HUMAN HERPESVIRUS-6 (HHV-6) ORF-1 TRANSACTIVATING GENE EXHIBITS MALIGNANT TRANSFORMING ACTIVITY AND ITS PROTEIN BINDS TO P53

The 357 amino acid open reading frame 1 (OFF-1), also designated DR7, within the SalI-L fragment of human herpesvirus 6 (HHV-6) exhibited tr ansactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter and increased HIV-1 replication (Kashan chi et al., Virology, 201, 95-106, 1994). Tn the current study, the Sa lI-L transforming region was localized to the SalI-L-SH subfragment, S everal ORFs identified in SalI-L-SH by sequence analysis were cloned i nto a selectable mammalian expression vector, pBK-CMV, Only pBK/ORF1 t ransformed NIH3T3 cells. Furthermore, cells expressing ORF-1 protein p roduced fibrosarcomas when injected into nude mice, whereas control ce lls, expressing either no ORF-1 protein or C-terminal truncated (after residue 172) ORF-1 protein, were not tumorigenic, Western blot analys is of proteins extracted from the tumors revealed ORF-1 protein. Addit ional studies indicated that ORF-1 was expressed in HHV-6-infected hum an T-cells by 18 h. Co-immunoprecipitation experiments showed that ORF -1 protein bound to tumor suppressor protein p53, and the OFF-1 bindin g domain on p53 was located between residues 28 and 187 of p53, overla pping with the specific DNA binding domain. Functional studies showed that p53-activated transcription was inhibited in ORF-1, but not in tr uncated OFF-1, expressing cells. Importantly, the truncated ORF-1 muta nt also failed to cause transformation. Analysis of several human tumo rs by PCR revealed OFF-1 DNA sequences in some angioimmunoblastic lymp hadenopathies, Hodgkin's and non-Hodgkin's lymphomas and glioblastomas . The detection of OFF-1 sequences in human tumors, while not proof pe r se, is a prerequisite for establishing its role in tumor development . Taken together, the results demonstrate that OFF-1 is an HHV-6 oncog ene that binds to and affects p53. The identification of both transfor ming and transactivating activities within ORF-1 is a characteristic o f other viral oncogenes and is the first reported for HHV-6.