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Baculovirus Expression Vector Systems Guide


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Baculovirus Expression Vector Systems Guide

Overview of Baculovirology

Baculoviruses are arthropod-specific viruses that contain large double-stranded circular DNA genomes of 80,000-180,000 bp. Baculovirus strains are highly species-specific and cannot propagate in any non-invertebrate host. The Baculovirus genome is replicated and transcribed in the nuclei of infected host cells where the large baculovirus DNA is packaged into rod-shaped nucleocapsids. Baculovirus nucleocapsids have a rod-shaped morphology with an average diameter of 30–60 nm and length of 250–300 nm, and are enclosed within an envelope to form a virion. Two forms of virions are produced during the course of infection: budded virions (BVs) and occlusion-derived virus (ODVs), which have identical genotypes, but distinct phenotypes (Figure 1). BVs and ODVs have the same nucleocapsid, but differ in their envelopes. The envelopes of former are derived from modified plasma membranes through nucleocapsid budding, but the envelopes of latter are formed from membranes assembled within the nucleus. There is no evidence that the hymenopteran baculoviruses produce a budded form.

Baculovirus Expression Vector Systems Guide Figure 1. Two distinct NPV phenotypes produced during lepidopteran NPV infection. (A) Budded virus (BV). (B) Occlusion-derived virus (ODV).

Because the size of these nucleocapsids is flexible, recombinant baculovirus particles can accommodate large amounts of foreign DNA. AcNPV is the most extensively studied baculovirus strain. Its entire genome has been mapped and fully sequenced. Infectious AcNPV particles enter susceptible insect cells via facilitated endocytosis or fusion, and viral DNA is uncoated in the nucleus (Figure 2). DNA replication starts about 6 h post-infection (pi). In both in vivo and in vitro conditions, the baculovirus infection cycle can be divided into two different phases, early and late. During the early phase, the infected insect cell releases extracellular virus particles (ECV) by budding off from the cell membrane of infected cells. During the late phase, occluded virus particles (OV) are assembled inside the nucleus. The OVs are embedded in a homogenous matrix made predominantly of a single protein, the polyhedrin protein. OVs are released when the infected cells lyse during the last phase of the infection cycle.

Baculovirus Expression Vector Systems Guide Figure 2. The Baculovirus life cycle in vivo and in vitro.

Baculoviruses as Expression Vectors

The main difference between the recombinant in vitro infection and the naturally occurring in vivo infection is that the naturally occurring polyhedrin gene within the wild-type baculovirus genome is replaced with a recombinant gene or cDNA. These genes are usually under the control of polyhedrin and p10 promoters. In the late phase of infection, the virions are assembled and budded recombinant virions are released. Nevertheless, during the very late phase of infection, the inserted heterologous genes are placed under the transcriptional control of the strong AcNPV polyhedrin promoter. Therefore, recombinant product is expressed in place of the naturally occurring polyhedrin protein. Commonly, the recombinant proteins are processed, modified, and targeted to the appropriate cellular locations.

The Baculovirus Expression Vector System (BEVS) is one of the most powerful and versatile eukaryotic expression systems available. The BEVS is a helper-independent viral system which has been used to express heterologous genes from a number of different sources, including fungi, bacteria, plants and viruses, in insect cells. The baculovirus vector system was originally developed for the production of high levels of recombinant proteins and vaccines using insect cell cultures. However, several studies have suggested that baculovirus can infect mammalian cells but is unable to replicate owing to transcriptional silencing of most of the baculoviral genes. Baculovirus vectors carrying mammalian regulatory elements can efficiently transduce and express transgenes in mammalian cells both in vitro and in vivo. Therefore, these properties make baculovirus vectors promising candidates for gene therapy application.

Baculoviruses infect a broad range of cell types including most of the mammalian cell lines, primary cells, adult, and embryonic stem cells. Several studies have revealed that the central nervous system, liver, prostate, eye, ovary, and testis can serve as targets for baculovirus-mediated gene delivery. Preclinical studies of cancer gene therapy have successfully been conducted in animal models with baculoviral vectors containing therapeutic genes, such as tumor suppressor genes, suicide genes, and genes encoding tumor-specific antigens.

Advantages of BEVS Technology

Baculoviruses have several distinct advantages as a gene transfer vector over other nonintegrating viral vectors such as adeno-associated virus (AAV) and adenovirus.

1) Safety

Baculoviruses are essentially nonpathogenic to plants and mammals. They have a restricted host range, which often is limited to specific invertebrate species. Because the insect cell lines are not transformed by pathogenic or infectious viruses, they can be cared for under minimal containment conditions. Helper cell lines or helper viruses are not required since the baculovirus genome contains all the genetic information.

2) Ease of scale up:

Baculovirus has a large packaging capacity (up to 38 kb). They have a broad host and tissue tropism. Baculoviruses have been reproducibly scaled up for the production of biologically active recombinant products.

3) High expression levels

Compared to other higher eukaryotic expression systems, the most distinguishing feature of the BEVS is its potential to achieve high levels of expression of a cloned gene. The BV system has been proven particularly useful in the generation of large quantities of proteins for structural analysis

4) Simultaneous expression of multiple genes

BEVS has the capability to express multiple genes simultaneously within single infected insect cells. Protein complexes that depend on dimer or multidimer formation for activity can be assembled.

5) Localization of recombinant proteins

Baculovirus-expressed recombinant proteins are commonly localized in the same subcellular compartment as the authentic protein. Nuclear proteins will be transported to the insect nucleus, membrane proteins will be anchored into the cell membrane, and secreted protein will be secreted by infected insect cells.

6) Accuracy

Baculoviruses can be propagated in insect hosts which post-translationally modify peptides in a manner similar to that of mammalian cells.

Currently, baculoviruses have been used widely in a number of biological applications such as protein expression systems, models of genetic regulatory networks and genome evolution, putative nonhuman viral vectors for gene delivery, and biological control agents against insect pests. Moreover, recombinant baculovirus vectors are attractive as novel gene therapy tools for transient and high-level transgene expression.


  1. Miele S A B, et al. Baculovirus: Molecular Insights on Their Diversity and Conservation. International Journal of Evolutionary Biology, 2011, 2011:1-15.
  2. Ikeda M, et al. Baculoviruses: diversity, evolution and manipulation of insects. Entomological Science, 2015, 18(1):1-20.
  3. Clem R J, et al. Baculoviruses: Sophisticated Pathogens of Insects. PLoS Pathogens, 2013, 9(11):e1003729.
  4. Nasimuzzaman M, et al. Purification of baculovirus vectors using heparin affinity chromatography. Molecular Therapy-Methods & Clinical Development, 2016, 3: 16071.
  5. Rohrmann G. Introduction to the baculoviruses, their taxonomy, and evolution. Baculovirus Molecular Biology, 2011: 9-22.

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