Suppression of oncogenic viral interferon regulatory factor (vIRF) of Kaposi's sarcoma-associated herpesvirus by ribozyme-mediated cleavage

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV8) has been etiologically associated with several malignancies including Kaposi's sarcoma and primary effusion lymphoma. Oncogenic viral interferon regulato ry factor (vIRF) encoded by KSHV ORF - K9 is a homologue of cellular interf eron regulatory factor (IRF), and has been demonstrated to inhibit type I! II interferon signal transduction and transform NIH3T3 cells through the in teractions with IRF-1, IRF-3, and CRP/p300 proteins. To counteract vlRF's p athogenic role, we have developed five ribozymes targeting ORF-K9 mRNA to s uppress vIRF expression. The vIRF RNA substrates were cleaved up to 80% in a substrate-specific manner in transcript cleavage assays in vitro. In a tr ansient transfection assay, two of the ribozymes efficiently suppressed the expression of vIRF protein measured by dual-color immunofluorescence assay that simultaneously detects the expression of both vIRF protein and ribozy me. Flow cytometry analysis showed that these ribozymes reduced vIRF expres sion up to 76%. A mutant ribozyme had no cleavage activity in vitro, but ex hibited antisense effect in vivo. These results suggest that the ribozymes may provide a new approach for functional knockout of vIRF gene, and are po tential candidates of antiviral therapy for KSHV- related malignancies.