Citation:
Methods Mol Med. 2004;106:309-22
Abstract:
Hepatitis C virus (HCV) contains a positive-sense, single-stranded RNA viral genome that encodes viral structural and nonstructural proteins. Initiation of translation is under control of the internal ribosome entry site (IRES) located within the viral 5'-nontranslated region (NTR) that is both highly conserved and structured. In contrast to cellular mRNAs that initiate translation at the extreme 5' terminus, IRES elements direct the translational machinery directly to the initiator AUG codon. Mutations to various regions of the HCV IRES have proven deleterious to translation in numerous in vitro studies. In addition, several antisense studies have demonstrated dramatic reduction in IRES activity. Therefore, downregulation of IRES function has emerged as a valid therapeutic target for controlling HCV infection. One general class of antisense oligonucleotides (AS-ODNs) are those that elicit cleavage of the mRNA-antisense complex by the cellular enzyme RNase H. These include unmodified DNA and phosphorothioate antisense (S-DNA). An alternative approach has been the use of AS-ODNs that contain modified backbone chemistries that do not elicit RNase H-mediated cleavage but, instead, block translation by steric mechanisms (RNase H independent). Both classes of oligonucleotides have been reported to be active against the HCV IRES. One representative RNase H-independent type is the morpholino antisense (M-AS) that contains a phosphorodiamidate-morpholino chemistry in place of the phosphodiester-ribose sugar backbone. These oligonucleotides have been shown previously to be highly specific compared to S-DNA. Because the M-AS does not lead to target degradation, it can be used to identify regions involved in RNA:RNA and RNA:protein interactions. This has been observed in HCV IRES antisense studies. AS-ODNs targeting stem loop (IIId) approx 60 nucleotides (nt) 5' of the viral open reading frame (ORF) inhibited translation at levels comparable with those directly targeting the initiator AUG. Subsequent studies identified this region as critical for IRES:40S ribosome subunit interaction.Another advantage of RNase H-independent AS-ODNs is their high degree of specificity. This can be attributed to their lack of negatively charged backbone. However, this property makes it difficult to introduce M-AS into cells using common methods such as cationic lipid transfer.