Vesicular stomatitis virus G-protein

986).

igure 6 illustrates 20 aa transmembrane domain, 29-amino acid cytoplasmic domain with the ontaining two N-linked glycosylation sites. A is located on the cytoplasmic domain (Schmidt and Schlesinger, 1979;

Retroviruses

d as a platform for display in baculoviruses (Chapple and Jone

cular stomatitis virus G-protein

One of the most widely used pseudotyping glycoproteins is the G-protein of vesicular stomatitis virus, VSV-G. The most studied vesicular stomatitis virus belongs to the Indiana strain. Vesicular stomatitis virus is a non-pathogenic ssRNA virus and belongs to the rhabdoviridae family. The VSV-G is a trimeric 500 aa membrane glycoprotein (Doms et al., 1987; Kreis and Lodish, 1 F

remaining amino acids displayed on the surface c single molecule of palmitate

Rose et al., 1984).

The VSV-G has been able to transduce all tested cell types (Coil and Miller, 2004), making it difficult to determinate what are the interactions between the cellular surface and VSV-G which make VSV-G pantropic. VSV-G has been used successfully to pseudotype various viruses, such as retroviruses (Watson et al., 2002a), herpes simplex virus (Watson et al., 2002b), baculoviruses (Tani et al., 2001) and even non-enveloped adenovirus (Yun et al., 2003).

and especially lentiviruses, routinely rely on the pantropic effect of VSV-G, that also stabilizes viral particles (Burns et al., 1993).

The membrane-proximal stem region of VSV-G protein ectodomain (GS i.e. G stem) with transmembrane and cytoplasmic domains can potentiate the membrane fusion activity when coexpressed with some heterologous viral fusion proteins (Jeetendra et al., 2002). It is also known that the membrane proximal region is non-essential for G protein oligomerization, transport to the cell surface, or incorporation into virus particles but essential for acid-induced membrane fusion activity and virus infectivity (Jeetendra et al., 2003). It has been reported that fusion activity is a response to conformational change to the pH decrease during endocytosis (Matlin et al., 1982).

However the exact region responsible for the fusion activity has not been determinated (Jeetendra et al., 2003). The VSV-GS has also been utilize

s, 2002; Ojala et al., 2004).

Figure 6. Schematic presentation of the vesicular stomatitis virus glycoprotein illustrating the functional domains (modified from (Rose and Whitt, 2001)).

signal sequence

1 16 463 482

transmembrane domain

462 451 budding domain

179 336

511 COOH NH₂

118 139 449 462

fusion domains

2.3.3 (Strept)avidin – biotin technology

Avidin was first discovered when raw egg whites, used as the sole source of protein in the diet, caused disorders in test animals (Steiniz, 1898). The reason was found to be avidin, which bound H-vitamin, (biotin) and caused deprivation of this vital vitamin. Bacterial-derived streptavidin also binds biotin with high affinity (Green, 1975). Together, the (strept)avidin-biotin bond has been found to be remarkably strong in vivo and in vitro, resulting in a great number of avidin-biotin applications in life sciences (Figure 7).

Figure 7. Rationale behind (strept)avidin-biotin technology (based on Wilchek and Bayer, 1990).

2.3.3.1 Avidin and streptavidin

Avidin is a tetrameric protein consisting of four identical 128 aa subunits, each capable of binding biotin with an affinity comparable to covalent bond (K ~10d

ins a glycosylation site, resulting in an increase to the molecular mass of tetramer after glycosylation from 57 120 to 62 400 Da (Wilchek and Bayer, 1990). The secondary structure of a single monomer consists of eight antiparaller β-strands, forming a tertiary structure with the biotin binding region on the end of barrel (Figure 8). Four monomers form a tetrameric quaternary structure with disulfide bridges (Livnah et al., 1993; Pugliese et al., 1993) .

Interestingly, avidin is highly thermostable, the temperature for denaturation without biotin is 85 °C and with biotin a remarkable 117 °C (Gonzalez et al., 1999). Furthermore, the complex is also highly resistant to strong denaturizing conditions over a wide pH range (Green, 1975). The overall charge of avidin is basic in physiological pH (pI~10.5), therefore causing possible charge-mediated binding

-15M) (DeLange and Huang, 1971). Each subunit conta

to negatively charged surfaces (Green, 1975).

The early attempts to produce recombinant avidin suffered from insolubility and incorrect folding, but later it was showed that production was possible using a baculovirus expression system (Airenne et al., 1997), in E.coli (Nardone et al., 1998; Hytonen et al., 2004), transgenic maize (Kusnadi et al., 1998) and later on with several viral expression systems, for example SFV (Juuti-Uusitalo et al., 2000).

The bacterial streptavidin has some major differences as compared to avidin: It has slightly acidic isoelectric point (pI) (Green, 1975) and does not have any disulfide bridges between the monomers. Depending on the application, the positive charge and sugar residues of avidin may hinder the use of avidin. Due to the bacterial origin, streptavidin is therefore often preferred in biotechnological applications, since the affinity of streptavidin is comparable to avidin, Kd~ 4x10^-14 M (Green, 1990; Wilchek and Bayer, 1999).

Figure 8 a) 3d-structure (http://www.ncbi.nlm.nih.gov/Structure/) and b) schematical structures of avidin tetramer, c) together with four biotinylated ligands.

2.3.3.2 Biotin

Biotin (vitamin H) is a sulphur-containing chiralic organic acid with a molecular weight of 244.2 g/mol (Figure 9). Biotin is known to be part of regulation of gene expression (McMahon, 2002) as well as having a known role in metabolism.

The attachment of the biologically active D-form of biotin, known as biotinylation, regulates the activity of enzymes involved in the central metabolism of a cell. The attached biotin serves as a carrier of an activated carboxyl group in carboxylation, decarboxylation and transcarboxylation reactions (Chapman-Smith and Cronan, Jr., 1999).

There are also several enzymes involved in the biotin metabolism in cells. Biotin protein ligase (BPL) and its bacterial analogue BirA are proteins specifically binding biotin and

attaching biotin to other proteins (Chapman-Smith and Cronan, Jr., 1999). Biotin carboxyl carrier protein (BCCP) is part of the acetyl coenzyme A carboxylase complex carrying the biotin cofactor 995). Biotinidase is a protein, which releases and a substrate of BPL (Athappilly and Hendrickson, 1

the biotin from biotinylated proteins (Hymes and Wolf, 1999). The use of these biotin processing enzymes has advanced the use of the avidin-biotin system and removed steps involving chemical attachment of biotin.

Figure 9. d-Biotin, 5-[(1R,2S,5S)-7-oxo-3-thia-6,8-diazabicyclo[3.3.0]oct-2-yl]pentanoic acid (http://pubchem.ncbi.nlm.nih.gov/)

2.3.3.3 (Strept)avidin-biotin technology in gene therapy

The avidin-biotin technology provides flexibility and increased compatibility for both therapeutic and research purposes. Applications are being developed to introduce new and more efficient, targetable vectors to the field of gene therapy (Laitinen et al., 2005b). This can be achieved either by biotinylation of gene therapy vectors chemically or metabolically (Barry et al., 2003) or by genetically attaching avidin to the viral surface, introduced in this thesis. Below are some examples of avidin-biotin technology within gene therapy.

Chemically biotinylated adenovirus vector was successfully targeted to haematopoietic cells through an avidin bridge carrying biotinylated c-Kit receptor ligand and

ilarly

pos et al., actor GF)-avidin fusion protein showed significant enhancement in transduction efficiency in EGF

able to diminish the native infectivity but guide the opism to targeted cell types (Purow and Staveley-O'Carroll, 2005). Altogether several viral ansduction systems have so far demonstrated the beneficial properties of avidin-biotin system for modifying viral properties for targeted delivery.

resulted in 2,440-fold increase in reporter gene expression (Smith et al., 1999). Sim biotinylated retrovirus coated with avidin-polylysin resulted in enhanced transduction and widened the ecotropic viral tropism to human cells (Zhong et al., 2001).

A photocleavable biotinylation reagent used with the adenovirus was reported to provide a way to control adenovirus infectivity by reversing a prior alkylation of the virus (Pandori et al., 2002b). Importantly, adenoviruses with biotin acceptor peptide in fibre capsid proteins have resulted in metabolical biotinylation and showed enhanced transduction with various targeting ligands (Parrott et al., 2003). However the use of biotin display in adenovirus capsid IX protein or hexon capsomeres together with targeting ligands reduced the transduction efficiency, possibly by preventing the disassembly of the delicate viral capsid structure. The authors conclude that adenovirus targeting would be most efficient through the capsid fibre biotinylation (Cam

2004).

Chemically biotinylated adeno-associated virus together with epidermal growth f (E

receptor- positive cells. (Ponnazhagan et al., 2002). Further on, biotinylated vaccinia virus with avidin crosslink to murine antibodies was

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