The adenovirus L1 52/55-kDa protein is required for viral DNA packaging and interacts with the viral IVa2 protein, which binds to the viral packaging sequence. Previous reports suggest that the IVa2 protein plays a role in viral DNA packaging and that this function of the IVa2 protein is serotype specific. To further examine the function of the IVa2 protein in viral DNA packaging, a mutant virus that does not express the IVa2 protein was constructed by introducing two stop codons at the beginning of the IVa2 open reading frame in a full-length bacterial clone of adenovirus type 5. The mutant virus, pm8002, was defective for growth in 293 cells, although it replicated its DNA and produced early and late viral proteins. Electron microscopic and gradient analyses revealed that the mutant virus did not assemble any viral particles in 293 cells. In 293-IVa2 cells, which express the IVa2 protein, infectious viruses were produced, although the titer of the mutant virus was lower than that of the wild-type virus, indicating that these cells may not fully complement the mutation. The mutant viral particles produced in 293-IVa2 cells were heterogeneous in size and shape, less stable, and did not traffic efficiently to the nucleus. Marker rescue experiments with a wild-type IVa2 DNA fragment confirmed that the only mutations present in pm8002 were in the IVa2 gene. The results indicate that the IVa2 protein is required for adenovirus assembly and suggest that virus particles may be assembled around the DNA rather than DNA being packaged into preformed capsids.The adenovirus capsid is an icosahedron containing three major proteins, hexon (720 molecules/virion), penton base (60 molecules/virion), and fiber (36 molecules/virion) (55, 61, 62), along with a series of minor components. Inside the capsid is the viral genome with associated core proteins (47). During assembly, hexon proteins trimerize to form hexon capsomers, which are the major structural units of the capsid (45). These capsomers then come together with the other capsid proteins and scaffolding proteins to form the capsid. The mechanisms of how adenovirus particles assemble and viral DNA is packaged are not yet fully understood. Empty capsids, which contain no viral DNA or core proteins, are produced in parallel to mature virions in infected cells (57). Intermediate particles with buoyant densities between those of empty capsids and mature virions have also been isolated from infected cells. These intermediates can be divided into light ( Ï 1.315 g/cm 3 ) particles containing small DNA fragments and heavy ( Ï 1.37 g/cm 3 ) particles containing full-length DNA (11). Kinetic radiolabeling and pulse-chase studies suggest that these empty capsids and intermediate particles are precursors of mature virions (34,35,57). On the basis of these early studies, it is generally believed that the viral genome and core proteins are inserted into preassembled empty capsids. Studies of the protein composition of empty capsids and intermediate particles also support the precursor-...