Requirement of the Adenovirus IVa2 Protein for Virus Assembly

Abstract

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 ( ϭ 1.315 g/cm 3 ) particles containing small DNA fragments and heavy ( ϭ 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-...

Dependence of the Encapsidation Function of the Adenovirus L1 52/55-Kilodalton Protein on Its Ability To Bind the Packaging Sequence

“…The IVa2 protein is present in assembly intermediates and mature virions (7,11,28). Characterization of a IVa2 mutant virus, pm8002, demonstrated that no viral particles were formed during infection although gene expression and viral DNA replication were not affected (31). These results support an essential role for the IVa2 protein in virus assembly and suggest that it is involved in DNA packaging.…”

“…For Western blot analysis, samples were resuspended in SDS sample buffer and separated on a 10% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose and probed for the L1 52/55-kDa protein with rabbit anti-L1 antibodies (8) and for the IVa2 protein with rabbit anti-IVa2 antibodies (31). Proteins were visualized using a secondary horseradish peroxidase-conjugated antibody and chemiluminescence as recommended by the manufacturer (Amersham Pharmacia Biotech).…”

“…293 cells that stably express the L1 52/55-kDa protein (293-L1) were used as a helper cell line to grow the pm8001 mutant virus, which fails to express the L1 52/55-kDa protein (7). 293 cells that stably express the IVa2 protein (293-IVa2) were used as a helper cell line to grow the pm8002 mutant virus, which fails to express the IVa2 protein (31). Wild-type Ad5 virus was propagated as previously described (5,30).…”

Viral DNA synthesis-dependent titration of a cellular repressor activates transcription of the human adenovirus type 2 IVa 2 gene

“…4. When the concentration of the IVa 2 protein incorporated into virions is reduced, entry of the viral genome into the infected cell nucleus and expression of all early genes is delayed (42). This property suggests that our Rep mutants that accelerate expression of the IVa 2 gene (Fig.…”

Genetic Identification of Adenovirus Type 5 Genes That Influence Viral Spread

“…The L1 52/55K protein, in association with a late protein, IVa2, plays a role in encapsidation of the viral DNA (42,83,84). Some of the mRNAs initiated from the major late promoter, particularly the L1 mRNAs, contain an additional leader (i-leader) spliced together between the second and third segments of the tripartite leader (17).…”