We wished to study the intracellular transport of adenoviruses. We constructed a novel recombinant adenovirus in which the structural protein IX was labeled with a mini-singlet oxygen generator (miniSOG). The miniSOG gene was synthesized by overlapping extension polymerase chain reaction (PCR), cloned to the pcDNA3 vector, and expressed in 293 cells. Activation of miniSOG generated sufficient numbers of singlet oxygen molecules to catalyze polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by transmission electron microscopy (TEM). To construct miniSOG-labelled recombinant adenoviruses, the miniSOG gene was subcloned downstream of the IX gene in a pShuttle plasmid. Adenoviral plasmid pAd5-IXSOG was generated by homologous recombination of the modified shuttle plasmid (pShuttle-IXSOG) with the backbone plasmid (pAdeasy-1) in the BJ5183 strain of Eschericia coli. Adenovirus HAdV-5-IXSOG was rescued by transfection of 293 cells with the linearized pAd5-IXSOG. After propagation, virions were purified using the CsC1 ultracentrifugation method. Finally, HAdV-5-IXSOG in 2.0 mL with a particle titer of 6 x 1011 vp/mL was obtained. Morphology of HAdV-5-IXSOG was verified by TEM. Fusion of IX with the miniSOG gene was confirmed by PCR. In conclusion, miniSOG-labeled recombinant adenoviruses were constructed, which could be valuable tools for virus tracking by TEM.
Adenoviruses, Human ; chemistry ; genetics ; metabolism ; Arabidopsis Proteins ; chemistry ; genetics ; metabolism ; Flavoproteins ; chemistry ; genetics ; metabolism ; Humans ; Phototropins ; chemistry ; genetics ; metabolism ; Singlet Oxygen ; chemistry ; Staining and Labeling ; Transfection

Truncated forms of gp91(phox) were expressed in E. coli in which the N-terminal hydrophobic transmembrane region was replaced with a portion of the highly soluble bacterial protein thioredoxin (TRX). TRX-gp91(phox) (306-569), which contains the putative FAD and NADPH binding sites, showed NADPH-dependent NBT (nitroblue tetrazolium) reductase activity, whereas TRX-gp91(phox) (304-423) and TRX-gp91(phox) (424-569) were inactive. Activity saturated at about a 1:1 molar ratio of FAD to TRX-gp91(phox) (306- 569), and showed the same Km for NADPH as that for superoxide generating activity by the intact enzyme. Activity was not inhibited by superoxide dismutase, indicating that it was not mediated by superoxide, but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). In the presence of Rac1, the cytosolic regulatory protein p67(phox) stimulated the NBT reductase activity, but p47(phox) had no effect. Truncated p67(phox) containing the activation domain (residues 199- 210) stimulated activity approximately 2-fold, whereas forms mutated or lacking this region failed to stimulate the activity. Our data indicate that: 1) TRX-gp91(phox) (306-569) contains the binding sites for both pyridine and flavin nucleotides; 2) this flavoprotein domain shows NBT reductase activity; and 3) the flavin-binding domain of gp91(phox) is the target of regulation by the activation domain of p67(phox).
Animals ; Cloning, Molecular ; DNA Primers ; Escherichia coli/genetics/metabolism ; Flavoproteins/chemistry/genetics ; Kinetics ; Membrane Glycoproteins/chemistry/*genetics/isolation & purification ; *NADPH Oxidase ; Neutrophils/physiology ; Polymerase Chain Reaction/methods ; Recombinant Fusion Proteins/chemistry/isolation & purification/metabolism ; Recombinant Proteins/chemistry/isolation & purification ; Restriction Mapping ; Sequence Deletion