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

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 bi nds to and inactivates the tumor suppressor protein p53, allowing efficient replication of the virus. During the late phase of infection, E1B-55K is r equired for efficient nucleocytoplasmic transport and translation of late v iral 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 p rotein. These mutants were characterized for their ability to modulate the p53 level, interact with the E4orf6 protein, mediate viral late-gene expres sion, and support virus replication in human cancer cells, Of the 26 mutant s, 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 i n the infected cells, In vitro binding assays indicated that R240A and H260 A bound p53 poorly compared to the wild-type protein,When interaction with another viral protein, E4orf6, was examined, H260A significantly lost its a bility 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 t hat the interaction between E1B-55K and E4orf6 was not required for efficie nt viral mRNA transport, Both R240A and H260A have retained, at least parti ally, the late functions of wild-type E1B-55K, as determined by the express ion of viral late proteins, host cell shutoff, and lack of a cold-sensitive phenotype, Virus expressing R240A (ONYX-051) replicated very efficiently i n human cancer cells, while virus expressing H260A (ONYX-053) was attenuate d 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 reta in the tumor selectivity of ONYX-015 but can replicate more efficiently tha n ONYX-015 in a broad spectrum of cell types.