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In Vitro Dynamic Visualization Analysis of Fluorescently Labeled Minor Capsid Protein IX and Core Protein V by Simultaneous Detection |
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| Authors: | Hideyo Ugai Long P Le Masato Yamamoto |
| Institution: | 1 Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, and Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, USA 2 Pathology Service, Massachusetts General Hospital, 55 Fruit Street GRJ 249, Boston, MA 02114, USA 3 Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK 4 Division of Basic and Translational Research, Department of Surgery, University of Minnesota, Minneapolis, MN 55455, USA 5 The Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA |
| Abstract: | 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. |
| Keywords: | pIX minor capsid protein IX mRFP1 monomeric red fluorescent protein 1 pV minor core protein V EGFP enhanced green fluorescent protein Ad5 adenovirus type 5 VP viral particle PFU plaque-forming unit FCU fluorescent cell unit TEM transmission electron microscopy MOI multiplicity of infection mpi minutes postinfection hpi hours postinfection qPCR quantitative PCR FBS fetal bovine serum PBS phosphate-buffered saline |
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