Inhibition of flavivirus infections by antisense oligomers specifically suppressing viral translation and RNA replication - PubMed (original) (raw)

Inhibition of flavivirus infections by antisense oligomers specifically suppressing viral translation and RNA replication

Tia S Deas et al. J Virol. 2005 Apr.

Abstract

RNA elements within flavivirus genomes are potential targets for antiviral therapy. A panel of phosphorodiamidate morpholino oligomers (PMOs), whose sequences are complementary to RNA elements located in the 5'- and 3'-termini of the West Nile (WN) virus genome, were designed to anneal to important cis-acting elements and potentially to inhibit WN infection. A novel Arg-rich peptide was conjugated to each PMO for efficient cellular delivery. These PMOs exhibited various degrees of antiviral activity upon incubation with a WN virus luciferase-replicon-containing cell line. Among them, PMOs targeting the 5'-terminal 20 nucleotides (5'End) or targeting the 3'-terminal element involved in a potential genome cyclizing interaction (3'CSI) exhibited the greatest potency. When cells infected with an epidemic strain of WN virus were treated with the 5'End or 3'CSI PMO, virus titers were reduced by approximately 5 to 6 logs at a 5 muM concentration without apparent cytotoxicity. The 3'CSI PMO also inhibited mosquito-borne flaviviruses other than WN virus, and the antiviral potency correlated with the conservation of the targeted 3'CSI sequences of specific viruses. Mode-of-action analyses showed that the 5'End and 3'CSI PMOs suppressed viral infection through two distinct mechanisms. The 5'End PMO inhibited viral translation, whereas the 3'CSI PMO did not significantly affect viral translation but suppressed RNA replication. The results suggest that antisense PMO-mediated blocking of cis-acting elements of flavivirus genomes can potentially be developed into an anti-flavivirus therapy. In addition, we report that although a full-length WN virus containing a luciferase reporter (engineered at the 3' untranslated region of the genome) is not stable, an early passage of this reporting virus can be used to screen for inhibitors against any step of the virus life cycle.

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Figures

FIG. 1.

FIG. 1.

Sequence selection and cellular delivery of PMOs. (A) WN virus genome. Terminal stem-loop structures and a potential genome cyclization are enlarged to display regions where antisense PMOs target to. Individual PMOs are indicated by their names and targeted regions (bracketed lines). WN virus genome cyclization is mediated through a perfect 12-nt base-pairing interaction (dotted lines) between the 5′CS and 3′CSI sequences (boxed sequence). A pseudoknot (Pskt) interaction within the 3′ stem-loop (45) is indicated by dashed lines. The major binding sites of host protein EF1α (2) is depicted by ovals. The flavivirus-conserved pentanucleotide located at the top of the 3′ stem-loop (55) is underlined. The AUG initiation codon of the ORF is in boldface type. (B) PMO sequences. All PMOs contain an Arg-rich peptide at their 5′ ends, designated P007. An extra C (italicized) was added to the 3′ end of the 5′End PMO to potentially interact with the 7-methylguanylate of the 5′ cap and to further stabilize its hybridization with the viral genome. (C) Structure of P007-PMOs. In a PMO, the ribose of RNA is replaced by a morpholine ring, and the phosphodiester internucleotide bond is replaced by a phosphorodiamidate linkage. The CH3CONH-(RAhxR)4-Ahx-βAla sequence was attached to each PMO through a piperazine linker, where R stands for arginine, Ahx stands for 6-aminohexanoic acid, and βAla stands for beta-alanine. (D) Enhancement of cellular delivery of PMOs by P007 conjugation. BHK cells were incubated with the fluorescein-labeled scramble PMO with or without a 5′ P007 conjugation at a concentration of 10 μM. After 15 min of incubation, cells incubated with the P007-conjugated PMO are fluorescence positive (left), whereas no fluorescence is observed in cells incubated with unconjugated PMOs (right).

FIG. 2.

FIG. 2.

Antiviral activities of PMOs in a WN virus replicon-reporting cell line. (A) An Rluc and Neo gene were inserted into a WN virus replicon, resulting in RlucNeoRep. A cell line containing persistently replicating RlucNeoRep was established previously (26). Antiviral activities were analyzed by incubation of the RlucNeoRep cells with PMOs at indicated concentrations for 24 h and assayed for Rluc activities. PMOs targeting the 5′ (B) and 3′ (C) regions of the genome are presented.

FIG. 3.

FIG. 3.

A full-length luciferase-reporting WN virus can be used for antiviral drug screening. (A) A full-length WN virus containing a luciferase reporter. An Rluc gene driven by an EMCV IRES was engineered at the upstream region of the 3 ′ UTR of the genome, resulting in RlucWN virus. The numbers indicate nucleotide positions in the WN virus genome (GenBank accession number AF404756). (B) RT-PCR analyses of RlucWN virus. Five sets of RT-PCR covering the entire WN virus genome were performed with template RNAs extracted from a wild-type isolate of WN virus (WT WN), the first passage of RlucWN virus possessing a high level of Rluc activity (RlucWN-P1), and the seventh passage of RlucWN virus lacking Rluc activity (RlucWN-P7). DNA fragments amplified from nt 8706 to 10515 indicate that the IRES-Rluc (1,520 bp) insertion was retained in RlucWN-P1 but was subsequently deleted at RlucWN-P7. The RT-PCR products were analyzed with a 1% agarose gel. (C) Antiviral assay with RlucWN virus. Vero cells were infected with RlucWN-P1 at an MOI of 0.5, immediately treated with known WN inhibitors as indicated, and assayed for Rluc activity at 24 h post infection. Rluc activities were plotted against compound concentrations. An average of two experiments is presented.

FIG. 4.

FIG. 4.

Antiviral and cytotoxic analyses of 5′End, 3′CSI, and AUG-I PMOs. (A) Antiviral activities of PMOs were analyzed by the RlucWN-infection assay. PMOs were added to Vero cells during inoculation of RlucWN-P1 virus at an MOI of 0.5. At 24 h postinfection, cells were lysed and assayed for Rluc activity. Antiviral activity was indicated by the reduction of Rluc signal upon PMO treatment. A scramble PMO was included as a negative control. The keys to PMO identity shown in panel B are the same in panel A. (B) Cytotoxicity was evaluated by incubation of Vero cells with the indicated concentration of 5′End, 3′CSI, AUG-I, or scramble PMO. At 24 h p.i., viability of treated cells was measured by an MTT assay and presented as a percentage of colorimetric absorbance derived from untreated cells (see details in Materials and Methods).

FIG. 5.

FIG. 5.

Inhibition of an epidemic strain WN virus infection by PMOs. Epidemic WN virus isolate 3356 was used to verify the potencies of the 5′End, 3′CSI, and AUG-I PMOs in a virus yield reduction assay. Each PMO was incubated with Vero cells infected with WN virus isolate strain 3356 at an MOI of 0.1. Antiviral activities of the PMOs were measured by virus titer reduction at 42 h postinfection. The scramble PMO was used as a negative control. One set of data representative of duplicate experiments is presented.

FIG. 6.

FIG. 6.

Mode-of-action analyses. (A) A WN virus reporting replicon containing an Rluc gene (fused in frame with the ORF; RlucRep) was used to analyze the modes of action of 5′End, 3′CSI, and AUG-I PMOs. BHK cells transfected with RlucRep exhibit two distinctive Rluc peaks at 2 to 10 h and after 24 h p.t., representing viral translation and RNA replication of input RlucRep, respectively (25). (B) Differentiation between PMO-mediated inhibition of viral translation and RNA synthesis. RlucRep-transfected BHK cells were incubated with 7.5 μM PMOs and assayed for Rluc activity at 2 h p.t. (viral translation) and 72 h p.t. (RNA replication). Values for PMO-mediated inhibition of viral translation and RNA replication are each presented as a percentage of the Rluc signal derived from the untreated cells at an equivalent time point.

FIG. 7.

FIG. 7.

Inhibition of viral translation by 5′End and AUG-I PMOs. (A) A reporting RNA (designated 5′ UTR-Rluc-3′ UTR) contains a Rluc gene (shaded box) fused in frame with the coding sequence of the N-terminal 31 amino acids of the C protein (open box). The ORF is flanked by WN virus 5′ and 3′ UTRs. (B) A time course of Rluc activity from BHK cells transfected with 5′ UTR-Rluc-3′ UTR RNA. (C) BHK cells were incubated with indicated PMOs (7.5 μM) immediately after transfection of 5′ UTR-Rluc-3′ UTR RNA, and assayed for Rluc activity at 5 h p.t. Inhibition of PMOs on translation is presented as percentage of the Rluc activity derived from the untreated cells.

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