Core labeling of adenovirus with EGFP

Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, University of Alabama at Birmingham, 901 19th Street South, BMR2-502, Birmingham, AL 35294, USA.
Virology (Impact Factor: 3.32). 09/2006; 351(2):291-302. DOI: 10.1016/j.virol.2006.03.042
Source: PubMed


The study of adenovirus could greatly benefit from diverse methods of virus detection. Recently, it has been demonstrated that carboxy-terminal EGFP fusions of adenovirus core proteins Mu, V, and VII properly localize to the nucleus and display novel function in the cell. Based on these observations, we hypothesized that the core proteins may serve as targets for labeling the adenovirus core with fluorescent proteins. To this end, we constructed various chimeric expression vectors with fusion core genes (Mu-EGFP, V-EGFP, preVII-EGFP, and matVII-EGFP) while maintaining expression of the native proteins. Expression of the fusion core proteins was suboptimal using E1 expression vectors with both conventional CMV and modified (with adenovirus tripartite leader sequence) CMV5 promoters, resulting in non-labeled viral particles. However, robust expression equivalent to the native protein was observed when the fusion genes were placed in the deleted E3 region. The efficient Ad-wt-E3-V-EGFP and Ad-wt-E3-preVII-EGFP expression vectors were labeled allowing visualization of purified virus and tracking of the viral core during early infection. The vectors maintained their viral function, including viral DNA replication, viral DNA encapsidation, cytopathic effect, and thermostability. Core labeling offers a means to track the adenovirus core in vector targeting studies as well as basic adenovirus virology.

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    • "Another approach recently employed by several research groups (including the experiments described here) involves generation of recombinant CRAds expressing fluorescent proteins (Borovjagin et al., 2010; Hofacre and Fan, Unpublished results; Le et al., 2006a; Ono et al., 2005; Ugai et al., 2010). Infected cells can be detected by fluorescence under UV illumination, and importantly cells or cultures can be sequentially imaged at different times, allowing temporal tracking of viral infection (Le et al., 2006b; Ono et al., 2005). We have used this approach to characterize very early events in CRAd virus infection and spread. "
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    • "TPL originates from the first three exons of Ad late genes, locates in the 5 noncoding region of every transcript of late genes, and contributes to selective exportation and translation of viral late mRNAs (Dolph et al., 1988; Huang and Flint, 1998). It has been proved that incorporation of TPL to the 5 terminal of transgene can increase significantly the expression of target gene (Le et al., 2006; Mariati et al., 2010; Sheay et al., 1993). In order to avoid introducing more exogenous sequence, TPL of HAdV-41 was cloned instead of that of Ad5. "
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    • "E3-encoded proteins include the adenovirus death protein (ADP) and at least six other proteins with varied effects including preventing lysis of Ad-infected cells by cytotoxic T lymphocytes (CTL) by down-modulation of MHC class I molecules and inhibition of the apoptosis process induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), TNF and Fas ligand [46] [47]. Robust expression of transgenes has been observed when they are inserted into the deleted E3 region, presumably driven via the major late promoter [48]. Taken together, removal of the E3 region may not only create space for a foreign gene, but also attenuate the resultant recombinant virus which will become more susceptible to host control. "
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