Analyses of single-amino-acid substitution mutants of adenovirus type 5 E1B-55K protein

Abstract

The E1B-55K protein plays an important role during human adenovirus type 5 productive infection. In the early phase of the viral infection, E1B-55K binds to and inactivates the tumor suppressor protein p53, allowing efficient replication of the virus. During the late phase of infection, E1B-55K is required for efficient nucleocytoplasmic transport and translation of late viral mRNAs, as well as for host cell shutoff. In an effort to separate the p53 binding and inactivation function and the late functions of the E1B-55K protein, we have generated 26 single-amino-acid mutations in the E1B-55K protein. These mutants were characterized for their ability to modulate the p53 level, interact with the E4orf6 protein, mediate viral late-gene expression, and support virus replication in human cancer cells. Of the 26 mutants, 24 can mediate p53 degradation as efficiently as the wild-type protein. Two mutants, R240A (ONYX-051) and H260A (ONYX-053), failed to degrade p53 in the infected cells. In vitro binding assays indicated that R240A and H260A bound p53 poorly compared to the wild-type protein. When interaction with another viral protein, E4orf6, was examined, H260A significantly lost its ability to bind E4orf6, while R240A was fully functional in this interaction. Another mutant, T255A, lost the ability to bind E4orf6, but unexpectedly, viral late-gene expression was not affected. This raised the possibility that the interaction between E1B-55K and E4orf6 was not required for efficient viral mRNA transport. Both R240A and H260A have retained, at least partially, the late functions of wild-type E1B-55K, as determined by the expression of viral late proteins, host cell shutoff, and lack of a cold-sensitive phenotype. Virus expressing R240A (ONYX-051) replicated very efficiently in human cancer cells, while virus expressing H260A (ONYX-053) was attenuated compared to wild-type virus dl309 but was more active than ONYX-015. The ability to separate the p53-inactivation activity and the late functions of E1B-55K raises the possibility of generating adenovirus variants that retain the tumor selectivity of ONYX-015 but can replicate more efficiently than ONYX-015 in a broad spectrum of cell types.

FIG. 1

Amino acid sequence of the E1B-55K protein of Ad5. The region for p53 binding (19) is underlined, the putative RNA-binding domains (K. Leppard, personal communication) are indicated by the solid boxes, and the region required for transcription repression (P. E. Branton, personal communication) is defined by a broken box. Asterisks indicate amino acids that are mutated.

FIG. 2

Effects of E1B-55K mutations on p53 accumulation and viral gene expression. A549 cells were either mock infected (mock) or infected with dl309, WtD, ONYX-015, or viruses harboring various E1B-55K mutants. All infections were performed at an MOI of 10. Cell extracts were prepared at 24 h postinfection and were separated by SDS-PAGE. Steady-state levels of E1B-55K mutants, p53, E2A, and fiber were determined by Western blotting with monoclonal antibodies 2A6, DO-1, B6-6, and a rabbit polyclonal anti-fiber antibody, respectively. Blots were visualized with ECL, as described in Materials and Methods.

FIG. 3

Coimmunoprecipitation of p53 by E1B-55K mutants. (A and B) Coimmunoprecipitation experiment. A549 cells were either mock infected (mock) or infected with various virus mutants at an MOI of 10. At 24 h postinfection, cells were metabolically labeled with [³⁵S]methionine-cysteine for a 3-h period. Cell extracts were immunoprecipitated with anti-p53 antibody Ab421 (A) or anti-E1B-55K antibody 2A6 (B) and analyzed by SDS-PAGE as described in Materials and Methods. E1B-55K mutant R240A was expressed from ONYX-051, and H260A was expressed from ONYX-053. (C and D) In vitro-translated (and ³⁵S-labeled) p53 was mixed with lysates prepared from infected A549 cells at 24 h post infection and immunoprecipitated with 2A6 anti-E1B-55K antibody. Immune complexes were separated by SDS-PAGE and visualized either directly by autoradiography (C) or by Western blotting with 2A6 antibody (D).

FIG. 4

Binding of E4orf6 by the E1B-55K mutants. A549 cells were either mock infected (mock) or infected with indicated virus mutants at an MOI of 10. At 24 h postinfection cells were lysed; cleared cell extracts were immunoprecipitated with anti-E1B-55K antibody 2A6. The precipitated materials were separated by SDS-PAGE and analyzed by Western blotting with a rat polyclonal antibody against E4orf6 (A) or with a rat anti-E1B-55K polyclonal antibody (B). (C) The level of E4orf6 expression in the infected cells by straight Western analysis using a polyclonal anti-E4orf6 antibody. dl355* is a dl309 derivative lacking the E4orf6 gene.

FIG. 5

Indirect immunofluorescent staining of adenovirus- and mock-infected (mock) cells. A549 cells grown on chamber slides were infected with dl309 or ONYX-015, -051, -052, or -053 at an MOI of 10 or were mock infected. At 24 h postinfection the cells were fixed, permeabilized, and analyzed by indirect immunofluorescent staining using the E1B-55K-specific monoclonal antibody 9C10 (αE1B), p53-specific monoclonal antibody DO-1 (αp53), and E4orf6-specific polyclonal antibody 1807-3 (αE4). Representative fields are shown for all cases.

FIG. 6

Replication of dl309, ONYX-015, ONYX-051, and ONYX-053 as a function of time postinfection in p53-null H1299 cells at 32 and 39°C. H1299 cells were infected and maintained at two temperatures, 32 and 39°C. Infections at 32°C were performed 1 h after the temperature shift from 39°C. All infections were performed at an MOI of 5. Infected cells were incubated at 32 and 39°C, respectively. Ninety-six hours postinfection cells and culture media were harvested, pooled, and freeze-thawed three times to release virus particles. Viral yields were determined by enzyme-linked immunosorbent assay on a 293 cell monolayer. Total viral yields were divided by the number of cells at the time of infection to determine viral production per cell. Results are the average from two independent experiments that are highly consistent with each other.

FIG. 7

Protein expression during the late phase of adenovirus infection. A549 cells were either mock infected (mock) or infected with various adenovirus mutants at an MOI of 10. At 24 h postinfection, cells were metabolically labeled with [³⁵S]methionine-cysteine for a 3-h period. Cell extracts were resolved by SDS-PAGE (4 to 20% gradient gel). The positions of the molecular mass markers are indicated at the right.

FIG. 8

Cytolytic activity in tumor cells. DU145 and U2OS cells were seeded into 96-well plates at a density of 2.5 × 10³ cells/well. Twenty-four hours after seeding, cells were infected with serial threefold dilutions of E1B-55K mutant viruses, ranging from an MOI of 30 to an MOI of 1.5 × 10⁻³. dl309 and ONYX-015 were included as controls. MTT assays were performed 6 days after infection as described in Materials and Methods. The MOIs that resulted in 50% cell killing were defined as IC50 and were plotted for each virus. Results from one of the two independent experiments are shown here. Similar results were obtained in the other experiment.