Gp64 and F protein) concentrate, and bud through obtaining their enve‐ lopes. Early in the systemic infection more BV are produced which spread the infection throughout the insect. Finally, late in infection, occluded virions are produced, and the cell dies releasing the occlusion bodies.

There are many biotechnological uses for baculoviruses. One of the most widespread is their use as insecticide agents. There have been much work on the development of baculoviruses to control insects but the acceptance and use of viruses for insect control has been limited. This can be attributed to their slow speed of kill and their limited host range. At present many research groups are working with the aim of overcoming these limitations developing novel strategies such as baculovirus-mediated expression of toxic proteins for insects. Moreover, recombinant baculoviruses have been extensively used as expression vectors in insect cell cultures. A variety of technological improvements have eliminated the tedious procedures to isolate the recombinant viruses turning the baculovirus-based expression system in a safe, easy to use and scale up system. (Kost et al., 2005).

Another application of baculovirus is their use as expression vectors for eukariotic proteins. Their ability to include quite large DNA extra fragments in their genomes and the possibility to use their very strong polihedryn promoter, which activates upon infection, make baculo‐ viruses a very useful tool in biotechnology for the production of recombinant proteins in insect cells.

In addition, protein expression in larvae or cell culture is not the only application of baculo‐ viruses. In fact, baculoviruses are widely used in the development of strategies for displaying foreign peptides and proteins on the virus surface as well as mammalian cell transduction using different mammalian expression cassettes. Baculovirus display consists of the expression of proteins or peptides in the surface of a baculovirus. This is achieved by fusing the protein of interest with the major baculoviral envelope glycoprotein Gp64, resulting in the localization of the chimeric protein on the viral envelope and the plasmatic membraneof infected cells. The surface displaying of antigenic epitopes make baculoviruses efficient vaccine vehicles capable of mounting a strong specific immune response.

**Figure 2. Baculovirus replication cycle.** Infection cycle initiates when a budded virus (BV) interacts with the cell membrane and is endocytosed. When the endocytic vesicle is acidified, GP64 fusion protein, located at the BV mem‐ brane, trigger the fusion of the plasma membrane and the BV envelope releasing the nucleocapsid in the cytoplasm. The nucleocapsid is then transported to the nuclus where it transcribes its genes, replicates its DNA in the virogenic stroma where new nucleocapsids are assembled. Nucleocapsids then egress from the nucleus, travel to the cytoplas‐

mic membrane and bud through aquiring an envelope containing the surface protein GP64.

**Figure 1.** Structure of the budded virus.

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

The aim of this chapter will be to describe the biotechnological utilities of baculovirus display. Particularly, it will describe this technique for vaccination and gene delivery. It will discuss the adjuvant effects of baculoviruses and the immunity response of recombinant viruses. Moreover, other applications of baculovirus display such as gene therapy and high throughput screening of antibodies and antigenic epitopes libraries will also be addressed.

aminoacids polypeptide with 15 cysteine residues. The resulting disulfide bonds participate

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As a member of the Class III fusion proteins, Gp64 is composed of five domains that result in a macromolecular structure very distinct from any reported class I or class II fusion protein. However, Gp64 conserves the typical characteristics of viral fusion proteins. It includes a fusion domain which mediates the fusion between the cell membrane and viral envelope; a transmembrane domain which anchors the protein in the lipidic bilayer and a multimerization domain that allows the protein to form trimmers. The detailed structure of AcMNPV Gp64 is shown in Figure 3 (Backovic & Jardetzky, 2009) Baculovirus gp64 also contains a seven residue C-terminal tail domain (CTD). Deletion of this domain does not significantly affect the ability to mediate fusion, but reduces the baculovirus titers to 50%. These data indicate that CTD is

**Figure 3. GP64 structure. a.** Trimmeric structure of baculovirus major surface glycoprotein Gp64 obtained using the Expasy tool *Make multimer.py* in www.expasy.org. **b.** Gp64 polypeptide scheme showing different functional domains

in the formation of the native structure.

involved in virus budding (Figure 3).

**a**.

**b**.

useful for antigen surface display.
