1. Baculovirus capsid display: a novel tool for transduction imaging
Sari P. Kukkonen, Kari J. Airenne, Varpu Marjomäki, Olli H. Laitinen, Pauliina Lehtolainen, Pasi Kankaanpää, Anssi J. Mähönen, Jani K. Räty, Henri R. Nordlund, Christian Oker-Blom, Markku S. Kulomaa, Seppo Ylä-Herttuala 
Molecular Therapy 
Volume 8, Issue 5, Pages (November 2003)
DOI: /j.ymthe
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Map of the capsid display plasmid pBACcap-1. The plasmid is designed for baculovirus capsid display by N-terminal or C-terminal fusion of peptides or proteins with the AcMNPV major capsid protein vp39 (for details, see Results).
Molecular Therapy 2003 8, DOI: ( /j.ymthe )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Characterization of the EGFP-displaying recombinant baculoviruses by immunoblotting. Vp39EGFP (lane 1) and EGFPvp39 (lane 3) virus-infected insect cells and gradient-purified Vp39EGFP (lane 2) and EGFPvp39 (lane 4) viruses were probed under reducing conditions with an anti-EGFP antibody. A major band of expected size (67 kDa) was detected in all lines. The results indicate that both fusion proteins are not only expressed efficiently in insect cells but also incorporated efficiently as parts of the intact baculovirus capsids.
Molecular Therapy 2003 8, DOI: ( /j.ymthe )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Immunoelectron microscopic analysis (on the left) and schematic presentation (on the right) of the vp39EGFP baculovirus. The typical rod-shaped morphology was detected for the anti-EGFP and immunogold-labeled viral capsid. The surface of the vp39EGFP baculovirus capsid was evenly stained by gold particles. The rod-shaped nucleocapsid of the virus surrounded by a lipid bilayer membrane and the surface projections, peplomers, at the one end formed by gp64 trimers are shown in the 3D picture. The principle of double-targeting is illustrated: lipid-embedded gp64 fusion proteins enable targeting at the cell surface level and vp39 fusion proteins at the cytoplasmic level. Original magnification: 50,000×.
Molecular Therapy 2003 8, DOI: ( /j.ymthe )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Imaging of the baculovirus entry and transport in HepG2 cells (top) compatible with the baculovirus-mediated gene transfer and in the incompatible EAHY (middle) and MG63 cells (bottom). The cells were treated with vp39EGFP virus and the course of the baculovirus was studied by detecting green fluorescence after the shown time periods posttransduction using confocal microscopy. The cells were labeled with antibody against the early endosome antigen 1 (EEA1) to visualize the localization of the baculovirus in the early endosomes (red). Yellow shows colocalization of the virus with early endosomes (merge). Note that the baculovirus capsids enter efficiently into the nuclei in HepG2 cells, but not in EAHY or MG63 cells, although they can escape from endosomes in both cell types. Bars, 10 μm.
Molecular Therapy 2003 8, DOI: ( /j.ymthe )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Electron microscopic pictures of the vp39EGFP virus-treated HepG2 and EAHY cells 4 h posttreatment. Free virus particles were detected in the cytoplasm of both cell types, further suggesting that the virus particles had escaped from the early endosomes. In HepG2 cells virus particles were present also in the nuclei, as opposed to EAHY cells, in which no virus particles were present in the nuclei. Arrows indicate the virus particles; n, nucleus; c, cytoplasm. Original magnification: 20,000–50,000×.
Molecular Therapy 2003 8, DOI: ( /j.ymthe )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Baculovirus enters mammalian cells by endocytosis. EAHY and HepG2 cells were transduced by vp39EGFP virus in the presence of 0.5 μM monensin and the fate of the virus was studied by confocal microscopy. Inhibition of the early endosome acidification by monensin led to a block of the baculovirus capsid entry into the cell cytoplasm in both cell types as seen by the lack of green spots in the nuclei of HepG2 cells and colocalization of the virus with early endosomes still at 4 h posttransduction (yellow). Bar, 10 μm.
Molecular Therapy 2003 8, DOI: ( /j.ymthe )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

8. FIG. 7
Visualization of the vp39EGFP virus distribution in the rat brain in vivo. At 7 h after vp39EGFP virus injection, virus particles were detected near the injection site of the right hemisphere of rat forebrain. (Top) Injections were performed into the right corpus callosum (blue arrowhead shows the injection site). (Bottom) vp39EGFP virus was clearly visible near the injection site above the corpus callosum. (Left) Bright-field image of the injection site. (Middle) Dark-field image of the same localization as on the left. (Right) Overlay of left and middle images. Red asterisk indicates the same location in all three images. Scale bars: top, 1000 μm; bottom, 500 μm. Abbreviations: cg, cingulum; fmi, forceps minor corpus callosum; gcc, genu of corpus callosum; IG, indusium griseum; L, left; LV, lateral ventricle; R, right; vhc, ventral hippocampal cortex.
Molecular Therapy 2003 8, DOI: ( /j.ymthe )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions