Oncolytic adenoviruses represent a promising therapeutic medicine for human
cancer therapy, but successful translation into human clinical trials requires careful evaluation of their viral characteristics. While the function of adenovirus
proteins has been analyzed in detail, the dynamics of
adenovirus infection remain largely unknown due to technological constraints that prevent adequate tracking of adenovirus particles after
infection. Fluorescence labeling of adenoviral particles is one new strategy designed to directly analyze the dynamic processes of
viral infection in virus-host cell interactions. We hypothesized that the double labeling of an adenovirus with fluorescent
proteins would allow us to properly analyze intracellular viruses and the fate of
viral proteins in a live analysis of an adenovirus as compared to single labeling. Thus, we generated a fluorescently labeled adenovirus with both a red fluorescent minor
capsid protein IX (pIX) [pIX monomeric
red fluorescent protein 1 (mRFP1)] and a green fluorescent minor
core protein V (pV) [pV
enhanced green fluorescent protein (EGFP)], resulting in Ad5-IX-mRFP1-E3-V-EGFP. The fluorescent signals for pIX-mRFP1 and pV-EGFP were detected within 10 min in living cells. However, a growth curve analysis of Ad5-IX-mRFP1-E3-V-EGFP showed an approximately 150-fold reduced production of the viral progeny at 48 h postinfection as compared to adenovirus type 5. Interestingly, pIX-mRFP1 and pV-EGFP were initially localized in the cytoplasm and nucleolus, respectively, at 18 h postinfection. These
proteins were observed in the nucleus during the late stage of
infection, and relocalization of the
proteins was observed in an adenoviral-replication-dependent manner. These results indicate that simultaneous detection of adenoviruses using dual-fluorescent
proteins is suitable for real-time analysis, including identification of infected cells and monitoring of viral spread, which will be required for a complete evaluation of oncolytic adenoviruses.