Adenovirus (Ad) assembly has been studied by using pulse-chase kinetic analyses, through the characterization of temperature-sensitive virus mutants blocked at different stages of assembly at the restrictive temperature, by studies of virus mutants defective in the expression of proteins involved in the packaging process, and by studies of mutants defective in the packaging sequences (2-4, 6-8, 12, 25, 31, 32). The results suggest that adenovirus assembly follows an ordered series of assembly and processing events analogous to those found in the assembly of bacteriophages. Whether the viral genome is involved in nucleating the initiating steps of capsid morphogenesis or whether viral DNA is inserted into an empty, preformed capsid has not been resolved. It is clear, however, that the viral genome contains cis-acting sequences that mediate the packaging process (reviewed in reference 20). Adenovirus DNA packaging occurs in a polar manner from the left end of the genome (1, 28). The genomes of adenovirus type 3 (Ad3), Ad5, and Ad16, representatives of adenovirus subgroups B and C, harbor conserved packaging signals (6, 10, 19, 22). These conserved cis-acting packaging domains suggest similar mechanisms of selective and polar DNA packaging for all adenovirus subgroups. Ad5 DNA encapsidation is dependent on cis-acting sequences located between nucleotides (nt) 230 and 380 (Fig. (Fig.1A)1A) (6, 7, 13, 25). Seven repeated sequences (termed A-repeats due to their AT-rich content) that contribute to viral DNA packaging are located within this domain. Although A-repeats are functionally redundant, they follow a hierarchy of importance: A1, A2, A5, and A6 are the most important repeats for packaging activity (6, 7, 25). A-repeats contain a bipartite consensus motif (5′-TTTG N8 CG-3′ [Fig. [Fig.1A]).1A]). Both the first and the second half-site of the consensus motif, as well as the 8-bp spacing between the half-sites, are critical for viral DNA packaging (25). Several lines of evidence suggest that a limiting trans-acting factor(s), which binds to the viral packaging sequences, plays a role in the packaging process (7, 25, 26). First, in vivo coinfection experiments show that viruses with a greater number of packaging repeats package more efficiently than viruses with fewer packaging repeats. Second, an isolated packaging domain on a multicopy plasmid represses packaging of a wild-type virus in vivo. Both results suggest competition for a limiting packaging factor(s) in vivo. Multimerized individual packaging repeats, termed minimal packaging domains, have been used to study Ad DNA packaging (5, 26). Minimal packaging sequences were shown to support packaging in vivo to various degrees when built into viruses lacking the authentic packaging domain. When minimal packaging sequences were used in binding assays in vitro as probes, three cellular transcription factors that bind to these sites were identified: chicken ovalbumin upstream promoter transcription factor (COUP-TF), OCT-1, and CCAAT displacement protein (CDP) (5, 26). It is very unlikely that COUP-TF and OCT-1 are required for Ad packaging, since viruses with mutated or synthetic packaging domains were identified that functioned efficiently in packaging in vivo but did not bind these transcription factors in vitro. However, a role for CDP in Ad packaging was suggested in earlier studies (5, 26). FIG. 1. Ad5 packaging sequences. (A) Schematic representation of the left end of the Ad5 genome. Nucleotide coordinates, relative to the left terminus, are indicated, and the inverted terminal repeat (ITR) is represented by a gray box. A-repeats 1 to 7 are represented ... Two viral proteins, L1 52/55K and IVa2, have been found to play important roles in Ad packaging and virus assembly (8, 12, 31, 32), although their exact roles in this process remain unclear. A null mutant and a temperature-sensitive mutant of the L1 52/55K protein, respectively, form empty capsids or capsids that contain only the left end of the viral genome (8, 12). The Ad L1 52/55K protein is found within immature virus particles (8, 12) and forms a physical complex with the Ad IVa2 protein (9). In turn, the Ad IVa2 protein is essential for virus assembly and the formation of empty viral capsids. Ad IVa2 is found in both empty and mature virus particles (31, 32). The IVa2 protein binds to packaging A-repeats 1 and 2 as well as to A-repeats 4 and 5 in vitro (30). Collectively, these data demonstrate that the Ad L1 52/55K and IVa2 proteins play a key role(s) in the very early stages of the virus assembly process. IVa2 appears to be a multifunctional protein during Ad infection. In addition to its role in viral assembly, IVa2 has been implicated in the transcriptional control of the major late promoter (MLP) (29) by binding to the DE elements within the MLP (17). In this study, we have examined the binding of the Ad IVa2 and L1 52/55K proteins to wild-type and mutant packaging sequences in vitro and in vivo in comparison to the growth properties of corresponding mutant viruses. Our results demonstrate that the binding of IVa2 to the packaging sequences in vitro correlates directly with the packaging efficiency of the virus in vivo. Furthermore, it appears that the higher-order IVa2-containing complexes that form on adjacent packaging A-repeats in vitro are the complexes required for the packaging activity of these sites in vivo. Further, both the IVa2 and L1 52/55K proteins associate with the packaging domain in vivo in direct correlation with packaging function. Finally, our results indicate that the cellular protein CDP does not play a role in packaging. These data are consistent with the known role of the IVa2 and L1 52/55K proteins in the Ad assembly process, and they provide strong evidence for a causal link between the binding of these proteins to packaging sequences and the assembly of virus particles.