Effective inhibition of human cytomegalovirus gene expression and replication by a ribozyme derived from the catalytic RNA subunit of RNase P from Escherichia coli

Abstract

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping exon 3 region of the mRNAs encoding the major transcription regulatory proteins IE1 and IE2 of human cytomegalovirus. A reduction of more than 80% in the expression levels of IE1 and IE2 and a reduction of about 150-fold in viral growth were observed in human cells that stably expressed the ribozyme. In contrast, a reduction of less than 10% in the IE1͞IE2 expression and viral growth was observed in cells that either did not express the ribozyme or produced a ''disabled'' ribozyme that carried mutations that abolished its catalytic activity. Examination of the expression of several other viral early and late genes in the cells that expressed the M1GS ribozyme further revealed an overall reduction of at least 80% in their expression. These results are consistent with the notion that the antiviral effects in these cells are due to the fact that the ribozyme specifically inhibits the expression of IE1 and IE2 and, consequently, abolishes the expression of viral early and late genes as well as viral growth. Our study is the first, to our knowledge, to use M1GS ribozyme for inhibiting human cytomegalovirus replication and demonstrates the utility of this ribozyme for antiviral applications. Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes mild or subclinical diseases in immunocompetent adults but may lead to severe morbidity or mortality in neonates and immunocompromised individuals (1). Infection by this virus accounts for one of the most common opportunistic diseases in patients with AIDS, CMV retinitis. The emergence of drug-resistant strains of HCMV has posed a need for the development of new drugs and novel treatment strategies (2, 3).Antisense nucleic acid molecules, including conventional antisense oligonucleotides and antisense ribozymes, are promising gene-targeting agents for specific inhibition of gene expression (4-8). Antisense molecules have been used as anti-HCMV agents to inhibit the expression of HCMV-essential genes and abolish viral replication (4, 9-12). External guide sequences (EGSs; refs. 13 and 14) are antisense oligoribonucleotides that have been used in conjunction with either ribonuclease P (RNase P) or the catalytic RNA subunit of RNase P from Escherichia coli (M1 RNA; ref. 15) to diminish the expression of several genes both in E. coli (16,17) and in mammalian cells (14,(18)(19)(20)(21). The EGS-based technology takes advantage of RNase P or M1 RNA to cleave a targeted mRNA when the EGS hybridizes to the target RNA (refs. 13 and 14; Fig. 1A). The EGSs, when expressed separately from the enzyme, have been shown recently to be effective in inhibiting the gene expression of herpes simplex virus 1 (HSV-1) and influenza virus and, in addition, in abolishing the replication of influenza virus (20,21). To increase the targeting efficiency, the EGS can be covalently linked to M1 RNA (i.e., its 3Ј end) to generate a sequence-specific...

Characterization of the structure and variability of an internal region of hepatitis C virus RNA for M1 RNA guide sequence ribozyme targeting

“…M1 RNA may be converted to an endoribonuclease that specifically cleaves other RNAs through the covalent attachment of a guide sequence (GS) which is complementary to the target RNA and to the 39 end of the M1 RNA. Reduced levels of virus replication of herpes simplex virus (Trang et al, 2000a) and cytomegalovirus (Trang et al, 2000b) in cell culture have been reported with the use of M1GSs.…”

Oral delivery of RNase P ribozymes by Salmonella inhibits viral infection in mice

“…We constructed functional ribozyme M1-A by linking the 3′ terminus of M1 RNA with a guide sequence of 18 nucleotides that is complementary to the targeted M80.5 mRNA sequence. Control "inactive" ribozyme M1-B was constructed to contain the same guide sequence and derived from C102 RNA, an M1 mutant that contained point mutations at the active P4 domain abolishing its catalytic activity (9). To determine if M1GS ribozyme with an incorrect guide sequence could affect the level of the target mRNA, ribozyme M1-TK1, which was derived from M1 RNA and targeted the HSV-1 thymidine kinase (TK) mRNA (9), was also used in the analysis.…”

“…Control "inactive" ribozyme M1-B was constructed to contain the same guide sequence and derived from C102 RNA, an M1 mutant that contained point mutations at the active P4 domain abolishing its catalytic activity (9). To determine if M1GS ribozyme with an incorrect guide sequence could affect the level of the target mRNA, ribozyme M1-TK1, which was derived from M1 RNA and targeted the HSV-1 thymidine kinase (TK) mRNA (9), was also used in the analysis. We observed in vitro cleavage of an M80.5 mRNA substrate by M1-A, but not M1-B or M1-TK1 (Fig.…”

“…The DNA sequence for the M80.5 mRNA substrate was constructed by annealing primers AF25 (5′-GGAATTCTAATAC-GACTCACTATAG-3′) and sm80.5 (5′-CGGGATCCGCCCGACTGAGGTAGACGC-GGTGGTTCATCCTATAGTG AGTCGTATTA-3′), followed by PCR. Mutant ribozyme C102 contains several point mutations (e.g., A 347 C 348 → C 347 U 348 , (9). The DNA sequences that encode ribozymes M1-A and M1-B were constructed by PCR using constructs pFL117 and pC102 (9), which contained the DNA sequences of the M1 and C102 ribozymes, as the templates and primers AF25 and M1m80.5 (5′-CCCGCTCGAGAAAAAATGGTGCGTCTACCTCAGTC-GGGTGTGGAATTGTG-3′) as 5′ and 3′ primers, respectively.…”

“…1 A and B) (6, 7). M1GS RNAs efficiently cleave target cellular and viral mRNAs in vitro and block their expression in cultured cells (8,9). The M1GS-based strategy represents a distinctive nucleic-acid-based interference approach because of the use of M1 RNA, an efficient naturally occurring RNA catalyst found in nature (4).…”