Core labeling of adenovirus with EGFP

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.

Stepwise Loss of Fluorescent Core Protein V from Human Adenovirus during Entry into Cells

“…6), as reported for wild-type pV (29,32). The absence of pV or GFP-pV on the viral cores in the nucleus is not surprising, since earlier studies showed that the viral DNA and pVII alone can be a template for replication and transcription without pV (9,28,67).…”

“…For adenoviruses, GFP had been fused to the capsid-stabilizing protein pIX (240 copies per virion) (51), and purified virions were observed at the plasma membrane and the cytoplasm (39). In addition, adenoviruses encoding either pV-GFP or pre-pVII-GFP and a dual-color Ad5 with pIX-RFP (red fluorescent protein) and pV-GFP were also reported (32,61). The utility of these viruses was, however, limited.…”

“…Both pre-pVII-GFP and pV-GFP were expressed from an artificial promoter in the deleted E3 region of the genome, while endogenous pre-pVII and pV were also expressed, which resulted in low incorporation of pV-GFP in virus particles (61). In addition, the pV-GFP-expressing Ad5 stocks contained significant amounts of disrupted virions (32). The pIX-RFP/pV-GFP-labeled virions on the other hand were heterogeneous with respect to green and red fluorescence, and it remained unknown how these viruses enter or exit cells.…”

Construction and expression of hepatitis B virus vector encoding TC-tagged core protein

“…Structural proteins of viruses were frequently tagged with GFP to form fluorescent virions for visualizing and studying these viruses in living cells [3][4][5][6][7][8][9]. However, GFP is not suitable for fluorescently labeling HBV virion [3].…”

“…The capsid has a diameter of 30 nm and is formed by either 180 or 240 copies of core protein [1,2]. Although recombinant virions whose structural proteins are tagged with green fluorescent protein (GFP) allow us to visualize and study virus in living cells [3][4][5][6][7][8][9], GFP tag is not suitable for labeling HBV, because HBV virion, as a small and densely packed virion, does not have enough space for incorporation of GFP tag that is as large as 238 amino acids (aa) fragment [3]. So far, visualizing and studying HBV in living cells has still been hampered by the lack of a perfect labeling approach.…”