The packaging of adenovirus (Ad) DNA into virions is dependent upon cis-acting sequences and trans-acting proteins. We studied the involvement of Ad packaging proteins in the serotype specificity of packaging. Both Ad5 and Ad17 IVa2 and L4-22K proteins complemented the growth of Ad5 IVa2 and L4-22K mutant viruses, respectively. In contrast, the Ad5 L1-52/55K protein complemented an Ad5 L1-52/55K mutant virus, but the Ad17 L1-52/55K protein did not. The analysis of chimeric proteins demonstrated that the N-terminal half of the Ad5 L1-52/55K protein mediated this function. Finally, we demonstrate that the L4-33K and L4-22K proteins have distinct functions during infection.The adenovirus (Ad) packaging domain is a cis-acting region located at the left end of the genome that directs viral DNA encapsidation (reviewed in reference 13). The packaging domain consists of seven A repeats, each sharing the consensus motif 5Ј-TTTG-N 8 -CG-3Ј (13). The Ad5 protein IVa2 binds to the CG portion of A repeats (15,17,22). The IVa2 protein is necessary for both Ad virion assembly and viral DNA packaging (23, 24). The IVa2 protein binds to the Ad5 L1-52/55 K protein (8), which also is involved in Ad5 DNA packaging (7, 9). The L1-52/55K protein is recruited to the packaging domain in vivo, although this does not require the IVa2 protein (15,17). It is unclear how this protein is recruited to the packaging domain. The Ad5 L4-22K and L4-33K proteins are splice variants of an Ad late region 4 transcript (11). The L4-33K protein was found to be involved in the early-to-late switch of the infectious cycle (4, 21) and also is necessary for virus assembly (5, 6, 10). The Ad5 L4-22K protein binds to the TTTG portion of A repeats (3,12). Binding of the IVa2 protein is a prerequisite for the specific interaction of the L4-22K protein with DNA (3,12). An Ad5 L4-22K viral mutant displays a late defect in the production of infectious virus (12).Although Ad DNA packaging is largely serotype specific (24), it is possible to pseudopackage the DNA from one Ad serotype into the capsid of a different serotype (14). These results imply that recognition exists between the viral proteins bound to the packaging domain and the capsid of the virus. Here, we provide evidence that the L1-52/55K protein is involved in the serotype specificity of Ad packaging. In contrast, the IVa2 and L4-22K proteins of two different Ad serotypes have interchangeable functions. The amino-terminal half of the L1-52/55K protein mediates this function. Finally, we demonstrate that the Ad5 L4-33K and L4-22K proteins have distinct functions during viral infection.We analyzed the Ad packaging proteins to determine which protein(s) may be implicated in the serotype specificity of DNA packaging. Viral mutants that are unable to express functional IVa2 (pm8002) (23), L1-52/55K (pm8001) (7), or L4-22K (v22K Ϫ ) (12) proteins were used in complementation experiments to determine if these mutants could be rescued when the corresponding proteins of Ad5 or Ad17 were provided in trans. pTG36...