Direct interaction of baculovirus capsid proteins VP39 and EXON0 with kinesin-1 in insect cells determined by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replicates in the nucleus of insect cells to produce nucleocapsids, which are transported from the nucleus to the plasma membrane for budding through GP64-enriched areas to form budded viruses. However, little is known about the anterograde trafficking of baculovirus nucleocapsids in insect cells. Preliminary confocal scanning laser microscopy studies showed that enhanced green fluorescent protein (EGFP)-tagged nucleocapsids and capsid proteins aligned and colocalized with the peripheral microtubules of virus-infected insect cells. A colchicine inhibition assay of virus-infected insect cells showed a significant reduction in budded virus production, providing further evidence for the involvement of microtubules and suggesting a possible role of kinesin in baculovirus anterograde trafficking. We investigated the interaction between AcMNPV nucleocapsids and kinesin-1 with fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) and show for the first time that AcMNPV capsid proteins VP39 and EXON0, but not Orf1629, interact with the tetratricopeptide repeat (TPR) domain of kinesin. The excited-state fluorescence lifetime of EGFP fused to VP39 or EXON0 was quenched from 2.4 ± 1 ns to 2.1 ± 1 ns by monomeric fluorescent protein (mDsRed) fused to TPR (mDsRed-TPR). However, the excited-state fluorescence lifetime of an EGFP fusion of Orf1629 remained unquenched by mDsRed-TPR. These data indicate that kinesin-1 plays an important role in the anterograde trafficking of baculovirus in insect cells.

Baculovirus as delivery system for gene transfer during hypothermic organ preservation.

Concerns over the safety of conventional viral vectors have limited the translation of gene transfer from an exciting experimental procedure to a successful clinical therapy in transplantation. Baculoviruses are insect viruses, but have the ability to enter mammalian cells and deliver potential therapeutic molecules with no evidence of viral replication. This study provides evidence of the ability of recombinant baculovirus to enter mammalian kidneys and livers during cold preservation. Six kidneys and six liver lobules retrieved from large pigs were perfused with University of Wisconsin (UW) solution containing a baculovirus tagged with green fluorescent protein and preserved for 8 h. In addition, six kidneys were perfused with UW containing a baculovirus expressing red fluorescent protein and preserved for 24 h. Green fluorescent virus particles were detected within transduced kidneys and livers after 8 h standard cold storage and red fluorescent protein mRNA was detected in kidneys after 24 h of cold preservation. There were no significant differences in tissue architecture, cell morphology or ATP content between experimental organs and their controls. Ex vivo transduction of organs with recombinant baculovirus during conventional cold preservation was demonstrated with no evidence of additional injury or reduction in cell viability.

Improved expression of secreted and membrane-targeted proteins in insect cells.

Secretory and membrane-bound proteins are generally produced in lower amounts in insect cells compared with cytoplasmic and nuclear proteins. There may be many reasons for this, including degradation of recombinant proteins by proteases, competition for cellular resources between native and recombinant proteins, and physical blockage of the secretory pathways. In the present study, we describe the construction of a baculovirus in which chiA (chitinase) and cath (cathepsin) genes have been deleted and show improved recombinant protein expression using this vector. We confirmed the complete removal of both genes by PCR, restriction enzyme analysis and enzyme assays, and the modified virus DNA was shown to be stable in bacterial cells over multiple passages. A selection of recombinant genes were inserted into the double-deletion virus and their expression levels compared with recombinant viruses that had single or no gene deletions. In all instances, the double-deletion viruses showed greatly enhanced levels of protein production for both secreted and nuclear/cytoplasmic proteins. In summary, we have conclusively demonstrated the importance of this deletion vector for the high-level production of recombinant proteins.