**1. Introduction**

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

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Baculoviruses are arthropod‐specific, enveloped viruses with circular, supercoiled double‐ stranded DNA genomes [1]. They infect *Lepidoptera* (butterflies and moths), *Hymenoptera* (sawflies) and *Diptera* (mosquitoes) [2]. While many viruses are studied because of their damaging effects, the study of baculoviruses was stimulated by their potential utility to control insect pests [3]. Later, the utility of baculovirus as gene expression vectors was evidenced and a new research area emerged [4]. A major step forward was the development of bacmid technology [5] (the construction of bacterial artificial chromosomes containing the genome of the baculovirus) which allows the manipulation of the baculovirus genome in bacteria. With this technology, foreign genes can be introduced into the bacmid by site‐ directed recombina‐ tion or by transposition. Baculoviruses have been used to explore fundamental questions in molecular biology such as the nature of programmed cell‐death [6]. Moreover, the ability of baculoviruses to transduce mammalian cells led to the consideration of their use as gene therapy and vaccine vectors. Strategies for genetic engineering of baculoviruses have been developed to meet the requirements of new application areas, and the establishment of new genetic modification systems is still necessary when an unexplored experimental system is to be addressed. The aim of this chapter is to detail the areas of application of the baculovirus in basic molecular biology and applied biotechnology and the strategies used to generate genetically modified baculoviruses according to each area of study.

#### **1.1. Molecular biology of baculoviruses**

Baculovirus genomes consist of a circular, double‐stranded DNA molecule with a size ranging from 80 to over 180 kbp and encoding 90 to 180 genes.

© 2013 Haase 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.

The name "baculovirus" is derived from the latin "*baculum*" (stick), denoting the rod‐shaped nucleocapsids that are 230– 385 nm in length and 40–60 nm in diameter [1]. The virions are enveloped and present two phenotypes: occluded virions (OVs) and budded virions (BVs). These two types of virions differ in the origin and composition of their envelopes and their functions in the virus life cycle. In both, the genome is complexed with multiple copies of a small basic protein (p 6.9) which neutralizes the negative charge of the DNA and this structure is protected by other proteins forming the nucleocapsid. The OVs are enclosed in a paracrys‐ talline matrix forming occlusion bodies (OBs), which are orally infectious. Their morphology was initially used to define two major groups or genera of the Baculoviridae: the Nucleopo‐ lyhedrovirus (NPVs) and the Granulovirus (GVs). NPV OBs, called polyhedra, are about 0.6– 2 μm in size and their major occlusion protein is called polyhedrin. GV OBs, also known as granules or capsules, are oval‐shaped with diameters in the range of 0.2–0.4 μm (Figure 1). OBs are highly stable and can resist most normal environmental conditions thereby allowing virions to remain infectious for very long periods of time.

virions enter midgut cells after fusion with membrane epithelial cells. The virions are uncoated

Genetic Engineering of Baculoviruses http://dx.doi.org/10.5772/56976 81

The gene transcription of baculovirus has been divided in sequential phases: immediate early, delayed early, late and very late. Immediate early genes are recognized by host transcription factors and viral proteins are not necessary at this stage. Transcription of delayed early genes requires activation by products of immediate early genes. The delayed early phase is followed by the synthesis of DNA and the late gene products of the virus [14]. There is a close relation‐ ship between the DNA replication and the switch to late gene transcription, and it is believed that these events are physically connected. In the late phase, that occurs following the initiation of viral DNA replication, nucleocapsid structural proteins are synthesized, including glyco‐ protein GP64 playing a crucial role in the horizontal infection by BV [15]. During the very late phase the production of infectious BV is greatly reduced. Nucleocapsids interact with nuclear membranes and eventually become enveloped usually in groups of a few particles. Envelop‐ ment of the nucleocapsids appears to be an essential primary step in the process of occlusion of nucleocapsids by the very late protein‐polyhedrin. The occlusion continues until eventually the nucleus becomes filled with occlusion bodies. As occlusion proceeds, fibrillar structures begin to accumulate in the nucleus (sometimes also in the cytoplasm). These structures are

composed mostly of a single polypeptide named p10, which is a very late protein [16]

infection, OBs may account for over 30% of the dry weight of the larvae [20].

used for functional studies, vaccine preparations or diagnostics.

**2. Baculovirus expression vectors**

**2.1. Introduction**

The function of fibrillar structures is not clear but they may play a role in the controlled cellular disintegration in caterpillars [17,18]. In the terminal stages of infection two viral proteins, chitinase and cathepsin, act together to facilitate host cuticle breakdown [19]. After death the caterpillar liquefies and releases polyhedra which can infect other insects. At the end of the

The high levels of expression of the very late genes has been exploited to design the first vectors for foreign gene expression based on baculoviruses. They are especially suitable regarding safety (not harmful to non‐target organisms) and easy containment in the laboratory. Baculo‐ virus vectors are used to infect insect cells or larvae where high levels of recombinant proteins are produced; the eukaryotic environment provides appropriate post-translational modifica‐ tions in comparison with prokaryotic expression systems. Insect cells to be used in the baculovirus expression system are derived from lepidopteran insects and are relatively easy to grow. No control of oxygen atmosphere is required. Moreover, insect cells can be adapted to serum‐free media and production of recombinant protein can be scaled up to pilot plant or larger bioreactors [21, 22]. The lepidopteran insect cells used are also normally free of human pathogens. Thereby, the proteins produced in the baculovirus virus expression system can be

and enter the nucleus where viral genes are expressed.

**Figure 1.** On the basis of the OB morphology, baculovirus were originally divided in two major groups: the Nucleopo‐ lyhedrovirus (NPVs) and the Granulovirus (GVs). NPVs occlusion bodies are called polyhedra and their major occlusion protein is called polyhedrin and GV occlusion bodies and granules or capsules for GVs.

Based on the fusion protein present in the BVs NPVs have been further classified into two groups: type I NPVs contain GP64, a low‐pH‐dependent membrane fusion protein required for virus entry and cell‐to‐cell transmission [7,8,9,10], BVs of group II NPVs and GVs lack a homolog of GP64, and membrane fusion during viral entry is triggered by F protein [11,12]. It was found that the entry of baculoviruses in mammalian cells is mediated by GP64. In contrast, baculoviruses with F protein cannot transduce these cells [13].

The natural cycle of infection by AcMNPV in insect larvae is summarized in Figure 2. Caterpillars ingest polyhedra that contaminate their food. The polyhedrin matrix is dissolved in the alkaline environment of the larvae midgut releasing ODVs (occlusion derived virions). These virions enter midgut cells after fusion with membrane epithelial cells. The virions are uncoated and enter the nucleus where viral genes are expressed.

The gene transcription of baculovirus has been divided in sequential phases: immediate early, delayed early, late and very late. Immediate early genes are recognized by host transcription factors and viral proteins are not necessary at this stage. Transcription of delayed early genes requires activation by products of immediate early genes. The delayed early phase is followed by the synthesis of DNA and the late gene products of the virus [14]. There is a close relation‐ ship between the DNA replication and the switch to late gene transcription, and it is believed that these events are physically connected. In the late phase, that occurs following the initiation of viral DNA replication, nucleocapsid structural proteins are synthesized, including glyco‐ protein GP64 playing a crucial role in the horizontal infection by BV [15]. During the very late phase the production of infectious BV is greatly reduced. Nucleocapsids interact with nuclear membranes and eventually become enveloped usually in groups of a few particles. Envelop‐ ment of the nucleocapsids appears to be an essential primary step in the process of occlusion of nucleocapsids by the very late protein‐polyhedrin. The occlusion continues until eventually the nucleus becomes filled with occlusion bodies. As occlusion proceeds, fibrillar structures begin to accumulate in the nucleus (sometimes also in the cytoplasm). These structures are composed mostly of a single polypeptide named p10, which is a very late protein [16]

The function of fibrillar structures is not clear but they may play a role in the controlled cellular disintegration in caterpillars [17,18]. In the terminal stages of infection two viral proteins, chitinase and cathepsin, act together to facilitate host cuticle breakdown [19]. After death the caterpillar liquefies and releases polyhedra which can infect other insects. At the end of the infection, OBs may account for over 30% of the dry weight of the larvae [20].
