| Literature DB >> 16997010 |
Anna R Mäkelä1, Christian Oker-Blom.
Abstract
For over a decade, phage display has proven to be of immense value, allowing selection of a large variety of genes with novel functions from diverse libraries. However, the folding and modification requirements of complex proteins place a severe constraint on the type of protein that can be successfully displayed using this strategy, a restriction that could be resolved by similarly engineering a eukaryotic virus for display purposes. The quite recently established eukaryotic molecular biology tool, the baculovirus display vector system (BDVS), allows combination of genotype with phenotype and thereby enables presentation of eukaryotic proteins on the viral envelope or capsid. Data have shown that the baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is a versatile tool for eukaryotic virus display. Insertion of heterologous peptides and/or proteins into the viral surface by utilizing the major envelope glycoprotein gp64, or foreign membrane-derived counterparts, allows incorporation of the sequence of interest onto the surface of infected cells and virus particles. A number of strategies are being investigated in order to further develop the display capabilities of AcMNPV and improve the complexity of a library that may be accommodated. Numerous expression vectors for various approaches of surface display have already been developed. Further improvement of both insertion and selection strategies toward development of a refined tool for use in the creation of useful eukaryotic libraries is, however, needed. Here, the status of baculovirus display with respect to alteration of virus tropism, antigen presentation, transgene expression in mammalian cells, and development of eukaryotic libraries will be reviewed.Entities:
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Year: 2006 PMID: 16997010 PMCID: PMC7112267 DOI: 10.1016/S0065-3527(06)68003-2
Source DB: PubMed Journal: Adv Virus Res ISSN: 0065-3527 Impact factor: 9.937
Fig 1A generalized schematic outline of the multifunctional baculovirus display technology used for eukaryotic library development and mammalian gene delivery. Entry of baculovirus into mammalian cells is thought to be similar to that for insect cells. The receptor molecule(s) for baculovirus binding and entry are unknown, but they have been suggested to involve common cell surface components. The virus enters mammalian cells by endocytosis followed by low‐pH–induced membrane fusion with endosomes and capsid release into the cytoplasm. The capsid is then transported toward the nucleus along actin filaments and enters the nucleus presumably through nuclear pores followed by uncoating and release of the genome. Through incorporation of ligands with high and specific affinities into the virus envelope, it is possible to target baculoviral transduction to desired cell types expressing the receptor molecule for the displayed ligand. Foreign peptides or proteins can be displayed on the baculovirus envelope as N‐terminal (A) or internal (B) fusions to gp64, using a heterologous membrane anchor derived from VSV‐G (C) for example, or by fusion to the major capsid protein, vp39 (D). The displayed proteins are directed to the surface of the recombinant baculovirus‐infected insect cells using a signal sequence derived from the major AcMNPV envelope protein gp64 (gp64ss) for example. Genes encoding the fusion proteins are then coupled with a strong baculoviral promoter (e.g., polh or p10) for strong expression in insect cells. The vectors can be further equipped with an expression cassette encoding a reporter or a suicide gene under a mammalian promoter (e.g., CMV or SV40), enabling transduction monitoring in mammalian cells. Cell surface display can be applied in library screening for studying ligand–receptor interactions and antigen recognition. Display on the viral envelope provides possibilities for transductional targeting, whereas capsid display rather facilitates studies on intracellular trafficking as well as nuclear targeting of the virus.