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 primary-effusion lymphoma cells infected with KSHV. A reduction of 90% in Rta expression and a reduction of approximately 150-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 P-mediated cleavage represents an approach for inhibiting specific gene expression and for treating human diseases, including KSHV-associated tumors.

Fig. 1.

Schematic representation of substrates for RNase P. (A) A natural substrate precursor tRNA for RNase P. (B and C) Hybridized complex of a target RNA (e.g., mRNA) and an EGS that resembles the structure of a tRNA and can be cleaved by RNase P. C results from B by deletion of the anticodon domain of the EGS, which is dispensable for EGS-targeting activity (21). Arrowheads indicate the site of cleavage by RNase P. (D and E) Complexes between the Rta mRNA sequence and EGS R1 and R2, respectively. The sequence of these EGSs equivalent to the tRNA sequence was derived from tRNA^ser and resembles the T-stem and loop and a variable region of the tRNA molecule.

Fig. 2.

Stability of 2′-O-methyl-modified EGS R1 and R2 in the presence of human plasma and HeLa nuclear extracts. EGSs were either loaded directly on polyacrylamide denaturing gels (-, lanes 1 and 4) or incubated in either DMEM containing 10% human plasma (Plasma, lanes 2 and 5) or in HeLa nuclear extracts (Promega) (HeLa, lanes 3 and 6) at 37°C for 24 h before loading on the gels.

Fig. 3.

Cleavage of ³²P-labeled substrate rta-S by human RNase P in the presence of different EGSs. No RNase P was added to the reaction mixture in lane 1. We incubated 5 nM of Rta-S substrate alone (lanes 1-2) or in the presence of 5 nM R1 (lane 3) or R2 (lane 4) in the presence of 2 units of RNase P. Cleavage reactions were carried out in buffer A (50 mM Tris, pH 7.0/100 mM NH₄Cl/10 mM MgCl₂) at 37°C for 1 h.

Fig. 4.

Internalization of EGSs in human cells. We complexed 20 nM 5′-fluorescein-labeled TK1 with 10 μg/ml Lipofectamine 2000 either in the absence or presence of R1 or R2 (80 nM), and it was then transfected into BCBL-1 cells. The transfected cells were isolated by using FACS analysis at 7 h after infection, and nuclear and cytoplasmic RNA fractions were purified. Northern blot analyses were carried out by using nuclear RNA fractions isolated from parental BCBL-1 cells (-, lanes 1 and 5) and cells that were treated with R1 (lanes 2, 4, 6, and 8) and R2 (lanes 3 and 7). We separated 30-μg (lanes 1-3, 5-7) and 60-μg RNA samples (2×, lanes 4 and 8) on 0.8% (B) and 2.5% (A) agarose gels that contained formaldehyde, and they were then transferred to a nitrocellulose membrane and hybridized to a ³²P-radiolabeled probe that contained the DNA sequence coding for R1 (A) or RNase P H1 RNA (B). The RNase P RNA sequence was used as the internal control.

Fig. 5.

Expression of KSHV mRNAs in EGS-treated cells. We first treated 2 × 10⁵ BCBL-1 cells with liposome complexes containing EGSs and isolated them by using FACS analysis, and then we incubated them either in the absence (lanes 1, 5, 9, and 13) or presence (lanes 2-4, 6-8, 10-12, and 14-16) of 20 ng/ml TPA and finally harvested them at 24 h (A and B)or72h(C and D) after TPA induction. RNA samples were isolated from cells treated with liposome complexes in the absence of EGSs (-, lanes 1-2, 5-6, 9-10, and 13-14) or in the presence of R1 (lanes 3, 7, 11, and 15) and R2 (lanes 4, 8, 12, and 16). Equal amounts of each RNA sample (30 μg) were separated on agarose gels, transferred to a nitrocellulose membrane, and hybridized to a ³²P-radiolabeled probe that contained the cDNA sequence of the actin mRNA (lanes 1-4), KSHV Rta (lanes 5-8), polyadenylated nuclear (PAN; lanes 9-12), and vIL6 transcripts (lanes 13-16).

Fig. 6.

Expression of KSHV proteins in EGS-treated cells. We first treated 1 × 10⁵ BCBL-1 cells with liposome complexes containing EGSs, isolated them by using FACS analysis, and then incubated them either in the absence (lanes 1, 5, 9, and 13) or the presence (lanes 2-4, 6-8, 10-12, and 14-16) of 20 ng/ml TPA, and we finally harvested them at either 24 (A and B)or72h(C and D) after TPA induction. Protein samples were isolated from cells treated with liposome complexes in the absence of EGSs (-, lanes 1-2, 5-6, 9-10, and 13-14) or in the presence of R1 (lanes 3, 7, 11, and 15) and R2 (lanes 4, 8, 12, and 16). The samples were separated by SDS/PAGE, transferred to membranes, and reacted with antibodies against human actin (A), KSHV Rta (B), ORF59 (C), and ORF65 proteins (D).

Fig. 7.

The level of extracellular KSHV DNAs isolated from supernatants of the culture of cells that were first treated with liposome complexes containing TK1 and in the presence of R1 or R2, isolated by FACS analysis, and then either incubated in the absence or presence of TPA. Supernatants were collected from cells at days 1-4, 6, 8, and 10 after TPA induction, and the levels of KSHV DNA were determined by dot-blot analysis. The values, which are the means from triplicate experiments, represent the folds of increase in KSHV virion DNA level in samples, as compared with the level of virion DNA found in cells that were treated with liposome complexes in the absence of R1 or R2 and were incubated in the absence of TPA (BCBL-1/no TPA). Bars indicate SD.