**2. Baculoviral fusion proteins**

Entry of enveloped viruses into host cells requires fusion of the viral envelope with the cytoplasmic membrane by the action of viral envelope fusion proteins. If the fusion occurs at the cell surface, viral fusion proteins typically act at neutral pH. On the other hand, in receptormediated endocytosis the major fusion protein activity is most often observed at the acidic endosomal pH (Monsma & Blissard, 1995).

In general, baculovirus fusion proteins mediate the membrane fusion at the late endosomal phase. For this reason, the major fusogenic activity was observed at low pH. Although it has been possible to identify which are the proteins that build fusogenic function, which is the cell receptor that recognizes these proteins remains a mystery.

Baculoviruses can be divided into two different groups according to the surface glycoprotein they use to mediate the fusion between the endosomal membrane and the viral envelope. One group is composed by viruses that use Gp64 as its fusogenic protein whereas the other group uses the F protein to mediate membrane fusion. This division is coincident with a phylogenetic separation of lepidopteran NPVs into the two major Groups I and II. These two groups differ significantly in gene content, most notably Group I NPVs use GP64 as their BV fusion protein, whereas Group II NPVs lack gp64 and utilize F protein (Zanotto et al., 1993).

AcMNPV is one of the most widely described baculovirus and belongs to Group I. It presents on its surface the major glycoprotein Gp64 and the residual F protein. While the F protein does not develop any specific function, Gp64 has been identified as the glycoprotein responsible for membrane fusion.

In this section it will be described the structure and function of glycoprotein Gp64 as respon‐ sible for the fusion of membranes and its biotechnological applications for the presentation of foreign antigens.

#### **2.1. Gp64: Structure and function**

Three classes of viral membrane fusion proteins havebeen identified. Class I which contain Nterminalhydrophobic fusion peptides, Class II, which fusion peptides are located in internal loops, and Class III that exhibit distinctstructural features in their architectures as well as in theirmembrane interacting fusion loops. Gp64 belongs to this latter group.

The major envelope protein of the budded virions, GP64, has been shown to mediate acidtriggered membrane fusion both in virions and when expressed alone in transfected cells. The native GP64 is a phosphoglycoprotein fatty acid acylated near the transmembrane domain (Monsma & Blissard, 1995). The Gp64 open reading frame (ORF) of AcMNPV encodes a 512 aminoacids polypeptide with 15 cysteine residues. The resulting disulfide bonds participate in the formation of the native structure.

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

Entry of enveloped viruses into host cells requires fusion of the viral envelope with the cytoplasmic membrane by the action of viral envelope fusion proteins. If the fusion occurs at the cell surface, viral fusion proteins typically act at neutral pH. On the other hand, in receptormediated endocytosis the major fusion protein activity is most often observed at the acidic

In general, baculovirus fusion proteins mediate the membrane fusion at the late endosomal phase. For this reason, the major fusogenic activity was observed at low pH. Although it has been possible to identify which are the proteins that build fusogenic function, which is the cell

Baculoviruses can be divided into two different groups according to the surface glycoprotein they use to mediate the fusion between the endosomal membrane and the viral envelope. One group is composed by viruses that use Gp64 as its fusogenic protein whereas the other group uses the F protein to mediate membrane fusion. This division is coincident with a phylogenetic separation of lepidopteran NPVs into the two major Groups I and II. These two groups differ significantly in gene content, most notably Group I NPVs use GP64 as their BV fusion protein,

AcMNPV is one of the most widely described baculovirus and belongs to Group I. It presents on its surface the major glycoprotein Gp64 and the residual F protein. While the F protein does not develop any specific function, Gp64 has been identified as the glycoprotein responsible for

In this section it will be described the structure and function of glycoprotein Gp64 as respon‐ sible for the fusion of membranes and its biotechnological applications for the presentation of

Three classes of viral membrane fusion proteins havebeen identified. Class I which contain Nterminalhydrophobic fusion peptides, Class II, which fusion peptides are located in internal loops, and Class III that exhibit distinctstructural features in their architectures as well as in

The major envelope protein of the budded virions, GP64, has been shown to mediate acidtriggered membrane fusion both in virions and when expressed alone in transfected cells. The native GP64 is a phosphoglycoprotein fatty acid acylated near the transmembrane domain (Monsma & Blissard, 1995). The Gp64 open reading frame (ORF) of AcMNPV encodes a 512

whereas Group II NPVs lack gp64 and utilize F protein (Zanotto et al., 1993).

theirmembrane interacting fusion loops. Gp64 belongs to this latter group.

screening of antibodies and antigenic epitopes libraries will also be addressed.

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

**2. Baculoviral fusion proteins**

endosomal pH (Monsma & Blissard, 1995).

membrane fusion.

foreign antigens.

**2.1. Gp64: Structure and function**

receptor that recognizes these proteins remains a mystery.

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 involved in virus budding (Figure 3).

**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 useful for antigen surface display.

Budded virions of baculoviruses enter cells by endocytosis. Gp64 is the major compo‐ nent of the viral envelope, and the unique protein with fusogenic activity in AcMNPV. Gp64 is triggered to induce the fusion at the low pH of endosomes. In addition Gp64 is distinguished from any other fusion protein in its ability of going through a reversi‐ ble conformational change, unlike class I and class II fusion proteins, for which the postfusion conformation is thermodynamically more stable and the conformational rearrangement is irreversible.

TLR9 was shown to be responsible in vivo for immune system stimulation by oligodeox‐ ynucleotides containing unmethylated CpG motifs. Like bacteria, AcMNPV contains a significant number of potentially bioactive CpG motifs. Indeed, a number of studies demonstrate that AcMNPV can stimulate professional Antigen Presenting Cells (APCs) by this pathway. Furthermore, Abe et al. demonstrated that internalization and endoso‐ mal maturation are required for TLR9 activation by CpG-rich DNA. They showed that the inhibition of endosomal maturation abolishes the immune system activation of AcMNPV in a dose-dependent manner. These results imply that immune system activation by AcMNPV through TLR9 requires membrane fusion via Gp64 as well as the liberation of the viral genome into cytoplasmic TLR9-containing vesicles (Figure 4.a)

Baculovirus Display: A Novel Tool for Vaccination

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On the other hand, despite BVs cannot replicate in mammalian or other vertebrate animal cells (Via et al., 1983), recent studies showed that BVs have strong adjuvant properties in mice, promoting potent humoral and CD8+ T cell adaptive responses (Abe et al., 2003; Gronowski et al., 1999). In addition, BVs induce the production of inflammatory cytokines by the *in vivo*

Zoth et al. evaluated the effect of baculovirus administration on the innate immune response of chickens. They found an upregulation of IFN-γ and IL-6 in the baculovirustreated chicken spleens and a decrease of the TGF-β gene expression. These facts indicated a strong proinflammatory immune response. Moreover, they demonstrated that BV induced modifications

The duration of the BV-induced response is very limited. This fact represents one of the many interesting benefits of the use of baculovirus for stimulating innate immunity, because the potential damage for a strong inflammatory immune response on an extended time period

On the other hand, it could be presumed that baculovirus inoculation produced an indirect effect on monocytes/macrophages. Zoth et al. also showed an increase of both the mRNA and the protein levels of IFN-γ, and a priming effect of Nitric Oxyde (NO) response in splenocytes of chickens treated with baculoviruses. NO acts as a multi-functional mediator with diverse physiological and pathological roles in host defense, (MacMicking et al., 1997). The production of NO by activated monocytes/macrophages is an important innate immune response sign of

Moreover, Kitajima et al. demonstrated that AcMNPV inoculation of mice induced NK cells activation. They observed that in AcMNPV inoculated animals there was up to fourfold increase in the number of NK cells in spleen, liver, bone marrow and thymus. Furthermore, it was analyzedt he antitumor ability of AcMNPV-induced NK cells and they concluded that AcMNPV injection induces a NKT cell and IFN-γ independent NK cell cytotoxicity against tumor cells in mice (Kitajima et al., 2007) These findings will be approached in section 7.

In conclusion, the strong immune response induced by AcMNPV makes it a promising candidate for a novel, adjuvant- containing vaccine vehicle against infectious diseases

in the mononuclear cells pattern of different organs using flow cytometry.

maturation of dendritic cells (Figura 4.c).

could be avoided (Chimeno Zoth et al., 2012)

cellular antiviral and bactericidal activity.

(Abe et al., 2005).

#### **2.2. Gp64 for protein display**

Gp64 is expressed early and late in the infection of an insect cell. It is a 64 kDa protein which forms trimmers and locates in the BV envelope with a polarized distribution. As Gp64 is a transmembrane protein that exposes an outer domain, it can be used to display a selected protein on the BV surface. A chimeric Gp64 can be constructed to contain the protein of interest allowing it to be incorporated in the BV structure upon infection of insect cells (Grabherr & Ernst, 2010).

In order to facilitate the construction of a chimeric protein it was shown that is not necessary to conserve the complete structure of Gp64. The signal peptide (SP), the multimerization domain, the transmembrane (TM) and the cytoplasmic tail domain (CTD)were shown to be enough for the surface display, whereas the rest of the protein can be eliminated. This strategy avoids the need of dealing with large transfer vectors as well as permitting to increase the number of displayed proteins.
