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.

The adenovirus (Ad) protein IX (pIX) is a minor component of the Ad capsid and is in part responsible for virion stability; virions lacking pIX are heat labile and lose their infectivity if the DNA content is greater than ϳ35 kb. More recently, pIX has been identified as a transcriptional activator and, in transient-transfection assays, was shown to enhance expression from the E1A, E4, and major late Ad promoters by as much as 70-fold. In this study, we examined the role of pIX's ability to activate transcription during Ad replication. In transient-transfection assays, pIX had a minimal effect on expression from the E1A promoter, increasing expression by only 1.4-fold. We used helper-dependent Ad vectors, which had all Ad protein coding sequences deleted with the exception of E1A and which had capsids that either contained or lacked pIX, to show that pIX derived from decapsidation of the infecting virion does not influence expression of E1A. Similarly, expression of pIX from the Ad genome did not alter the expression levels of E1A. Viruses that had pIX deleted showed a threefold reduction in virus yield and expression of late genes compared to those of a similar virus which encoded pIX. This phenotype could not be rescued by growing the virus in cells which constitutively express pIX. Our results indicate that, although pIX can affect transcription from a variety of viral promoters, it does not appear to play a significant role in activation of Ad promoters during normal Ad replication.The adenovirus (Ad) protein IX (pIX) is a minor component of the Ad virion that associates with the hexons that make up the facets of the icosahedron (1). There are 240 molecules of pIX per Ad virion, and within the virion, pIX is found as a trimer. pIX is expressed at an intermediate time during infection (after initiation of early gene expression but before expression of late genes); however, the levels of pIX increase dramatically after DNA replication has initiated (1). Although originally thought to be dispensable for virion formation (4), pIX was shown to be essential for the packaging of full-length viral DNA molecules (6). Deletion of pIX results in virions that are heat labile, with capsids that reportedly can no longer package full-length Ad DNA (36 kb) but, instead, can accommodate only 35 kb or less (ϳ97% of the wild-type genome). However, we have recently shown that large DNA molecules (up to at least 37.2 kb) can be accommodated in capsids deficient in pIX but that these virions are noninfectious (i.e., do not form plaques) for reasons which are still unclear (16).In addition to its role as a structural protein, pIX has been implicated as a transcriptional activator and appears to be involved in virus-induced nuclear reorganization (10,14). Whereas the N-terminal region of pIX is required for incorporation of the protein into virions, the C terminus, specifically a leucine repeat, is important for its transcriptional activity (14). In transient-transfection assays, pIX was shown to enhance expression from the Ad earl...