Analysis of regions of the 5 and 3 UTRs involved in DENV translation and RNA synthesis

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Viral translation and RNA synthesis of positive-sense RNA viruses is often regulated by the 5' and 3' UTRs. Previously, we had demonstrated a role for the DENV-2 terminal 3'SL in stimulating viral translation via deletion analysis in RNA reporter constructs (Holden & Harris 2004). To examine other regions of the DENV-2 UTRs, we targeted conserved domains in the 5' and 3' UTRs by complementary peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMOs), as depicted in Fig. 3. The P-PMOs most effective in inhibiting viral replication were those complementary to the DENV 5' stem-loop ('5'SL') and to the DENV 3' cyclization sequence ('3'CS'), consistent with recent results with DENV and WNV (Deas et al 2005, Kinney et al 2005). We also identified a third P-PMO, complementary to the top of the 3' stem-loop ('3'SLT'), that inhibited DENV replication in BHK cells. Presumably, these P-PMOs interfere with DENV replication by blocking critical RNA-RNA or RNA-protein interactions involved in viral translation and/or RNA synthesis.

To distinguish between effects of DENV-specific P-PMOs on viral translation and/or RNA synthesis, a DENV-2 reporter replicon was constructed (Fig. 4A) using the DENV-2 infectious clone (pD2/IC) as a backbone, with the firefly luci-ferase gene replacing most of the structural genes. The non-infectious DENV-2 reporter replicon should translate and replicate in a manner similar to wild-type DENV RNA. The DENV-2 reporter replicon was transcribed in vitro and trans-

Denv Utr Structure

FIG. 3. Schematic diagrams of the targeted locations in the DENV 5'- and 3'-UTRs. (A) Locations of P-PMO target sequences in the DENV-2 5' UTR. Indicated by lines are target sequences for the DENV 5'SL P-PMO and the 5' AUG P-PMO. The predicted secondary structure of the DENV-2 5' UTR was determined using the mFold web server (Zuker 2003). (B) Locations of P-PMO target sequences in the DENV 3' UTR. Indicated by lines adjacent to the 3' UTR sequence are target sequences for the DENV 3' PKIIA, 3' PKIIB, 3'CS2/RCS2, 3'CS, and 3'SLT P-PMOs. The shaded regions indicate the interactions involved in forming the PKIIA and PKIIB pseudoknot secondary structures; the shaded loop sequence is predicted to interact with the shaded region downstream. The secondary structures of the DENV-2 3' UTR are based on computer predicted secondary structures (Hahn et al 1987, Mohan & Padmanabhan 1991, Olsthoorn & Bol 2001, Proutski et al 1999, Shi et al 1996a) and chemical probing of the terminal 100 nucleotides (Shi et al 1996a). Reprinted with permission from Elsevier (Holden et al 2006).

FIG. 3. Schematic diagrams of the targeted locations in the DENV 5'- and 3'-UTRs. (A) Locations of P-PMO target sequences in the DENV-2 5' UTR. Indicated by lines are target sequences for the DENV 5'SL P-PMO and the 5' AUG P-PMO. The predicted secondary structure of the DENV-2 5' UTR was determined using the mFold web server (Zuker 2003). (B) Locations of P-PMO target sequences in the DENV 3' UTR. Indicated by lines adjacent to the 3' UTR sequence are target sequences for the DENV 3' PKIIA, 3' PKIIB, 3'CS2/RCS2, 3'CS, and 3'SLT P-PMOs. The shaded regions indicate the interactions involved in forming the PKIIA and PKIIB pseudoknot secondary structures; the shaded loop sequence is predicted to interact with the shaded region downstream. The secondary structures of the DENV-2 3' UTR are based on computer predicted secondary structures (Hahn et al 1987, Mohan & Padmanabhan 1991, Olsthoorn & Bol 2001, Proutski et al 1999, Shi et al 1996a) and chemical probing of the terminal 100 nucleotides (Shi et al 1996a). Reprinted with permission from Elsevier (Holden et al 2006).

fected into BHK cells, and luciferase activity was monitored for 96 h post-transfec-tion. Two peaks of luciferase activity were generated by the DENV-2 reporter replicon: an early peak between 4 and 8 h post-transfection and a later peak between 48 and 96 h post-transfection (Fig. 4B). By using mycophenolic acid (MPA), a potent inhibitor of DENV RNA synthesis (Diamond et al 2002), it was determined that the first peak of luciferase activity (4—8 h) correlates with translation of the DENV-2 reporter replicon, whereas the later peak of luciferase activity

Luciferase E

m7GpppA-

2Apro

m7GpppA-

2Apro

FIG. 4. Effects of the DENV-specific P-PMOs on translation and RNA synthesis of the DENV-2 reporter replicon. (A) Schematic diagram of the DENV-2 reporter replicon. Indicated are the DENV-2 5' UTR (black line), first 72 nucleotides of C (grey box) fused to the firefly luciferase gene (black box), the FMDV 2Apro (dotted box), the last 90 nucleotides of E (striped box), the entire NS region (white box), and the DENV-2 3' UTR (black line). (The pD2/IC infectous clone of DENV-2 that was used as the backbone for the replicon was a gift from R. Kinney, Center for Disease Control and Prevention, Fort Collins, CO.) (B) The DENV-2 reporter replicon distinguishes between viral translation and RNA synthesis. Cells were trans-fected with the DENV-2 reporter mRNA, then treated with medium or MPA (3 (iM). Luciferase activity was monitored at 4, 8, 24, 54, 72 and 96 h after transfection. Data shown are representative of three independent experiments. Error bars indicate the SD of duplicate samples. The amount of transfected RNA was not impacted by the presence of the P-PMOs, as determined by quantitative real-time RT-PCR (data not shown). (C) DENV-specific P-PMOs inhibit translation and/or RNA synthesis of the DENV-2 reporter replicon. Cells were treated with the DENV-specific P-PMOs (5'SL, 3'CS, and 3'SLT) or the DScr P-PMO and transfected with the DENV-2 reporter replicon. Luciferase activity was monitored at 4, 6, 8, 12, 24, 48, 72 and 96 h after transfection. Data shown are representative of three independent experiments. Error bars indicate the SD of duplicate samples. The amount of transfected RNA was not impacted by the presence of P-PMO, as determined by quantitative real-time RT-PCR (data not shown). Reprinted with permission from Elsevier (Holden et al 2006).

(48-96 h) —which was inhibited by MPA—is dependent upon RNA synthesis (Fig. 4B). The DENV-2 reporter replicon was then used to distinguish between the effect of the DENV-specific P-PMOs on viral translation and RNA synthesis. Cells were treated with the different P-PMOs before transfection of the DENV-2 reporter replicon and again after RNA transfection; no effect on RNA transfection efficiency was observed, as determined by real-time (RT) -PCR (data not shown).

A 'scrambled' P-PMO ('DScr') was used to control for any non-specific effect of the P-PMOs. The 5'SL P-PMO, which inhibited translation of the DENV reporter mRNA (data not shown), also inhibited translation of the DENV-2 reporter replicon by 95% and consequently, reduced the RNA synthesis peak by over 10 000fold (Fig. 4C). The 3'CS P-PMO strongly inhibited the appearance of the RNA synthesis peak of the DENV-2 reporter replicon (>10 000-fold) with no significant effect on the first peak of translation, indicating that the 3'CS P-PMO inhibited only viral RNA synthesis (Fig. 4C). The 3'SLT P-PMO reduced translation of the DENV-2 reporter replicon by 40%o and inhibited the second peak of luciferase activity that is primarily dependent on RNA synthesis by over 10 000-fold, indicating that it may directly inhibit RNA synthesis in addition to its effects on translation.

Thus, using a novel DENV-2 reporter replicon and a DENV-2 reporter mRNA, we determined that the 5'SL P-PMO inhibited viral translation, the 3'CS P-PMO blocked viral RNA synthesis but not viral translation, and the 3'SLT P-PMO inhibited both viral translation and RNA synthesis. These results show that the 3'CS and the 3'SL domains regulate DENV translation and RNA synthesis and further demonstrate that P-PMOs are potentially useful as antiviral agents.

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