Insect-derived baculoviruses have emerged as versatile and safe workhorses of biotechnology. Baculovirus expression vectors (BEVs) have been applied widely for crop and forest protection, as well as safe tools for recombinant protein production in insect cells. However, BEVs ability to efficiently transduce noninsect cells is still relatively poorly recognized despite the fact that efficient baculovirus-mediated in vitro and ex vivo gene delivery into dormant and dividing vertebrate cells of diverse origin has been described convincingly by many authors. Preliminary proof of therapeutic potential has also been established in preclinical studies. This review summarizes the advantages and current status of baculovirus-mediated gene delivery. Stem cell transduction, preclinical animal studies, tissue engineering, vaccination, cancer gene therapy, viral vector production, and drug discovery are covered.

Baculoviruses are enveloped insect viruses that can carry large quantities of foreign DNA in their genome. Baculoviruses have proved to be very promising gene therapy vectors but little is known about their transduction mechanisms in mammalian cells. We show in this study that Autographa californica multiple nuclear polyhedrosis virus capsid is compatible with the incorporation of desired proteins in large quantities. Fusions can be made to the N-terminus or C-terminus of the major capsid protein vp39 without compromising the viral titer or functionality. As an example of the baculovirus capsid display we show a tracking of the baculovirus transduction in mammalian cells by an enhanced green fluorescent protein (EGFP)-displaying virus. Our confocal and electron microscopy results suggest that the transduction block in mammalian cells is not in the endosomal escape, as previously proposed, but rather in the cytoplasmic transport or nuclear entry of the virus capsid. Our results also suggest that the EGFP-tagged virus can be used for visualization of the virus biodistribution in vivo. Furthermore, capsid-modified baculoviruses hold great promise for the nuclear and subcellular targeting of transgenes and as a novel peptide display system for a variety of eukaryotic applications.

A recombinant non-glycosylated and acidic form of avidin was designed and expressed in soluble form in baculovirusinfected insect cells. The mutations were based on the same principles that guided the design of the chemically and enzymatically modified avidin derivative, known as NeutraLite Avidin. In this novel recombinant avidin derivative, five out of the eight arginine residues were replaced with neutral amino acids, and two of the lysine residues were replaced by glutamic acid. In addition, the carbohydrate-bearing asparagine-17 residue was altered to an isoleucine, according to the known sequences of avidin-related genes. The resultant mutant protein, termed recombinant NeutraLite Avidin, exhibited superior properties compared to those of avidin, streptavidin and the conventional NeutraLite Avidin, prepared by chemo-enzymatic means. In this context, the recombinant mutant is a single molecular species, which possesses strong biotin-binding characteristics. Due to its acidic pI, it is relatively free from non-specific binding to DNA and cells. The recombinant NeutraLite Avidin retains seven lysines per subunit, which are available for further conjugation and derivatization.z 2000 Federation of European Biochemical Societies.

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Sea urchin fibropellins are epidermal growth factor homologues that harbor a C-terminal domain, similar in sequence to hen egg-white avidin and bacterial streptavidin. The fibropellin sequence was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin-binding sites of the tetrameric proteins, avidin and streptavidin. Three different mutations of avidin, Trp-110-Lys, Trp-70-Arg and the double mutant, were expressed in a baculovirus-infected insect cell system. A mutant of streptavidin, Trp-120-Lys, was similarly expressed. The homologous tryptophan to lysine (WC CK) mutations of avidin and streptavidin were both capable of binding biotin and biotinylated material. Their affinity for the vitamin was, however, significantly reduced: from K d V V10 315 M of the wild-type tetramer down to K d V V10 38 M for both WC CK mutants. In fact, their binding to immobilized biotin matrices could be reversed by the presence of free biotin. The Trp-70-Arg mutant of avidin bound biotin very poorly and the double mutant (which emulates the fibropellin domain) failed to bind biotin at all. Using a gel filtration fast-protein liquid chromatography assay, both WC CK mutants were found to form stable dimers in solution. These findings may indicate that mimicry in the nature of the avidin sequence and fold by the fibropellins is not designed to generate biotin-binding, but may serve to secure an appropriate structure for facilitating dimerization.z 1999 Federation of European Biochemical Societies.