Article

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: 3.28). 09/2006; 351(2):291-302. DOI: 10.1016/j.virol.2006.03.042
Source: PubMed

ABSTRACT 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.

0 Followers
 · 
111 Views
  • Source
    • "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involves generation of recombinant CRAds expressing fluorescent proteins (Borovjagin et al., 2010; Hofacre and Fan, Unpublished results; Le et al., 2006a; Ono et al., 2005; Ugai et al., 2010).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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Conditionally-replicating adenoviruses (CRAds) and other oncolytic viruses replicate selectively in tumor cells, presenting a potential cancer treatment approach. To optimize application of these viruses, understanding of early spread of these viruses in target cells is important. Here we used a recombinant adenovirus expressing enhanced jellyfish green fluorescent protein (EGFP) in place of the EIA and EIB genes (AdEGFPuci). Infection of susceptible cells (AD-293) under plaque formation conditions (MOI<1) on gridded culture dishes and daily monitoring allowed visualization of initially infected cells, as well as spread to neighboring cells. We determined key parameters of early infection, including the rate and efficiency of spread from the initially infected cell to other cells. It was noteworthy that a minority of initially infected cells ultimately resulted in plaques. The approaches elucidated here will be useful for determining early infection parameters for CRAds of therapeutic interest.
    Virology 12/2011; 423(1):89-96. DOI:10.1016/j.virol.2011.11.014 · 3.28 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: An HIV Env immunogen capable of eliciting broad immunity is critical for a successful vaccine. We constructed and characterized adenovirus 5 host range mutant (Ad5hr) recombinants encoding HIV(SF162) gp160 (subtype B) and HIV(TV1) gp160 (subtype C). Immunization of mice with one or both induced cellular immunity to subtype B and C peptides by ELISpot, and antibody responses with high binding titers to HIV Env of subtypes A, B, C, and E. Notably, Ad5hr-HIV(TV1) gp160 induced better cellular immunity than Ad5hr-HIV(SF162) gp160, either alone or following co-administration. Thus, the TV1 Env recombinant alone may be sufficient for eliciting immune responses against both subtype B and C envelopes. Further studies of Ad5hr-HIV(TV1) gp160 in rhesus macaques will evaluate the suitability of this insert for a future phase I clinical trial using a replication-competent Ad4 vector.
    Vaccine 04/2010; 28(23):3963-71. DOI:10.1016/j.vaccine.2010.03.046 · 3.49 Impact Factor
Show more