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.

FIG. 1.

IVa2 mutations in pm8002. The black boxes represent the two exons of the IVa2 gene. The intron (INT) and the positions of the primers (A to D) described in Materials and Methods are indicated. The three point mutations, which generated stop codons at amino acids 17 and 19 (underlined), and two restriction enzyme sites, SpeI and AflII, are shown.

FIG. 2.

Gene expression of pm8002. A549 cells were infected with pm8002 or wild-type Ad5 at an MOI of 5. Protein lysates were collected at the indicated times (in hours) after infection. Immunoblotting was performed with antibodies against IVa2, the L1 52/55-kDa protein, 72-kDa DBP, hexon, and E1A, as indicated to the right of the blots.

FIG. 3.

DNA replication of pm8002. 293 (A) or C7 (B) cells were infected with pm8002 or wild-type Ad5 and harvested at the indicated times (in hours) after infection. Viral DNA was extracted, digested with KpnI and SpeI, and analyzed by Southern blotting using an Ad5 probe. In both panels, the black arrowhead and arrows indicate wild-type Ad5- and pm8002-specific bands, respectively. In panel B, the white arrow to the left of the blot points to the control band resulting from inclusion of digested plasmid DNA as an internal standard prior to DNA extraction (Materials and Methods).

FIG. 4.

pm8002 trafficking to the nucleus. Viral DNA was extracted from pm8002- or Ad5-infected 293 cells (A) or purified nuclei (B) at 1 or 4 h after infection. PCR was performed to quantify the DNA using primers for the IVa2 gene. Two different dilutions (1:1 and 1:10) of the template DNA were used in panel A, as described in Materials and Methods.

FIG. 5.

CsCl gradient analysis of viral particles. 293 cells or 293-IVa2 cells were infected with pm8002 or Ad5 at an MOI of 5. CsCl gradient centrifugation was performed 48 h postinfection, and fractions were collected from the bottom of the tube. (A) The density and absorbance of each fraction were measured. Note the difference in scale of the y axis between the top graph and the other two. (B) The amount of IVa2 protein in pm8002 viral particles was analyzed by immunoblotting with anti-IVa2 antibodies. Lane 1, 5 × 10⁹ viral particles from fraction 12 of the pm8002/293-IVa2 gradient; lane 5, 5 × 10⁹ viral particles from fraction 14 of the Ad5/293 gradient; lanes 2 to 4, 27-, 9-, and 3-fold dilutions of the sample in lane 5, respectively.

FIG. 6.

Electron microscopic analysis of virus-infected cells. A mock-infected 293 cell (A), an Ad5-infected 293 cell (B), a pm8002-infected 293 cell (C), and a pm8002-infected 293-IVa2 cell (D) are shown. nm, nuclear membrane. Bars, 0.3 μm.

FIG. 7.

pm8002 is not stable at 42°C. Viral lysates from pm8002-infected 293-IVa2 cells or Ad5-infected 293 cells were incubated at 42°C for various periods of time after which they were assayed on 293 cells in a fluorescent focus assay. The results are presented as percentages of the virus titer before incubation at 42°C.