**2.2. Historical perspective**

The major component of the polyhedrovirus OB is polyhedrin. This polypeptide comprises about 25% of total cell protein in the late phase of baculovirus infection [23]. This was the main property of baculovirus that led to their consideration as expression vectors. In the first studies of baculovirus, AcMNPV polyhedrin gene was located and cloned. Then, a plasmid containing the sequences of the polyhedrin gene (polh) and its flanking regions was constructed and subsequently the polyhedrin open reading frame (ORF) was replaced with the reporter gene β‐galactosidase [24]. This is the simplest version of a transfer plasmid, which contains the strong polh promoter and upstream and downstream flanking sequences, but lacks the polyhedrin ORF which is usually replaced with the coding sequence of a foreign gene of interest. Transfer plasmid and viral DNA are cotransfected into cultured insect cells, where allelic replacement of polyhedrin can occur via homologous recombination involving the flanking viral sequences present in both DNAs.

vector [32]. As linearized parental DNA has a reduced infectivity compared with its circular counterpart, frequencies of recombinant progeny rise to about 30%. The baculoviral genome can also be modified to contain restriction sites on both sides of a cassette containing a reporter gene coding sequence (such as β‐galactosidase) under the control of a baculoviral promoter, so that double digestion with this enzyme removes the reporter gene cassette. The presence of two restriction sites reduces the frequency of undigested circular DNA genome. Moreover, if undigested or single digested‐repaired parental DNA produces progeny, those few parental plaques stain blue in the presence of X‐gal and can be easily discarded. This strategy was exploited in the AcMNPV BEV system. In this virus, the genome was modified to contain Bsu36I sites on both sides of the β‐galactosidase sequences, so that digestion with this enzyme removed the gene and also part of a virus gene (ORF 1629) that encodes a structural protein [33]. By removing part of the essential ORF 1629 gene, the virus is unable to form infectious particles efficiently even if the double digested linear DNA is repaired and recircularized in insect cells. In contrast, a process of homologous recombination repairs the deletion of ORF 1629 while simultaneously inserting the foreign gene in place of β‐galactosidase. Several commercial systems make use of the repair of the deletion in ORF 1629. Among them, Bac‐to‐ Bac® (Invitrogen) and flashBacTM (Oxford Expression Technologies) quickly gained popu‐

Genetic Engineering of Baculoviruses http://dx.doi.org/10.5772/56976 85

A major step forward in the technology of baculovirus genetic engineering has been the development of baculovirus genomes capable of replicating in a bacterial host as bacterial artificial chromosomes (Figure 4). These recombinant baculoviruses are called bacmids, and they have been modified to contain classical bacterial artificial chromosomes replicons and selection markers for selection in bacteria. BAC vectors contain a fragment of *E. coli* fertility factor (F‐ factor) replicon (*miniF*) and are maintained as circular supercoiled extrachromosomal single copy plasmid in the bacterial host [57, 58]. BACs can accept inserts up to 300 Kb in length. The principal advantage BACs have over other high insert capacity vectors like yeast artificial chromosomes (YAC) and mammalian artificial chromosomes is stability of insert propagation

Once transferred into the bacterial host, the baculovirus genome can be manipulated easily through site‐specific recombination, Rec‐A mediated homologous recombination or transpo‐ sition. Once the recombinant bacmid is generated and the presence of transgene and the absence of the parental bacmid in the bacterial colonies are verified, e.g., by PCR, the DNA from those colonies is purified and used to transfect susceptible insect cells. As was mentioned above, naked genomic DNA from baculovirus can efficiently establish infection when it reaches the cell nuclei. BV particles can be recovered from culture supernatant and used as

Various commercial transfer vectors are available and compatible with bacmid systems to allow expression of one or two proteins (*e.g*., pFastBac1TM and pFastBacDualTM from InvitrogenTM). Some are designed to add tags and signal peptide sequences fused to the

larity in the scientific community.

**2.4. Bacmid technology**

over multiple generations.

inoculum to produce high titer stocks.

This strategy was devised because the baculovirus genome is large [25]; and in vitro ligation of foreign DNA fragments with restriction enzyme‐digested viral DNA has been successful only in few cases [26, 27]. Other strategies that have been explored include enzymatic recom‐ bination in vitro [28] and homologous recombination in yeast followed by selection [29]. Although these methods are ingenious, neither has become part of the mainstream baculovirus technology.

Allelic replacement is a consequence of double homologous recombination between viral DNA and transfer plasmid, and occurs at a frequency of only about 1% [30]. Thus, the viral progeny is a mixture of recombinant and wild type virus, which needs to be resolved in plaque assays. The recombinant progeny is occlusion negative (*occ‐*) and produces polyhedrin‐ negative plaques (cell plaques with no polyhedral OBs), whereas wild‐type progeny produces polyhe‐ drin‐positive plaques (*occ+*). Clonal purification requires several plaque passages. After this, viral stocks can be produced and amplified to infect cell cultures for recombinant protein production.

#### **2.3. Strategies to simplify the isolation of recombinant baculoviruses**

As mentioned before, following the strategy described above, the proportion of the progeny virus population derived from the cotransfection experiment is less than 1% [31]. Plaque purification of recombinant clones requires a tedious search for *occ‐* plaques. Good quality microscope and experienced eye are necessary, and few recombinant viruses can be screened simultaneously because of the number of titrations required. To circumvent these problems, several modifications in the parental viral genome were carried out in order to simplify the isolation of recombinant baculoviruses, with the aim of reducing the parental virus yields in the progeny of the co‐transfected insect cells.

One of the most successful strategies is the use of a linearized parental genome (in principle, no virus can be recovered) instead of the circular viral DNA (fully infectious). The addition of a unique naturally infrequent restriction site in the baculoviral genome allows the digestion with the adequate restriction enzyme and digested parental DNA is cotransfected with transfer vector [32]. As linearized parental DNA has a reduced infectivity compared with its circular counterpart, frequencies of recombinant progeny rise to about 30%. The baculoviral genome can also be modified to contain restriction sites on both sides of a cassette containing a reporter gene coding sequence (such as β‐galactosidase) under the control of a baculoviral promoter, so that double digestion with this enzyme removes the reporter gene cassette. The presence of two restriction sites reduces the frequency of undigested circular DNA genome. Moreover, if undigested or single digested‐repaired parental DNA produces progeny, those few parental plaques stain blue in the presence of X‐gal and can be easily discarded. This strategy was exploited in the AcMNPV BEV system. In this virus, the genome was modified to contain Bsu36I sites on both sides of the β‐galactosidase sequences, so that digestion with this enzyme removed the gene and also part of a virus gene (ORF 1629) that encodes a structural protein [33]. By removing part of the essential ORF 1629 gene, the virus is unable to form infectious particles efficiently even if the double digested linear DNA is repaired and recircularized in insect cells. In contrast, a process of homologous recombination repairs the deletion of ORF 1629 while simultaneously inserting the foreign gene in place of β‐galactosidase. Several commercial systems make use of the repair of the deletion in ORF 1629. Among them, Bac‐to‐ Bac® (Invitrogen) and flashBacTM (Oxford Expression Technologies) quickly gained popu‐ larity in the scientific community.

#### **2.4. Bacmid technology**

**2.2. Historical perspective**

technology.

production.

flanking viral sequences present in both DNAs.

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

the progeny of the co‐transfected insect cells.

The major component of the polyhedrovirus OB is polyhedrin. This polypeptide comprises about 25% of total cell protein in the late phase of baculovirus infection [23]. This was the main property of baculovirus that led to their consideration as expression vectors. In the first studies of baculovirus, AcMNPV polyhedrin gene was located and cloned. Then, a plasmid containing the sequences of the polyhedrin gene (polh) and its flanking regions was constructed and subsequently the polyhedrin open reading frame (ORF) was replaced with the reporter gene β‐galactosidase [24]. This is the simplest version of a transfer plasmid, which contains the strong polh promoter and upstream and downstream flanking sequences, but lacks the polyhedrin ORF which is usually replaced with the coding sequence of a foreign gene of interest. Transfer plasmid and viral DNA are cotransfected into cultured insect cells, where allelic replacement of polyhedrin can occur via homologous recombination involving the

This strategy was devised because the baculovirus genome is large [25]; and in vitro ligation of foreign DNA fragments with restriction enzyme‐digested viral DNA has been successful only in few cases [26, 27]. Other strategies that have been explored include enzymatic recom‐ bination in vitro [28] and homologous recombination in yeast followed by selection [29]. Although these methods are ingenious, neither has become part of the mainstream baculovirus

Allelic replacement is a consequence of double homologous recombination between viral DNA and transfer plasmid, and occurs at a frequency of only about 1% [30]. Thus, the viral progeny is a mixture of recombinant and wild type virus, which needs to be resolved in plaque assays. The recombinant progeny is occlusion negative (*occ‐*) and produces polyhedrin‐ negative plaques (cell plaques with no polyhedral OBs), whereas wild‐type progeny produces polyhe‐ drin‐positive plaques (*occ+*). Clonal purification requires several plaque passages. After this, viral stocks can be produced and amplified to infect cell cultures for recombinant protein

As mentioned before, following the strategy described above, the proportion of the progeny virus population derived from the cotransfection experiment is less than 1% [31]. Plaque purification of recombinant clones requires a tedious search for *occ‐* plaques. Good quality microscope and experienced eye are necessary, and few recombinant viruses can be screened simultaneously because of the number of titrations required. To circumvent these problems, several modifications in the parental viral genome were carried out in order to simplify the isolation of recombinant baculoviruses, with the aim of reducing the parental virus yields in

One of the most successful strategies is the use of a linearized parental genome (in principle, no virus can be recovered) instead of the circular viral DNA (fully infectious). The addition of a unique naturally infrequent restriction site in the baculoviral genome allows the digestion with the adequate restriction enzyme and digested parental DNA is cotransfected with transfer

**2.3. Strategies to simplify the isolation of recombinant baculoviruses**

A major step forward in the technology of baculovirus genetic engineering has been the development of baculovirus genomes capable of replicating in a bacterial host as bacterial artificial chromosomes (Figure 4). These recombinant baculoviruses are called bacmids, and they have been modified to contain classical bacterial artificial chromosomes replicons and selection markers for selection in bacteria. BAC vectors contain a fragment of *E. coli* fertility factor (F‐ factor) replicon (*miniF*) and are maintained as circular supercoiled extrachromosomal single copy plasmid in the bacterial host [57, 58]. BACs can accept inserts up to 300 Kb in length. The principal advantage BACs have over other high insert capacity vectors like yeast artificial chromosomes (YAC) and mammalian artificial chromosomes is stability of insert propagation over multiple generations.

Once transferred into the bacterial host, the baculovirus genome can be manipulated easily through site‐specific recombination, Rec‐A mediated homologous recombination or transpo‐ sition. Once the recombinant bacmid is generated and the presence of transgene and the absence of the parental bacmid in the bacterial colonies are verified, e.g., by PCR, the DNA from those colonies is purified and used to transfect susceptible insect cells. As was mentioned above, naked genomic DNA from baculovirus can efficiently establish infection when it reaches the cell nuclei. BV particles can be recovered from culture supernatant and used as inoculum to produce high titer stocks.

Various commercial transfer vectors are available and compatible with bacmid systems to allow expression of one or two proteins (*e.g*., pFastBac1TM and pFastBacDualTM from InvitrogenTM). Some are designed to add tags and signal peptide sequences fused to the protein of interest in order to facilitate their purification. In addition, transfer vectors that are compatible with GatewayTM and TOPO® cloning technologies have been developed.

A problem frequently found when working with BAC systems is the presence of parental bacmid background even in the same colony where the recombinant bacmid is found, despite the antibiotic‐based selection and blue‐white screening. This then requires a new transforma‐ tion of bacterial cells with mixed DNA and the screening of newly replated colonies. To avoid this requirement, a negative‐selection system has been developed that makes use of the *sacB* marker [59]. When the transposon is not integrated in the bacmid genome, the bacterial cell will be killed in presence of sucrose due to the expression of *sacB* gene, which encodes an enzyme that metabolizes sucrose to a toxic compound.

The first bacmid developed contained the AcMNPV genome. Later, bacmid systems were developed for *Bombyx mori* NPV, *Helicoverpa armigera* single‐nucleocapsid nucleopolyhedro‐ virus *(HearSNPV)* [61] and *Cydia pomonella* granulovirus (CpGV) [60] (the first report of a granulovirus bacmid).

Later, bacmid technology was exploited to develop a system that allows the generation of recombinant baculoviruses with negligible background. This system relies on homologous recombination in insect cells between a transfer vector containing a gene to be expressed and a replication‐deficient AcMNPV bacmid. The deficiency of AcMNPV is due to a deletion in the essential gene *orf1629*, and homologous recombination between bacmid DNA and transfer vector (containing *orf1629*) repairs this deleted gene [62, 63, 64]. Therefore, only recombinant virus can replicate and no further selection is required, facilitating the rapid production of multiple recombinant viruses on automated platforms in a one‐step procedure. Several commercial vectors (flashbackTM, Oxford Expression Technologies Ltd., BacMagicTM (Merck), BaculoOneTM (PAA), etc.) follows this principle. All these systems claim that no plaque purification of baculovirus is required, although it is recommended (there is a possi‐ bility that defective genomes can be replicated when a replication competent viral genome resides in the same cell). Later, bacmids using this selection system were improved for protein expression, carrying additional deletions in cathepsin (*v‐cath*) and p10 gene [65].

#### **2.5. Improving protein quality and quantity in baculovirus expression systems**

#### *2.5.1. Introduction*

The proteins to be expressed using recombinant baculovirus and insect cells are commonly of mammalian origin and, as it happens in other expression systems, the expression levels and the conformation and posttranslational modifications vary among individual proteins. The principal purpose of a protein expression system is not only the production of large quantities of recombinant protein, but also the production of a recombinant protein that resembles the native protein. One of the most difficult challenges in expression systems is the expression of transmembrane proteins. The correct expression of complex transmembrane proteins that cross the membrane several times is even more difficult. In order to improve the quality in the routing, the post-translational modifications and the stability of recombinant proteins, several modifications have been carried out that address these limitations of baculovirus expression

system. It is important to notice, however, that the protein is produced in the context of a viral infection. Since certain protein processing pathways are compromised by baculovirus infection, the capacity of host cells to correctly route, fold and modify the recombinant protein is affected. This intrinsic limitation must be recognized and baculovirus expression system

**Figure 4.** Baculovirus expression vectors over time. Various methods exist to generate recombinant baculoviruses ex‐ pressing a foreign gene (gene X). Historically, recombinant baculoviruses were generated through homologous re‐ combination (1). Subsequently, linearized vectors were developed to increase the percentage of recombinants (2). Bacmid technology allowed the maintenance of defective baculoviruses as bacterial artificial chromosomes. Homolo‐ gous recombination with transfer vector in cells repairs the essential gene (3). Bacmid technology also allowed the

X

Tn7 Tn7

Tn7 Tn7

miniF Tn7 Tn7

pPolh

X

lacZ

Genetic Engineering of Baculoviruses http://dx.doi.org/10.5772/56976 87

pPolh

pPolh

a b

X X

pPolh pPolh

c d

miniF

ORF 1629

miniF

polh

X

X

pPolh

pPolh

pPolh

polh

pPolh

ORF 1629

pPolh pPolh

<sup>X</sup> <sup>X</sup>

must be regarded as a transient expression system.

generation of recombinant baculovirus by in vitro transposition (4).

protein of interest in order to facilitate their purification. In addition, transfer vectors that are compatible with GatewayTM and TOPO® cloning technologies have been developed.

A problem frequently found when working with BAC systems is the presence of parental bacmid background even in the same colony where the recombinant bacmid is found, despite the antibiotic‐based selection and blue‐white screening. This then requires a new transforma‐ tion of bacterial cells with mixed DNA and the screening of newly replated colonies. To avoid this requirement, a negative‐selection system has been developed that makes use of the *sacB* marker [59]. When the transposon is not integrated in the bacmid genome, the bacterial cell will be killed in presence of sucrose due to the expression of *sacB* gene, which encodes an

The first bacmid developed contained the AcMNPV genome. Later, bacmid systems were developed for *Bombyx mori* NPV, *Helicoverpa armigera* single‐nucleocapsid nucleopolyhedro‐ virus *(HearSNPV)* [61] and *Cydia pomonella* granulovirus (CpGV) [60] (the first report of a

Later, bacmid technology was exploited to develop a system that allows the generation of recombinant baculoviruses with negligible background. This system relies on homologous recombination in insect cells between a transfer vector containing a gene to be expressed and a replication‐deficient AcMNPV bacmid. The deficiency of AcMNPV is due to a deletion in the essential gene *orf1629*, and homologous recombination between bacmid DNA and transfer vector (containing *orf1629*) repairs this deleted gene [62, 63, 64]. Therefore, only recombinant virus can replicate and no further selection is required, facilitating the rapid production of multiple recombinant viruses on automated platforms in a one‐step procedure. Several commercial vectors (flashbackTM, Oxford Expression Technologies Ltd., BacMagicTM (Merck), BaculoOneTM (PAA), etc.) follows this principle. All these systems claim that no plaque purification of baculovirus is required, although it is recommended (there is a possi‐ bility that defective genomes can be replicated when a replication competent viral genome resides in the same cell). Later, bacmids using this selection system were improved for protein

expression, carrying additional deletions in cathepsin (*v‐cath*) and p10 gene [65].

**2.5. Improving protein quality and quantity in baculovirus expression systems**

The proteins to be expressed using recombinant baculovirus and insect cells are commonly of mammalian origin and, as it happens in other expression systems, the expression levels and the conformation and posttranslational modifications vary among individual proteins. The principal purpose of a protein expression system is not only the production of large quantities of recombinant protein, but also the production of a recombinant protein that resembles the native protein. One of the most difficult challenges in expression systems is the expression of transmembrane proteins. The correct expression of complex transmembrane proteins that cross the membrane several times is even more difficult. In order to improve the quality in the routing, the post-translational modifications and the stability of recombinant proteins, several modifications have been carried out that address these limitations of baculovirus expression

enzyme that metabolizes sucrose to a toxic compound.

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

granulovirus bacmid).

*2.5.1. Introduction*

**Figure 4.** Baculovirus expression vectors over time. Various methods exist to generate recombinant baculoviruses ex‐ pressing a foreign gene (gene X). Historically, recombinant baculoviruses were generated through homologous re‐ combination (1). Subsequently, linearized vectors were developed to increase the percentage of recombinants (2). Bacmid technology allowed the maintenance of defective baculoviruses as bacterial artificial chromosomes. Homolo‐ gous recombination with transfer vector in cells repairs the essential gene (3). Bacmid technology also allowed the generation of recombinant baculovirus by in vitro transposition (4).

system. It is important to notice, however, that the protein is produced in the context of a viral infection. Since certain protein processing pathways are compromised by baculovirus infection, the capacity of host cells to correctly route, fold and modify the recombinant protein is affected. This intrinsic limitation must be recognized and baculovirus expression system must be regarded as a transient expression system.

#### *2.5.2. Heterologous DNA properties and codon usage*

Although the promoter elements that control the transcription of the heterologous gene are derived from baculovirus, it is important to consider the effect of introducing heterologous or artificial 5´ and 3´untranslated regions (UTR). The 5´UTRs of baculovirus are short AT‐rich sequences. Therefore, the introduction of GC‐rich sequences upstream of the ORF may have a negative effect on the heterologous gene transcriptional levels. The choice of 3´UTR, including polyA sequences may also determine the heterologous gene expression levels. As expected, p10 polyA signals are more efficient than the widely used SV40 terminator [66]. There are no in‐depth studies comparing the influence of codon usage on translation levels in baculovirus expression systems. However, no strong bias in alternative codon frequency has been observed in baculovirus coding sequences, suggesting that the codon optimization is unlikely to improve significantly the translation levels [67].

system is that it can produce glycosylated proteins. However, the protein glycosylation pathways of lepidopteran cells differ from those of higher eukaryotes [73]. N‐glycosylation begins in insect cells with the transfer of the oligosaccharide Glc3Man3GlcNAc2 (where Glc, Man and GlcNAc refer to glucose, mannose and N‐acetylglucosamine, respectively) from a lipid complex to an asparagine residue in the polypeptide chain in the ER lumen. As the protein passes through the ER and Golgi system, enzymes trim and add different sugar moieties to this N‐linked glycan. In this step is where insect and mammalian cells start to vary. This results in glycoproteins with simple oligomannose sugar chains in insects, while in mammals complex

Genetic Engineering of Baculoviruses http://dx.doi.org/10.5772/56976 89

Differences in glycosylation patterns may affect the folding and targeting of recombinant glycoproteins and their immunological properties. Moreover, differences in glycosylation may

To overcome the limitations of the baculovirus insect‐cell system in glycosylation, a series of transgenic cell lines derived from lepidopteran Sf9 and High Five cells expressing genes for the enzymes required to produce the complex mammalian glycosylation patterns were developed [75, 76]. Genes incorporated include bovine β‐1,4‐galactosyl transferase and rat α‐ 2,6 sialyltransferase. The introduction of these enzyme resulted in the incorporation of

Proteins to be secreted are translated in the cytosol and can translocate across the ER membrane either by a cotraslational or post‐traslational mechanism. Transport in mammalian cells is primarily cotraslational, and in yeast both post‐traslational and cotraslational mechanisms are used. In insect cells, the predominant mechanism is still not known. In mammalian cells, the cytosolic chaperone hsp70 is believed to contribute to the traslocation of proteins by interacting with nascent polypeptides and preventing their aggregation. Coexpression of immunoglobu‐ lin G (IgG) and human hsp70 resulted in higher levels of soluble IgG precursor. As a conse‐

In the ER chaperones also assist the folding of polypeptides by preventing improper aggre‐ gations and conformations. In mammalian cells, immunoglobulin heavy chain binding protein (BiP) is an ER chaperone that interacts with several polypeptides destined for secretion and may be involved in the translocation or proteins across the ER membrane. When recombinant BiP was coexpressed in insect cells with IgG the soluble and secreted IgG levels were increased [78]. Other additional chaperones, such as calnexin and calreticulin, can also assist folding and assembly of membrane proteins in BEVs. Catalytic enzymes in the ER also collaborate by accelerating the folding. Disulfide bond formation occurs in the oxidizing ER compartment, with the catalytic action of protein disulfide isomerase (PDI). Studies demonstrated that overexpression of PDI increases the folding and secretion of IgG in insect cells [79]. It has been observed that co‐expression of foldases appears to work more efficiently when the corre‐ sponding genes are provided by the baculovirus vector than those integrated in the genome of transgenic cell lines. This observation may be related to the phenomenon of host genome

transcriptional shut down known to occur during baculovirus infection.

sugar groups with terminal sialic acids are added.

galactosyl and sialyl residues in the produced proteins.

*2.5.6. Expression of cytosolic and ER processing enzymes*

quence, mature IgG secreted levels increased [77].

even lead to protein degradation [74].

#### *2.5.3. Deletion of baculovirus genes to prevent proteolytic cleavage*

Chitinase, the product of the gene *chiA*, is an enzyme that breaks down the chitin exoskeleton of the insect host, together with cathepsin (V‐CATH, encoded by *v‐cath*) at the end of the infection, ensuring the dispersal of the viral occlusion bodies [68]. As those genes have a specific function in the context of the infection in the insect, they are not required for the propagation of the virus in cultured insect cells. Chitinase is produced at high levels and stored in the endoplasmic reticulum, and it may interfere in the secretory apparatus of the host cell. On the other hand, cathepsin is a protease that is made as an inactive precursor (PRO‐V‐ CATH). PRO‐V‐CATH can be activated when preparing protein samples for SDS‐PAGE, leading to the degradation of the recombinant protein. Bacmids were developed with deletions in the genes *chiA* and *v‐ cath*, resulting in higher levels of secreted protein [69, 70].

#### *2.5.4. Secretion of proteins*

Many secretory pathway proteins have N‐terminal signal peptides that direct the protein correctly through the ER and the Golgi system and ultimately to the surface of the cell. If the signal peptide is not adequately recognized, the protein may be not targeted to the cell surface, and the misfolded protein is also prone to degradation as it may be recognized by quality control systems [71].

Native signal peptides of mammalian proteins may be replaced by signal peptides derived from insect proteins such as the signal peptide of honey bee melittin [72] or derived from baculovirus proteins, such as the GP64 signal peptide. Although the introduction of insect signal peptides normally targets the protein to cell surface, it does not always lead to a correct folding of the protein.

#### *2.5.5. Glycosylation of proteins in the baculovirus‐insect cell system*

Glycosylation is a common covalent chemical modification that can affect many protein properties, including intracellular trafficking, biological function, immunological properties and biochemical stability. One of the most advantageous features of the baculovirus‐insect cell system is that it can produce glycosylated proteins. However, the protein glycosylation pathways of lepidopteran cells differ from those of higher eukaryotes [73]. N‐glycosylation begins in insect cells with the transfer of the oligosaccharide Glc3Man3GlcNAc2 (where Glc, Man and GlcNAc refer to glucose, mannose and N‐acetylglucosamine, respectively) from a lipid complex to an asparagine residue in the polypeptide chain in the ER lumen. As the protein passes through the ER and Golgi system, enzymes trim and add different sugar moieties to this N‐linked glycan. In this step is where insect and mammalian cells start to vary. This results in glycoproteins with simple oligomannose sugar chains in insects, while in mammals complex sugar groups with terminal sialic acids are added.

Differences in glycosylation patterns may affect the folding and targeting of recombinant glycoproteins and their immunological properties. Moreover, differences in glycosylation may even lead to protein degradation [74].

To overcome the limitations of the baculovirus insect‐cell system in glycosylation, a series of transgenic cell lines derived from lepidopteran Sf9 and High Five cells expressing genes for the enzymes required to produce the complex mammalian glycosylation patterns were developed [75, 76]. Genes incorporated include bovine β‐1,4‐galactosyl transferase and rat α‐ 2,6 sialyltransferase. The introduction of these enzyme resulted in the incorporation of galactosyl and sialyl residues in the produced proteins.

#### *2.5.6. Expression of cytosolic and ER processing enzymes*

*2.5.2. Heterologous DNA properties and codon usage*

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

unlikely to improve significantly the translation levels [67].

*2.5.3. Deletion of baculovirus genes to prevent proteolytic cleavage*

*2.5.5. Glycosylation of proteins in the baculovirus‐insect cell system*

*2.5.4. Secretion of proteins*

control systems [71].

folding of the protein.

Although the promoter elements that control the transcription of the heterologous gene are derived from baculovirus, it is important to consider the effect of introducing heterologous or artificial 5´ and 3´untranslated regions (UTR). The 5´UTRs of baculovirus are short AT‐rich sequences. Therefore, the introduction of GC‐rich sequences upstream of the ORF may have a negative effect on the heterologous gene transcriptional levels. The choice of 3´UTR, including polyA sequences may also determine the heterologous gene expression levels. As expected, p10 polyA signals are more efficient than the widely used SV40 terminator [66]. There are no in‐depth studies comparing the influence of codon usage on translation levels in baculovirus expression systems. However, no strong bias in alternative codon frequency has been observed in baculovirus coding sequences, suggesting that the codon optimization is

Chitinase, the product of the gene *chiA*, is an enzyme that breaks down the chitin exoskeleton of the insect host, together with cathepsin (V‐CATH, encoded by *v‐cath*) at the end of the infection, ensuring the dispersal of the viral occlusion bodies [68]. As those genes have a specific function in the context of the infection in the insect, they are not required for the propagation of the virus in cultured insect cells. Chitinase is produced at high levels and stored in the endoplasmic reticulum, and it may interfere in the secretory apparatus of the host cell. On the other hand, cathepsin is a protease that is made as an inactive precursor (PRO‐V‐ CATH). PRO‐V‐CATH can be activated when preparing protein samples for SDS‐PAGE, leading to the degradation of the recombinant protein. Bacmids were developed with deletions

Many secretory pathway proteins have N‐terminal signal peptides that direct the protein correctly through the ER and the Golgi system and ultimately to the surface of the cell. If the signal peptide is not adequately recognized, the protein may be not targeted to the cell surface, and the misfolded protein is also prone to degradation as it may be recognized by quality

Native signal peptides of mammalian proteins may be replaced by signal peptides derived from insect proteins such as the signal peptide of honey bee melittin [72] or derived from baculovirus proteins, such as the GP64 signal peptide. Although the introduction of insect signal peptides normally targets the protein to cell surface, it does not always lead to a correct

Glycosylation is a common covalent chemical modification that can affect many protein properties, including intracellular trafficking, biological function, immunological properties and biochemical stability. One of the most advantageous features of the baculovirus‐insect cell

in the genes *chiA* and *v‐ cath*, resulting in higher levels of secreted protein [69, 70].

Proteins to be secreted are translated in the cytosol and can translocate across the ER membrane either by a cotraslational or post‐traslational mechanism. Transport in mammalian cells is primarily cotraslational, and in yeast both post‐traslational and cotraslational mechanisms are used. In insect cells, the predominant mechanism is still not known. In mammalian cells, the cytosolic chaperone hsp70 is believed to contribute to the traslocation of proteins by interacting with nascent polypeptides and preventing their aggregation. Coexpression of immunoglobu‐ lin G (IgG) and human hsp70 resulted in higher levels of soluble IgG precursor. As a conse‐ quence, mature IgG secreted levels increased [77].

In the ER chaperones also assist the folding of polypeptides by preventing improper aggre‐ gations and conformations. In mammalian cells, immunoglobulin heavy chain binding protein (BiP) is an ER chaperone that interacts with several polypeptides destined for secretion and may be involved in the translocation or proteins across the ER membrane. When recombinant BiP was coexpressed in insect cells with IgG the soluble and secreted IgG levels were increased [78]. Other additional chaperones, such as calnexin and calreticulin, can also assist folding and assembly of membrane proteins in BEVs. Catalytic enzymes in the ER also collaborate by accelerating the folding. Disulfide bond formation occurs in the oxidizing ER compartment, with the catalytic action of protein disulfide isomerase (PDI). Studies demonstrated that overexpression of PDI increases the folding and secretion of IgG in insect cells [79]. It has been observed that co‐expression of foldases appears to work more efficiently when the corre‐ sponding genes are provided by the baculovirus vector than those integrated in the genome of transgenic cell lines. This observation may be related to the phenomenon of host genome transcriptional shut down known to occur during baculovirus infection.

#### **2.6. Improving baculovirus genome stability**

A major drawback that limits the application of baculovirus for large‐scale production is the accumulation of defective interfering (DI) particles upon serial viral cell culture passages. DI particles are not able to propagate autonomously due to deletion of large portions of their genomic DNA, but can co‐propagate in the presence of viable virus [80]. Since deletion often includes the inserted foreign gene of interest, when DI particles proportion increases, re‐ combinant protein expression levels decreases. The accumulation of DI particles can be reduced by the practice of infecting at low multiplicities of infection (MOI).

**3.2. Genetic improvement of baculovirus insecticides**

virus causes the infected larvae to rapidly lose water.

killing of larvae and significant reduction in food consumption.

developed.

specificity.

In the search of increasing the commercial fitness of baculovirus as bioinsecticides, strategies to improve the baculovirus pesticide parameters by means of genetic engineering were

Genetic Engineering of Baculoviruses http://dx.doi.org/10.5772/56976 91

Slow action of baculoviruses often limits its practical application and many strategies aimed to improving the timing of the pest killing or paralyzation by baculovirus. The first strategies were based on the interference of host physiology with insect hormones. When a diuretic hormone gene was introduced into *B. mori* baculovirus genome, recombinant BmNPV killed larvae about 20% faster than wild type virus [38]. The expression of this hormone by baculo‐

Another strategy was based upon the control of juvenile hormone. In lepidoptera, this hormone controls the onset of metamorphosis at the final molt. The expression of juvenile hormone esterase decreases the concentration of the hormone [39, 40]. A reduction in the levels of juvenile hormone (JH) early in the last larval instar has been shown to initiate metamor‐ phosis and lead to a cessation of feeding behavior. If this juvenile hormone esterase (JHE) is inhibited, the concentration of JH remains high enough to keep the larva in the feeding stage, resulting in giant insects. Another approach used consists in the deletion of the virus‐encoded ecdysteroid glucosyltransferase gene [41]. The product of the *egt* gene normally prevents larval molting during infection increasing feeding activity of infected larvae. The EGT enzyme inactivates hormone ecdysone by transferring sugar molecules. The inactivation of this hormone results in an increased food consumption, allowing the virus to maximize the viral progeny. The infection with an *egt* defective recombinant AcMNPV resulted in a 30% faster

The degree of improvement that can be achieved by gene deletion alone appears limited. For this reason, several research lines have focused on the use of gene insertion technology in order

Among the strategies that have been explored to date, the insertion of insect‐specific toxins is the most promising one for development of commercially viable baculovirus insecticides [42]. In nature, insect predators and parasites use venoms to immobilize their prey. Although arthropod venoms are composed of a mixture of toxins that may have activity against organisms other than insects, it is possible to isolate genes that target insects with high

Although the first experiments using an insect‐specific toxin of the scorpion *Buthus eupeus* [42] did not show an improvement in the speed of action of the recombinant baculovirus, the use of other scorpion toxin genes resulted in significant enhancement of virus insecticidal per‐ formance. One of the most promising insect‐specific toxins used for the generation of recombi‐ nant baculovirus is the product of the gene AaIT of the scorpion *Androctonus australis*. The product of this gene is a small peptide (70 amino acids) that interacts with voltage‐ dependent sodium channels causing rapid paralysis in insects. Moreover, AaIT has no activity on vertebrate nervous tissue and is nontoxic to mice. When AaIT toxin was introduced into

to achieve more substantial improvement in the performance of viral insecticides.

Genetic engineering strategies have been developed to prevent the accumulation of DI particles. In *Spodoptera exigua* MNPV (SeMNPV), it has been observed that DI particles are enriched in a non‐*hr ori* fragment. Removal of this *non‐hr ori* from the genome of the baculovirus prevented the formation of DI particles up to 20 cell culture passages [81]. Removal of an AcMNPV *non‐ hr ori* had the same effects on genomic stability. It was also observed that when a large foreign fragment of DNA is cloned in baculovirus DNA genome in which no selection pressure exists, the addition of an *hr* (which functions as origin of replication) may prevent the loss of the foreign DNA [82].

## **3. Baculoviruses as bioinsecticides**

#### **3.1. Introduction**

The basis of modern baculovirology was stimulated by the potential utility of baculoviruses to control insect pests [34]. Baculoviruses are highly infectious and selective pathogens (their host range is usually limited to one species), are very safe to people and wildlife and long term crop protection can be established [35]. Despite these advantageous features, the application of baculovirus as bioinsecticides has not still matched their potential. Although the use of baculovirus bioinsecticides was hampered by their slow speed of action when compared with fast‐killing chemical insecticides, they gained increasing acceptance as they were considered for long term protection of crops, in the framework of integrated pest management.

Up to date, the most successful project was implemented in Brazil where over two million hectares of soybean were controlled by baculovirus AgMNPV [36, 37]. However, it is important to notice that a series of factors contributed to the success of AgMNPV as bioinsecticide. First, AgMNPV is highly pathogenic and only one application is sufficient to control the pest over the production cycle. In second place, *Anticarsia gemmatalis* was the most important plague in soybean crops in Brazil, and other plagues did not cause significant economic damage. Finally, the application of AgMNPV was promoted by Brazilian state and the integrated pest man‐ agement governmental programs facilitated the public acceptance of alternatives to chemical insecticides. Despite this favorable unique context, the success of Brazilian project revitalized the interest in baculovirus as bioinsecticides and many countries and private companies begun to develop new programs of baculovirus control and the search of novel baculoviruses.

## **3.2. Genetic improvement of baculovirus insecticides**

**2.6. Improving baculovirus genome stability**

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

loss of the foreign DNA [82].

**3.1. Introduction**

**3. Baculoviruses as bioinsecticides**

A major drawback that limits the application of baculovirus for large‐scale production is the accumulation of defective interfering (DI) particles upon serial viral cell culture passages. DI particles are not able to propagate autonomously due to deletion of large portions of their genomic DNA, but can co‐propagate in the presence of viable virus [80]. Since deletion often includes the inserted foreign gene of interest, when DI particles proportion increases, re‐ combinant protein expression levels decreases. The accumulation of DI particles can be

Genetic engineering strategies have been developed to prevent the accumulation of DI particles. In *Spodoptera exigua* MNPV (SeMNPV), it has been observed that DI particles are enriched in a non‐*hr ori* fragment. Removal of this *non‐hr ori* from the genome of the baculovirus prevented the formation of DI particles up to 20 cell culture passages [81]. Removal of an AcMNPV *non‐ hr ori* had the same effects on genomic stability. It was also observed that when a large foreign fragment of DNA is cloned in baculovirus DNA genome in which no selection pressure exists, the addition of an *hr* (which functions as origin of replication) may prevent the

The basis of modern baculovirology was stimulated by the potential utility of baculoviruses to control insect pests [34]. Baculoviruses are highly infectious and selective pathogens (their host range is usually limited to one species), are very safe to people and wildlife and long term crop protection can be established [35]. Despite these advantageous features, the application of baculovirus as bioinsecticides has not still matched their potential. Although the use of baculovirus bioinsecticides was hampered by their slow speed of action when compared with fast‐killing chemical insecticides, they gained increasing acceptance as they were considered

Up to date, the most successful project was implemented in Brazil where over two million hectares of soybean were controlled by baculovirus AgMNPV [36, 37]. However, it is important to notice that a series of factors contributed to the success of AgMNPV as bioinsecticide. First, AgMNPV is highly pathogenic and only one application is sufficient to control the pest over the production cycle. In second place, *Anticarsia gemmatalis* was the most important plague in soybean crops in Brazil, and other plagues did not cause significant economic damage. Finally, the application of AgMNPV was promoted by Brazilian state and the integrated pest man‐ agement governmental programs facilitated the public acceptance of alternatives to chemical insecticides. Despite this favorable unique context, the success of Brazilian project revitalized the interest in baculovirus as bioinsecticides and many countries and private companies begun to develop new programs of baculovirus control and the search of novel baculoviruses.

for long term protection of crops, in the framework of integrated pest management.

reduced by the practice of infecting at low multiplicities of infection (MOI).

In the search of increasing the commercial fitness of baculovirus as bioinsecticides, strategies to improve the baculovirus pesticide parameters by means of genetic engineering were developed.

Slow action of baculoviruses often limits its practical application and many strategies aimed to improving the timing of the pest killing or paralyzation by baculovirus. The first strategies were based on the interference of host physiology with insect hormones. When a diuretic hormone gene was introduced into *B. mori* baculovirus genome, recombinant BmNPV killed larvae about 20% faster than wild type virus [38]. The expression of this hormone by baculo‐ virus causes the infected larvae to rapidly lose water.

Another strategy was based upon the control of juvenile hormone. In lepidoptera, this hormone controls the onset of metamorphosis at the final molt. The expression of juvenile hormone esterase decreases the concentration of the hormone [39, 40]. A reduction in the levels of juvenile hormone (JH) early in the last larval instar has been shown to initiate metamor‐ phosis and lead to a cessation of feeding behavior. If this juvenile hormone esterase (JHE) is inhibited, the concentration of JH remains high enough to keep the larva in the feeding stage, resulting in giant insects. Another approach used consists in the deletion of the virus‐encoded ecdysteroid glucosyltransferase gene [41]. The product of the *egt* gene normally prevents larval molting during infection increasing feeding activity of infected larvae. The EGT enzyme inactivates hormone ecdysone by transferring sugar molecules. The inactivation of this hormone results in an increased food consumption, allowing the virus to maximize the viral progeny. The infection with an *egt* defective recombinant AcMNPV resulted in a 30% faster killing of larvae and significant reduction in food consumption.

The degree of improvement that can be achieved by gene deletion alone appears limited. For this reason, several research lines have focused on the use of gene insertion technology in order to achieve more substantial improvement in the performance of viral insecticides.

Among the strategies that have been explored to date, the insertion of insect‐specific toxins is the most promising one for development of commercially viable baculovirus insecticides [42]. In nature, insect predators and parasites use venoms to immobilize their prey. Although arthropod venoms are composed of a mixture of toxins that may have activity against organisms other than insects, it is possible to isolate genes that target insects with high specificity.

Although the first experiments using an insect‐specific toxin of the scorpion *Buthus eupeus* [42] did not show an improvement in the speed of action of the recombinant baculovirus, the use of other scorpion toxin genes resulted in significant enhancement of virus insecticidal per‐ formance. One of the most promising insect‐specific toxins used for the generation of recombi‐ nant baculovirus is the product of the gene AaIT of the scorpion *Androctonus australis*. The product of this gene is a small peptide (70 amino acids) that interacts with voltage‐ dependent sodium channels causing rapid paralysis in insects. Moreover, AaIT has no activity on vertebrate nervous tissue and is nontoxic to mice. When AaIT toxin was introduced into AcMNPV, the speed of kill increased by about 40% and the feeding damage was reduced by about 60% [43].

nonpermissive insect cells, reporter gene expression was observed from early baculovirus promoters, but expression from very late baculovirus promoters was limited. Expression from late baculovirus promoters varied among nonpermissive insect cell lines. As mentioned before, it was established that the transcription from late baculovirus promoters requires the viral DNA replication [53]. These findings indicate that in nonpermissive insect cells viral DNA is delivered to the nucleus, the site of baculovirus replication, although replication is restricted in a cell specific manner. For this reason, the viral genes that determine the host range are likely

Genetic Engineering of Baculoviruses http://dx.doi.org/10.5772/56976 93

One of the first steps forward in baculovirus host range alteration was the generation of a recombinant AcMNPV capable of replicate in nonpermissive *B. mori* cells and larvae. This was achieved by replacing the endogenous *p143* gene, which encodes an essential protein with homology to DNA helicases by a hybrid *p143* gene [54]. The hybrid *p143* gene resulted from the homologous recombination between AcMNPV and BmNPV *p143* genes, and differed from AcMNPV *p143* only in four amino acids. How these changes in *p143* affected AcMNPV host range is still not well understood. Infection of *B. mori* BmN cells by wild type AcMNPV induces protein synthesis arrest [55]. This suggests that AcMNPV *p143* or perturbations in the cell caused by AcMNPV *p143* or its activity may induce a cellular response. This example dem‐ onstrates that baculovirus host range can be manipulated through genetic engineering. However, it is important to notice that BmNPV and AcMNPV are closely related baculoviruses showing on average ORF amino acid sequence identities of about 93%. Although deletion of a gene critical for replication in one host can reduce the virus host range, in many cases, the insertion or modification of a single gene will not be sufficient to expand host range. The expanded AcMNPV host range resulting from *p143* recombination with BmNPV is probably a singular case. Functional complementation studies have conducted to the identification of other viral elements that may result in host‐ specific interaction. Those elements include the homologous regions (*hrs). Hrs* consist of repeated units of about 70 base pairs with an imperfect 30 base pairs palindrome near their center, and have been implicated both as transcriptional enhancers and origins of DNA replication for a number of baculoviruses. It was demonstrated that *hrs* interact with host and viral factors in a species‐specific way. In an interesting work baculoviruses were analyzed by bioinformatics in the search of genes subject to positive selection pressure (when the rate of nonsynonymous substitutions per potential nonsynony‐ mous site in a gene is greater than the rate of synonymous substitutions per potential synon‐ ymous site, the gene is said to be undergoing positive selection). Since most genes appear to be subject to negative selection most of the time, this method can be used to identify viral genes

Another relevant topic to be addressed in the development of baculovirus recombinants with expanded host range is the selection of appropriate promoters for the expression of heterolo‐ gous genes. If selected candidate genes for expansion of the host range are to be incorporated in the baculovirus genome under the control of their own promoters, it is necessary to evaluate

to be related with the process of DNA replication.

involved in adapting to new or current hosts [56].

the functionality of these promoters in this context.

Another paralytic toxin that holds promise is the TxP‐I toxin, a component of the venom of the predatorystrawitchmite*Pyemotestritici*[44,45].Themechanismof actionthis toxinhasnotbeen studied in depth, although it is related to voltage‐dependent calcium channels (VDCC). The mean time to death of larvae infected with AcMNPV recombinant baculovirus expressing TxP‐ Iunderthe controlofp10verylatepromoterwas reducedby50‐60%comparedtolarvae infected with the wild‐type strain, depending on virus dose and larval instar [46].

The choice of the promoter that controls the transcription of the heterologous toxic gene is very important. Although *polh* and *p10* very late promotes provides high levels of transcription, early and late viral promoters or constitutive promoters can result in an earlier accumulation of the toxin, causing more significant reductions in the speed of paralysis of the larvae. A chimeric promoter constructed by insertion of the p6.9 promoter downstream of the *polh* promoter was found to be more effective than *polh* promoter alone [47]. Another promoter tested was the constitutive *Drosophila hsp70* heat‐shock protein gene promoter [48]. Despite the lower levels of toxin accumulation, the results obtained with this promoter were comparable to those obtained with the p6.9 promoter. The choice of the promoter must be considered from a biosafety perspective. Evidence indicates that recombinant baculoviruses expressing toxin genes are not pathogenic to vertebrates, and that the probability of horizontal transfer of the toxin gene to vertebrates is very low. Moreover, as it was mentioned above, specific arthropod toxins have no effect on vertebrate neural system. Despite these arguments, it is desirable to select promoters that are not functional in vertebrates.

#### **3.3. Strategies for modifying host range of baculoviruses**

A primary advantage of baculovirus bioinsecticides is their host specificity. In contrast to chemical insecticides that may harm vertebrates or kill arthropods indiscriminately, baculo‐ viruses target specific populations of insect pests. This feature makes them compatible with classical biological controls in integrated pest management strategies and makes particularly useful for controlling insect pests in environmentally sensitive areas. Although bioinsecticides are attractive from an ecological perspective, their limited host range is undesirable from an economical point of view. Since many different baculoviruses may be needed to control complexes of simultaneous insect pests, costs would be excessively high. For this reason, many researchers have studied the possibility of modifying the baculovirus host range while maintaining their safety for vertebrates and nontarget arthropods.

#### **3.4. Determinants of virus host range**

The host range of any virus is determined by its ability to enter the cells and tissues of a host organism, replicate and release new infectious virus particles. The virus host range is fre‐ quently determined by the presence of suitable receptors that facilitate virus attachment and entry into a host cell. This does not appear to be the case for baculoviruses. Baculoviruses are able to enter nonpermissive insect and even mammalian cells. This indicates that if receptors are used by baculoviruses, they are common to insect and mammalian cells [49, 50, 51, 52]. In nonpermissive insect cells, reporter gene expression was observed from early baculovirus promoters, but expression from very late baculovirus promoters was limited. Expression from late baculovirus promoters varied among nonpermissive insect cell lines. As mentioned before, it was established that the transcription from late baculovirus promoters requires the viral DNA replication [53]. These findings indicate that in nonpermissive insect cells viral DNA is delivered to the nucleus, the site of baculovirus replication, although replication is restricted in a cell specific manner. For this reason, the viral genes that determine the host range are likely to be related with the process of DNA replication.

AcMNPV, the speed of kill increased by about 40% and the feeding damage was reduced by

Another paralytic toxin that holds promise is the TxP‐I toxin, a component of the venom of the predatorystrawitchmite*Pyemotestritici*[44,45].Themechanismof actionthis toxinhasnotbeen studied in depth, although it is related to voltage‐dependent calcium channels (VDCC). The mean time to death of larvae infected with AcMNPV recombinant baculovirus expressing TxP‐ Iunderthe controlofp10verylatepromoterwas reducedby50‐60%comparedtolarvae infected

The choice of the promoter that controls the transcription of the heterologous toxic gene is very important. Although *polh* and *p10* very late promotes provides high levels of transcription, early and late viral promoters or constitutive promoters can result in an earlier accumulation of the toxin, causing more significant reductions in the speed of paralysis of the larvae. A chimeric promoter constructed by insertion of the p6.9 promoter downstream of the *polh* promoter was found to be more effective than *polh* promoter alone [47]. Another promoter tested was the constitutive *Drosophila hsp70* heat‐shock protein gene promoter [48]. Despite the lower levels of toxin accumulation, the results obtained with this promoter were comparable to those obtained with the p6.9 promoter. The choice of the promoter must be considered from a biosafety perspective. Evidence indicates that recombinant baculoviruses expressing toxin genes are not pathogenic to vertebrates, and that the probability of horizontal transfer of the toxin gene to vertebrates is very low. Moreover, as it was mentioned above, specific arthropod toxins have no effect on vertebrate neural system. Despite these arguments, it is desirable to

A primary advantage of baculovirus bioinsecticides is their host specificity. In contrast to chemical insecticides that may harm vertebrates or kill arthropods indiscriminately, baculo‐ viruses target specific populations of insect pests. This feature makes them compatible with classical biological controls in integrated pest management strategies and makes particularly useful for controlling insect pests in environmentally sensitive areas. Although bioinsecticides are attractive from an ecological perspective, their limited host range is undesirable from an economical point of view. Since many different baculoviruses may be needed to control complexes of simultaneous insect pests, costs would be excessively high. For this reason, many researchers have studied the possibility of modifying the baculovirus host range while

The host range of any virus is determined by its ability to enter the cells and tissues of a host organism, replicate and release new infectious virus particles. The virus host range is fre‐ quently determined by the presence of suitable receptors that facilitate virus attachment and entry into a host cell. This does not appear to be the case for baculoviruses. Baculoviruses are able to enter nonpermissive insect and even mammalian cells. This indicates that if receptors are used by baculoviruses, they are common to insect and mammalian cells [49, 50, 51, 52]. In

with the wild‐type strain, depending on virus dose and larval instar [46].

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

select promoters that are not functional in vertebrates.

**3.3. Strategies for modifying host range of baculoviruses**

maintaining their safety for vertebrates and nontarget arthropods.

**3.4. Determinants of virus host range**

about 60% [43].

One of the first steps forward in baculovirus host range alteration was the generation of a recombinant AcMNPV capable of replicate in nonpermissive *B. mori* cells and larvae. This was achieved by replacing the endogenous *p143* gene, which encodes an essential protein with homology to DNA helicases by a hybrid *p143* gene [54]. The hybrid *p143* gene resulted from the homologous recombination between AcMNPV and BmNPV *p143* genes, and differed from AcMNPV *p143* only in four amino acids. How these changes in *p143* affected AcMNPV host range is still not well understood. Infection of *B. mori* BmN cells by wild type AcMNPV induces protein synthesis arrest [55]. This suggests that AcMNPV *p143* or perturbations in the cell caused by AcMNPV *p143* or its activity may induce a cellular response. This example dem‐ onstrates that baculovirus host range can be manipulated through genetic engineering. However, it is important to notice that BmNPV and AcMNPV are closely related baculoviruses showing on average ORF amino acid sequence identities of about 93%. Although deletion of a gene critical for replication in one host can reduce the virus host range, in many cases, the insertion or modification of a single gene will not be sufficient to expand host range. The expanded AcMNPV host range resulting from *p143* recombination with BmNPV is probably a singular case. Functional complementation studies have conducted to the identification of other viral elements that may result in host‐ specific interaction. Those elements include the homologous regions (*hrs). Hrs* consist of repeated units of about 70 base pairs with an imperfect 30 base pairs palindrome near their center, and have been implicated both as transcriptional enhancers and origins of DNA replication for a number of baculoviruses. It was demonstrated that *hrs* interact with host and viral factors in a species‐specific way. In an interesting work baculoviruses were analyzed by bioinformatics in the search of genes subject to positive selection pressure (when the rate of nonsynonymous substitutions per potential nonsynony‐ mous site in a gene is greater than the rate of synonymous substitutions per potential synon‐ ymous site, the gene is said to be undergoing positive selection). Since most genes appear to be subject to negative selection most of the time, this method can be used to identify viral genes involved in adapting to new or current hosts [56].

Another relevant topic to be addressed in the development of baculovirus recombinants with expanded host range is the selection of appropriate promoters for the expression of heterolo‐ gous genes. If selected candidate genes for expansion of the host range are to be incorporated in the baculovirus genome under the control of their own promoters, it is necessary to evaluate the functionality of these promoters in this context.
