Developing Novel Oncolytic Adenoviruses through Bioselection

Mutants of human adenovirus 5 (Ad5) with enhanced oncolytic activity were isolated by using a procedure termed bioselection. Two mutants, ONYX-201 and ONYX-203, were plaque purified from a pool of randomly mutagenized Ad5 that was repeatedly passaged in the human colorectal cancer cell line HT29, and they were subsequently characterized. ONYX-201 and ONYX-203 replicated more rapidly in HT29 cells than wild-type Ad5, and they lysed HT29 cells up to 1,000-fold more efficiently. The difference was most profound when cells were infected at a relatively low multiplicity of infection, presumably due to the compounding effects of multiple rounds of infection. This enhanced cytolytic activity was observed not only in HT29 cells but also in many other human cancer cell lines tested. In contrast, the cytotoxicity of the bioselected mutants in a number of normal primary human cells was similar to that of wild-type Ad5, thus enhancing the therapeutic index (cytotoxicity in tumor cells versus that in normal cells) of these oncolytic agents. Both ONYX-201 and -203 contain seven single-base-pair mutations when compared with Ad5, four of which were common between ONYX-201 and -203. The mutation at nucleotide 8350, shared by both mutant viruses, was shown to be essential for the observed phenotypes. This mutation was mapped to the i-leader region of the major late transcription unit, resulting in the truncation of 21 amino acids from the C terminus of the i-leader protein. This work demonstrates that bioselection is a powerful tool for developing novel tumor-selective oncolytic viruses. Other potential applications of this technology are discussed

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

Interactions of SV40 large T antigen and other viral proteins with retinoblastoma tumour suppressor

Simian virus 40 large T antigen, human papilloma virus E7 and adenovirus E1A are all potent oncoproteins that can induce several types of tumours. One of the major functions of these oncoproteins is to interact with the retinoblastoma tumour suppressor protein, Rb, a master switch of the mammalian cell cycle, and to inactivate its function. Rb promotes cell-cycle arrest by recruiting and regulating proteins involved in the transcription of cell proliferation genes. The binding of viral oncoproteins to Rb disrupts the Rb-E2F complex, a central component in the Rb-mediated cell-cycle network. The crystal structures of Rb pocket-viral oncoprotein complexes indicate that the viral proteins recognise a highly conserved region in the Rb pocket through a common motif, LxCxE, and through other unique regions within each viral protein, Although the mechanism of Rb inactivation by viral proteins is not fully understood, information at the atomic level about the interactions between the Rb pocket and viral proteins is providing some insights into how viral proteins dissociate E2F from Rb and thus how they deregulate the cell cycle.close303