Genetic incorporation of a herpes simplex virus type 1 thymidine kinase and firefly luciferase fusion into the adenovirus protein IX for functional display on the virion

Abstract

An advantage of the adenoviral vector is its molecular flexibility, which allows for vector tropism modifications for the purpose of cell targeting. In addition to targeting ligands, the capacity to incorporate heterologous peptides has allowed capsid incorporation of other functionalities. We have defined the minor capsid protein IX (pIX) as a locus capable of presenting incorporated ligands on the virion surface. Thus, we sought to exploit the possibility of incorporating functional proteins at pIX. In our current study, we sought to expand the potential utility of our capsid labeling strategy by developing simultaneous imaging capacity for dedicated small animal positron emission tomography and bioluminescence imaging on a single adenoviral vector. Therefore, we constructed an adenovirus that incorporates a fusion protein of herpes simplex virus type 1 thymidine kinase and firefly luciferase (Luc) (TK-Luc) into adenovirus capsid pIX. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional TK-Luc as a component of their capsid surface. Most importantly, Ad-pIX-TK-Luc retained dual enzymatic functions in vitro and in vivo. This dual-modality approach will allow dynamic or real-time imaging analysis of adenovirus-based interventions with maximized analytic flexibility and enhanced resolution potential.

Figure 1

Strategy for the construction of an adenovirus with a herpes simplex virus 1 thymidine kinase and firefly luciferase (TK-Luc) fusion reporter incorporated at the pIX capsid protein. Human adenovirus serotype 5 was genetically engineered to express a modified capsid protein IX. An E1/E3 deleted adenovirus was constructed containing a pIX-TK-Luc carboxy-terminal fusion gene in place of wild-type pIX (Ad-pIX-TK-Luc). Between the pIX and TK-Luc coding regions is an incorporated 18–amino acid linker SADDYKDDDDKLAGSGSG containing the octapeptide FLAG-tag sequence (underlined).

Figure 2

Expression of capsid-incorporated TK-Luc fusion protein. A, Western blot analysis of thymidine kinase (TK) expression in cells infected with TK-expressing virions. HEK293 cells were infected with 100 viral particles (VPs)/cell of Ad-CMV-TK, Ad-wt-pIX-TK, or Ad-pIX-TK-Luc. Cell lysates were collected and resolved on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene difluoride (PVDF) membrane. Staining was performed with a rabbit polyclonal anti-TK antibody. The arrow indicates pIX-TK-Luc protein. B, Western blot analysis of Ad-pIX-TK-Luc for TK-Luc protein incorporation. In this assay, 10¹⁰ VPs of CsCl gradient purified Ad-wt-pIX-TK and Ad-pIX-TK-Luc were resolved on an SDS-PAGE gel and transferred to PVDF membrane. Staining was performed with a monoclonal anti-FLAG antibody. The arrow indicates pIX-TK-Luc protein. MW = molecular weight.

Figure 3

In vitro analysis of luciferase activity of an adenovirus with a capsid-incorporated TK-Luc fusion protein. Luciferase activities were measured from 10¹⁰ viral particles of CsCl gradient purified Ad5Luc1 virions, Ad-wt-pIX-TK virions, and Ad-pIX-TK-Luc virions. Values are expressed as mean 6 standard deviation of three replicates.

Figure 4

Validation of an adenovirus with a capsid-incorporated TK-Luc fusion protein via bioluminescent imaging. HEK 293 cells were infected with 10⁸ viral particles of (A) Ad-pIX-TK-Luc, (B) Ad-CMVluc, or (C) Ad-CMV-GFP. After 24 hours, 1 mM d-luciferin was added to each dish and luciferase activity was detected using a cooled bioluminescence camera. The signal bars indicate 0 to 100% activity.

Figure 5

Validation of an adenovirus with a capsid-incorporated TK-Luc fusion protein via micro–positron emission tomography (microPET). HEK 293 cells were infected with 10⁸ viral particles of (A) Ad-pIX-TK-Luc, (B) Ad-CMV-TK, or (C) Ad-CMV-GFP and after 24 hours were labeled with 50 μCi of [¹⁸F]-FHBG. The quantitative labeling through the planes of the dishes is shown. MicroPET image data were acquired for 15 minutes. The signal bars indicate 0 to 100% activity.

Figure 6

Micro–positron emission tomography (microPET) and bioluminescent imaging analysis of mice infected with an adenovirus containing a capsid-incorporated TK-Luc fusion protein. Athymic nude mice were implanted with subcutaneous xenografts of the human head and neck squamous cell carcinoma cell line (FaDu). Tumor nodules were injected with adenovirus and then subjected to imaging analysis. A, MicroPET. Animals were injected intratumorally with control adenovirus encoding TK, Ad-CMV-TK (1 × 10¹⁰ viral particles [VPs] in 0.1 mL phosphate-buffered saline [PBS]) and in an opposing tumor with the dual-modality imaging Ad-pIX-TK-Luc (1 × 10¹⁰ VPs in 0.1 mL PBS). Mice were subjected to microPET analysis 48 hours after the injection. B, Bioluminescence imaging. Animals were injected intratumorally on one flank with control adenovirus encoding luciferase, Ad-CMV-luc (1 × 10¹⁰ VPs in 0.1 mL PBS), and intratumorally on the other flank with the dual-modality