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 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.
Shen
Y
Y
ONYX Pharmaceuticals, Inc., Richmond, California 94806, USA. jshen@onyx-pharm.com
Kitzes
G
G
Nye
J A
JA
Fattaey
A
A
Hermiston
T
T
D017501:Adenovirus E1B Proteins / Q000235:genetics / Q000378:metabolism*; D017504:Adenovirus E4 Proteins / Q000378:metabolism*; D000260:Adenoviruses, Human / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D004279:DNA, Viral; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008969:Molecular Sequence Data; D013194:Staining and Labeling / Q000379:methods; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D017501:Adenovirus E1B Proteins; D017504:Adenovirus E4 Proteins; D004279:DNA, Viral; D016159:Tumor Suppressor Protein p53; D014764:Viral Proteins; C056461:terminal protein, adenovirus