Effective inhibition of Rta expression and lytic replication of Kaposi's sarcoma-associated herpesvirus by human RNase P

Abstract

Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a nucleic acid-based gene interference approach to knock-down gene expression. Unlike other strategies, such as antisense oligonucleotides, ribozymes, and RNA interference, the RNase P-based technology is unique because a custom-designed EGS molecule can bind to any complementary mRNA sequence and recruit intracellular RNase P for specific degradation of the target mRNA. In this study, we demonstrate that the RNase P-based strategy is effective in blocking gene expression and growth of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), the causative agent of the leading AIDS-associated neoplasms, such as KS and primary-effusion lymphoma. We constructed 2-O-methyl-modified EGS molecules that target the mRNA encoding KSHV immediate-early transcription activator Rta, and we administered them directly to human primaryeffusion lymphoma cells infected with KSHV. A reduction of 90% in Rta expression and a reduction of Ϸ150-fold in viral growth were observed in cells treated with a functional EGS. In contrast, a reduction of <10% in the Rta expression and viral growth was found in cells that were either not treated with an EGS or that were treated with a disabled EGS containing mutations that preclude recognition by RNase P. Our study provides direct evidence that EGSs are highly effective in inhibiting KSHV gene expression and growth. Exogenous administration of chemically modified EGSs in inducing RNase Pmediated cleavage represents an approach for inhibiting specific gene expression and for treating human diseases, including KSHVassociated tumors.gene targeting ͉ gene inactivation ͉ antisense ͉ cancer therapy

Inhibition of gene expression in human cells using RNase P-derived ribozymes and external guide sequences

“…Thus, the EGS-based technology is highly specific and does not generate nonspecific "irrelevant cleavage" that is observed in RNase H-mediated cleavage induced by conventional antisense phosphorothioate molecules [36,38]. Third, EGSs exhibit little sign of cytotoxicity because cells expressing these molecules for more than 40 days appear to be normal [36,38,44,81].…”

“…For stability, the ribozymes and EGSs could be chemically synthesized with 2′ hydroxyl modification and/or phosphorothioates to resist cellular endonucleases [87]. As an alternative to the viral vector approach, smaller ribozymes can be delivered ex vivo by encapsulating them in liposomes or other biodegradable polymeric matrix [37,38,44]. In the case of M1GS, chemical synthesis of a functional active ribozyme is at present technically difficult and economically impractical due to its large size (~400 nucleotides).…”

“…Utilization of RNase P and M1 RNA for the development of antiviral agents has been extensively pursued in several laboratories, including ours, for blocking infection of human immunodeficiency virus (HIV) [72,73], human influenza virus [74], and three human herpesviruses such as human cytomegalovirus (HCMV) [41,69], herpes simplex virus 1 (HSV-1) [65,70,75], and Kaposi's sarcoma-associated herpesvirus (KSHV) [44]. Herpes simplex virus 1 (HSV-1) is a causative agent of cold sores and encephalitis in newborns [76].…”

“…More recently, EGS-based gene interference strategy has been attempted in blocking gene expression and growth of yet another member of the herpesvirus family, namely Kaposi's sarcoma-associated herpesvirus (KSHV). Chemically modified EGS molecules were constructed to target the mRNA encoding KSHV immediate-early transactivator called Rta [44]. Exogenous administration of 2′-O-methyl-modified EGS to KSHV-infected human primary-effusion lymphoma cells significantly inhibited Rta expression and subsequently resulted in 150-fold reduction in viral growth [44].…”

“…Accessibility of the target mRNAs should be mapped in vitro and in vivo, using numerous methods. For example, we have utilized in vivo mapping of accessible regions in some target mRNAs with dimethyl sulfate (DMS) [41,44]. DMS methylates N7 of guanine, N1 of adenine, and N3 of cytosine, of which the latter two can be examined by primer extension assay (transcription terminates at the base immediately before the modified one) [34,45].…”