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 regulatory factor (vIRF) encoded by KSHV ORF-K9 is a homologue of cellular interferon regulatory factor (IRF), and has been demonstrated to inhibit type I/II interferon signal transduction and transform NIH3T3 cells through the interactions with IRF-1, IRF-3, and CBP/p300 proteins. To counteract vIRF's pathogenic role, we have developed five ribozymes targeting ORF-K9 mRNA to suppress vIRF expression. The vIRF RNA substrates were cleaved up to 80% in a substrate-specific manner in transcript cleavage assays in vitro. In a transient 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 ribozyme. Flow cytometry analysis showed that these ribozymes reduced vIRF expression up to 76%. A mutant ribozyme had no cleavage activity in vitro, but exhibited antisense effect in vivo. These results suggest that the ribozymes may provide a new approach for functional knockout of vIRF gene, and are potential candidates of antiviral therapy for KSHV-related malignancies.
Zhang
Y J
YJ
Department of Pediatrics, The University of Texas Health Science Center at San Antonio, 78229, USA.
Wang
X P
XP
Deng
J H
JH
Salinas
R A
RA
Oishi
N
N
Gao
S J
SJ
D000818:Animals; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D019288:Herpesvirus 8, Human / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D050835:Interferon Regulatory Factors; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D016337:RNA, Catalytic / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014764:Viral Proteins
D004268:DNA-Binding Proteins; D050835:Interferon Regulatory Factors; D016337:RNA, Catalytic; D012333:RNA, Messenger; D014157:Transcription Factors; D014764:Viral Proteins; C427204:hammerhead ribozyme; C413486:viral interferon regulatory factors