Use of adenovirus protein IX (pIX) to display large polypeptides on the virion—generation of fluorescent virus through the incorporation of pIX-GFP

Abstract

The adenovirus (Ad) protein IX (pIX) is a minor component of the Ad capsid and associates with the hexons that make up the facets of the icosahedron. In this study, we investigated whether a large protein tag could be fused to pIX without compromising the Ad vector itself. As proof-of-principle, we generated a pIX-green fluorescent protein (GFP) fusion protein. We show that a virus encoding the pIX-GFP can be generated and that pIX-GFP fusion protein was incorporated into the Ad capsid as efficiently as native pIX. In tissue culture, translocation of Ad/pIX-GFP from the outside of the cell to the nucleus could be followed using fluorescence microscopy, and the timing of migration to the nucleus was similar to that previously reported for Ad. We also could track the virus after injection into the tibialis anterior muscle of mice. Shortly after injection, the majority of the Ad/pIX-GFP accumulated in pockets adjacent to the muscle fibers, with some migration of the virus between fibers. Our ability to attach GFP to the Ad virion, through fusion to pIX, provides a valuable tool for virus tracking in vitro and in vivo. Moreover, our data indicate that pIX can be used as a platform to anchor proteins to the Ad capsid, such as large ligands for cell-type-specific targeting of the vector.

Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping

“…Besides its role as a structural protein, it is a transcriptional activator (Rosa-Calatrava et al, 2001;Sargent et al, 2004a) and reorganizes promyelocytic leukaemia (PML) nuclear bodies (Rosa-Calatrava et al, 2003) (reviewed by Parks, 2005). Protein IX has been extensively studied as a platform to express foreign peptides on the surface of the capsid (Dmitriev et al, 2002;Le et al, 2004;Li et al, 2005;Meulenbroek et al, 2004;Vellinga et al, 2004Vellinga et al, , 2007. Since protein IX has been so well characterized, we selected it as a fusion partner to test whether 2A sequences could be used to express foreign genes in adenoviruses.…”

Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

“…We and others have shown that pIX can be modified genetically to incorporate large polypeptides into the Ad capsid (38,47,53,85). pIX is a minor capsid protein that stabilizes the hexon on the facets of the capsid (14,23).…”

Transductional targeting of adenovirus vectors for gene therapy

“…Of note, the presence of the pIX-eGPF fusion in purified Ad virions did not appreciably decrease virus viability or capsid stability, and has allowed monitoring of Ad localization in vitro and in vivo. 132,133 Further, other Ad vectors harboring complex ligands at the pIX locale have been reported. [134][135][136] As a whole, these studies have established pIX as a highly relevant capsid locale marked by the highest structural compatibility, with diverse targeting and imaging ligands observed to date.…”