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.
Bibliographical noteFunding Information:
We thank Dr. Roger Y. Tsien (University of California at San Diego) for providing the mRFP1 construct and Dr. Igor P. Dmitriev (University of Alabama at Birmingham) for providing a rabbit polyclonal pIX antibody. We also thank Drs. Anton V. Borovjagin, Erin E. Thacker, George C. Dobbins, Joel N. Glasgow, Matthew S. Beatty, Qiana L. Matthews, and Yizhe Tang for useful advice and fruitful discussions. We are grateful to Cynthia M. Rodenburg for technical support with TEM at the High Resolution Imaging Facility of the University of Alabama at Birmingham. This work was supported by grants from the National Institutes of Health (grant 5R01CA111569 to Dr. David T. Curiel and grant 5R56CA094084 to Drs. Masato Yamamoto and David T. Curiel) and by a grant from Susan G. Komen for the Cure (grant PDF0707736 to Dr. Hideyo Ugai).
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- fluorescent protein