Baculoviruses are large, rod-shaped DNA viruses that replicate in the nucleus of insects cells.
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Baculoviruses are large, rod-shaped DNA viruses that replicate in the nucleus of insects cells. Their double-stranded, circular genomes are 80-180 kbp in size depending on the virus species and the nonoverlapping open reading frames (ORFs) are present in about equal proportions on both DNA strands. Baculoviruses are characterized by having two different virion types (Figure 1A and B): budded virions (BVs) that bud from the cell membrane and spread infection from cell to cell in an infected host insect and a second type, called occlusion-derived virus (ODV), which is assembled entirely in the nucleus of infected cells and is occluded in large proteinaceous occlusion bodies (OBs). Depending on the genus, baculoviruses occlude their nucleocapsids in granular OBs carrying only one virion (granuloviruses, genus Betabaculovirus) or in large polyhedral shaped OBs that may harbor over 100 virions (NPVs, genera Alphabaculovirus, Gammabaculovirus and Deltabaculovirus).
Figure 1. Two different virion types of baculoviruses.
In contrast to most other DNA viruses, baculovirus gene expression occurs in four phases: immediate-early, delayed-early, late and very late. In the very early phase, host-encoded RNA polymerase II transcribes immediate early-genes that activate delayed-early and late genes. In the early phase, genes are transcribed, which encode proteins required for DNA replication and late gene expression (including DNA polymerase, helicase, a four-subunit viral RNA polymerase, and the late essential factors). Proteins that prevent host defense mechanisms, such as inhibitors of apoptosis are also made in the early phase. In the late phase, the DNA is being replicated and proteins needed for virion assembly and virus budding, are produced including nucleocapsid and viral envelope proteins. In the very late phase, the virions are occluded and two proteins, polyhedrin (ca. 33 kDa) and a 10 kDa protein (P10), are produced in very high amounts. Polyhedrin forms the matrix of OBs in which the virions are embedded, while P10 is involved in release of OBs from the nucleus of infected cells toward the end of infection.
The principle of the baculovirus expression system
The P10 and polyhedrin proteins are needed to complete the infection cycle in a larval population via horizontal transmission, but are not required to produce BVs, the form of the virus that is responsible for the systemic infection of the larva and for infection of insect cells in culture. As a consequence, the polh and p10 promoters can be used to drive the expression of foreign genes in cultured cells (Figure 2). This forms the basis of the baculovirus insect cell expression system. These very late promoters, containing a canonical TAAG transcription initiation site, can also be exploited in the presence of the polh and p10 genes, by adding single or multiple promoter constructs with foreign genes into the baculovirus genome, which facilitates protein production in insect larvae. The two baculovirus species used most commonly for foreign gene expression are the above mentioned AcMNPV, the type species of the genus Alphabaculovirus, and BmNPV, another alphabaculovirus, that replicates in the silkworm. For protein production with AcMNPV vectors, insect cell cultures are used up to a 1000 L scale, while for BmNPV, cell cultures or silkworm larvae are used.
Figure 2. The principle of the baculovirus expression system.
BES as a platform for protein expression
The baculovirus expression system (BES) has been one of the versatile platforms for the production of recombinant proteins requiring multiple post-translational modifications, such as folding, oligomerization, phosphorylation, glycosylation, acylation, disulfide bond formation and proteolytic cleavage. Advances in recombinant DNA technology have facilitated application of the BES, and made it possible to express multiple proteins simultaneously in a single infection and to produce multimeric proteins sharing functional similarity with their natural analogs. Therefore, the BES has been used for the production of recombinant proteins and the construction of virus-like particles (VLPs), as well as for the development of subunit vaccines, including VLP-based vaccines. The VLP, which consists of one or more structural proteins but no viral genome, resembles the authentic virion but cannot replicate in cells. The high-quality recombinant protein expression and post-translational modifications obtained with the BES, along with its capacity to produce multiple proteins, imply that it is ideally suited to VLP production.
Challenges for protein quantity and quality
The proteins expressed in the baculovirus expression system are often of mammalian or avian origin, and the expression levels vary considerably between individual proteins. In general cytoplasmic proteins are expressed at much higher levels than secreted glycoproteins. Transmembrane proteins are even more difficult to express, let alone complex transmembrane proteins that cross the membrane several times.
Chitinase (encoded by chiA) is an enzyme that breaks down the chitin skeleton of the insect host, together with cathepsin (v-CATH), towards the end of infection, thereby liquefying the insect and releasing viral occlusion bodies. However, chitinase and cathepsin are not needed for efficient replication and high-level gene expression in cultured insect cells.
In many cases the native signal peptides of mammalian (glyco-) proteins direct the recombinant protein correctly through the ER and the Golgi system to the surface of insect cells. The recombinant proteins may be misrouted or folded incorrectly and subsequently are degraded by cellular proteases with consequent low yields.
Differences in glycosylation may affect the folding and targeting of recombinant glycoproteins and may even lead to their degradation. As a consequence of differences in glycosylation patterns the biological properties and/or the immunogenicity of the recombinant proteins may differ from native mammalian proteins. Differences in glycosylation may also induce allergic reactions.
Baculovirus infection is a lytic process, which finally leads to cell death. The fact that the virus replication cycle is lytic means that protein production is not continuous, requiring infection of new cultures frequently in fed-batch or semi-continuous bioreactor systems.
The co-expression of foldases appears to work more efficiently when performed from the same or a second baculovirus backbone than when expressed from the genome of transformed cells. This may be related to the fact that expression from the host genome is shut down late in infection when the foldases are needed. Insect foldase levels also decrease during infection.
The baculovirus expression system has grown over the years from an exotic system into mainstream technology and has become a powerful tool for recombinant protein production. The availability of baculovirus bacmids and the convenient engineering of baculovirus recombinants in E. coli have greatly added to the familiarity of the system. Fundamental research on the baculovirus itself and in the field of insect cell biology has proven very important and has allowed for tailoring of the baculovirus expression system to our needs. On the other hand, a lot has been learned about the baculovirus and the insect cells having expressed hundreds if not thousands of foreign genes. A remaining challenge is to combine various tailoring techniques for improved protein quality (folding, glycosylation, preventing degradation) and stabilizing the expression of the heterologous genes over longer periods of time.
Creative Biogene is a biotechnology company specializing in custom baculovirus production service. Our advanced technologies and highly experienced staffs are available to assist in all aspects of baculovirus transfer vector construction, recombinant bacmid DNA preparation, as well as the generation of the baculovirus in high titer.