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The envelope protein gp64 of the baculovirus Autographa californica nuclear polyhedrosis virus is essential for viral entry into insect cells, as the glycoprotein both mediates pHdependent membrane fusion and binds to host cell receptors. Surface modification of baculovirus particles by genetic engineering of gp64 has been demonstrated by various strategies and thus has become an important and powerful tool in molecular biology. To improve further the presentation of peptides on the surface of baculovirus particles, several insertion sites within the gp64 envelope protein were selected by their theoretical maximum surface probability and investigated for efficient peptide presentation. The ELDKWA peptide of the gp41 of HIV-1, specific for the human mAb 2F5, was inserted into 17 different positions of the glycoprotein gp64. Propagation of viruses was successful in 13 cases, mutagenesis at four positions did not result in production of intact virus particles. Western blotting, FACS analysis and ELISA were used for characterization of the different binding properties of the mutants. Insertion of this peptide into the native envelope protein resulted in high avidity display on the surface of baculovirus particles. This approach offers the possibility of effective modification of surface properties in regard to host range specificity and antigen display.

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To create a tool for eukaryotic surface display, this approach is aimed at demonstrating a direct modification of the native envelope protein gp64 of Autographa californica NPV without disturbing viral infectivity. Short affinity-tag peptides, the biotin mimic streptagII, and the gp41 amino-acid motif ELDKWA of HIV-1, specific for the human monoclonal antibody 2F5, were engineered into the baculovirus major coat protein gp64 and presented on the viral surface. Two different streptag peptides were inserted at the naturally occurring Not I site at aminoacid 278 of gp64. Additionally, the ten-amino-acid peptide GG-ELDKWA-GG, containing the epitope of mAb 2F5, was introduced into gp64 envelope protein at the same position. In all cases we were able to propagate viable virus-achieving infectious titers in the range of wild-type AcMNPV. Streptag and ELDKWA-epitope surface localization on purified virus particles was demonstrated by flow cytometry and Western blot analysis. We could also show selective retention of mutant viruses by specific interaction between chimeric virions and their target counterparts, recognizing the epitope or the streptag peptide in the viral envelope. These data provide evidence that altering the surface properties of the baculovirus virion could be of value in improving baculovirus display technology and developing new applications.Keywords: baculovirus; Autographica californica nuclear polyhedrosis virus; insect cells; gp64 envelope protein; streptag peptide.To generate baculovirus-budded virions as a tool for eukaryotic surface display, different strategies have been investigated. When a foreign gene is fused to a second copy of the entire coat protein of AcMNPV, gp64 [1], or alternatively to the membrane anchor domain of gp64, simultaneous expression of the fusion construct and the native gp64 envelope protein results in packaging of both proteins into the viral coat and its presence on the surface of baculovirus-infected insect cells and budded virions [2±4]. Boublik and colleagues demonstrated the display of glutathione-S-transferase/gp64 fusion protein on the baculoviral surface, forming a hetero-oligomer with the wild-type gp64 coat protein [2]. Also, they targeted the gp120 surface glycoprotein of HIV-1 to the viral surface and showed that gp120 was still active, using a CD4 binding assay. Similar proteins such as the gp41 ectodomain of HIV-1 were directed to the baculoviral envelope thereby investigating the influence of different viral promoters on the efficiency of the process of viral packaging [3], indicating that only a minor portion of the target protein was incorporated and could be detected on budded virus.The native gp64 is abundantly expressed during the infection cycle and transported to the cell surface to be incorporated into budded virions. Two functional domains have been characterized, an oligomerization domain within a predicted a helix, necessary for oligomerization and transport, and a fusion domain as part of a hydrophobic cluster [5]. The essential role of gp64 ...