Inhibition of 5'-UTR RNA conformational switching in HIV-1 using antisense PNAs

Abstract

Background: The genome of retroviruses, including HIV-1, is packaged as two homologous (+) strand RNA molecules, noncovalently associated close to their 5'-end in a region called dimer linkage structure (DLS). Retroviral HIV-1 genomic RNAs dimerize through complex interactions between dimerization initiation sites (DIS) within the (5'-UTR). Dimer formation is prevented by so calledLong Distance Interaction (LDI) conformation, whereas Branched Multiple Hairpin (BMH) conformation leads to spontaneous dimerization.

Methods and results: We evaluated the role of SL1 (DIS), PolyA Hairpin signal and a long distance U5-AUG interaction by in-vitro dimerization, conformer assay and coupled dimerization and template-switching assays using antisense PNAs. Our data suggests evidence that PNAs targeted against SL1 produced severe inhibitory effect on dimerization and template-switching processes while PNAs targeted against U5 region do not show significant effect on dimerization and template switching, while PNAs targeted against AUG region showed strong inhibition of dimerization and template switching processes.

Conclusions: Our results demonstrate that PNA can be used successfully as an antisense to inhibit dimerization and template switching process in HIV -1 and both of the processes are closely linked to each other. Different PNA oligomers have ability of switching between two thermodynamically stable forms. PNA targeted against DIS and SL1 switch, LDI conformer to more dimerization friendly BMH form. PNAs targeted against PolyA haipin configuration did not show a significant change in dimerization and template switching process. The PNA oligomer directed against the AUG strand of U5-AUG duplex structure also showed a significant reduction in RNA dimerization as well as template- switching efficiency.The antisense PNA oligomers can be used to regulate the shift in the LDI/BMH equilibrium.

Figure 1. Proposed stem-loop structure of the 5′untranslated region of HIV-1.

Target loop regions are highlighted. PNAs targeted against different sites of 5′UTR of HIV-1 are shown in the diagram (Abbink et. al. 2003).

Figure 2. Schematic representation of RNA transcripts showing motifs within the 5′ UTR of HIV-1 RNA genome.

(A)5′ leader HIV-1 RNA transcripts including the 5' end of the gag gene (position +1 to 447 relative to the transcriptional start site +1). (B) Acceptor and donor RNA templates used in the template-switching assays.

Figure 3. UV T_m curves.

UV T_m curves (A)PNA :RNA antiparallel duplexes of (a)PNA 72, (b)PNA 98, (c) PNA 102,(d) PNA 110, (e) PNA 237,(f) PNA 251,(g) PNA 271, (h) PNA 332, (B) PNA :DNA antiparallel duplexes of (a) PNA 72,(b) PNA 98,(c) PNA 102,(d) PNA 110, (e) PNA 237,(f) PNA 251,(g) PNA 271,(h) PNA 332. 10 mM sodium phosphate buffer, pH 7 containing NaCl (100 mM) and EDTA (0.1 mM) buffer was used for UV T_m melting studies.

Figure 4. Dimerization efficiency of the HIV-1 RNA (1–447) transcript.

The RNA transcripts were incubated in monomer (no MgCl₂) and dimerization buffer with varying concentration of MgCl₂. All samples were analyzed on TBM gels. Lane1: monomer buffer, Lane 2: Dimerization buffer with 1 mM MgCl₂ Lane 3: Dimerization buffer with 2 mM MgCl₂, Lane 4: Dimerization buffer with 5 mM MgCl₂, Lane 5: Dimerization buffer with 10 mM MgCl_2.

Figure 5. Effects of PNA oligomers on gel mobility of HIV-1 leader RNA (447nts).

The RNA transcript was incubated in dimerization buffer (5 mM MgCl₂) at 65°C in the absence or presence of increasing concentration of PNAs. The samples were analyzed on TBM gels. Each gel has the following loading pattern - Lane 1: Dimerization buffer without PNA oligomer, Lane 2–6: Dimerization buffer with the increasing template ratios, as indicated.

Figure 6. Effects of PNA oligomers on the efficiency of dimerization of HIV-1 RNA transcripts.

The samples from dimerization assays were analyzed on the 4% non-denaturing gels and the intensity of monomer and dimer bands on each gel was quantified. The dimerization efficiency was calculated as d/D × 100%, where d is the percentage intensity of dimer with different concentration of PNA and D is the percentage intensity of dimer in the absence of PNAs. Error bars indicate standard deviation from triplicate runs.

Figure 7. Efficiency of heterodimerization between the acceptor and donor transcripts.

The acceptor and donor transcripts were incubated in Formamide, monomer and dimerization buffers, similar to the dimerization assay. The efficiency of heterodimerization between the donor and acceptor was performed by adding molar excess (as indicated at the top of the gel) of unlabelled acceptor transcript to labeled donor transcript under optimal dimerization conditions. All samples were analyzed on the 4% non-denaturing TBE gel. Lane 1: Donor transcript, Lane 2: Acceptor transcript, Lane 3: Donor transcript, Lane 4: Acceptor transcript, Lane 5: Donor transcript, Lane 6: Acceptor transcript, Lane 7–9: Donor transcript with increasing concentration of unlabeled acceptor transcript (1∶1, 1∶2, and 1∶5). F- Formamide buffer, M-monomer buffer and D-dimerization buffer (with 5 mM MgCl₂).

Figure 8. Effect of PNA oligomers on HIV-1 template-switching.

Samples were analyzed on the 6% denaturing TBE gel. Each gel has the following loading pattern- Lane 1: HIV-1 leader transcripts including the AUG codon of gag gene, Lane 2: Donor transcript, Lane 3–7: Donor and acceptor transcripts with increasing concentration of anti-sense PNAs in molar ratio (with respect to the concentration of donor transcript) as indicated on top of each gel (Reverse transcribed with 0.2 unit of HIV-1RT/µl), Lane 8- Donor and acceptor transcripts without PNA oligo {Reverse transcribed with 0.2 unit of HIV-1RT/µl (from Lane 3 to 8 in each panel )},. FT- Full-length transcript, DT- Donor transcript, T- Template-switched product (Lane 3 to 8), F- Full-length donor product (Lane 3 to 8).

Figure 9. Effects of PNA on the template-switching efficiency between the acceptor and donor HIV-1 RNA templates.

The RNA samples from the coupled dimerization-template switching experiments were analyzed on the 6% TBE denaturing gels and the intensity of bands corresponding to the template-switched products and the full-length product of the donor on each gel was quantified The template-switching efficiency for each sample was calculated as t/T × 100%, where ‘t’ is the % intensity of template-switching product in the primer extension reaction with different concentration of PNA oligomer and ‘T’ is the % intensity of the template-switching product in the primer extension reaction without PNA oligomers. Error bars indicate standard deviation of triplicate runs.

Figure 10. Mobility of HIV-1 leader RNA (447nts) in different buffers.

The RNA transcripts were incubated in different buffer conditions and were analyzed on 4% non-denaturing TBE gel. Lane 1: Formamide buffer, Lane 2: Tris-Cl buffer, Lane 3: TN buffer, Lane 4: TN buffer with 0.1 mM MgCl₂, Lane 5: TN buffer with 1.0 mM MgCl₂, Lane 6: TN buffer with 5 mM MgCl₂, Lane 7: TEN buffer and Lane 8: Dimerization buffer with 5 mM MgCl₂.

Figure 11. RNA (447nts) in presence of increasing concentration of PNA oligomers.

The radiolabeled HIV-1 leader transcript was incubated in TEN buffer with increasing concentration of respective PNA as well as in formamide and dimerization buffer. The samples were analyzed on non-denaturing TBE gels. Each gel has the same loading pattern. Lane 1: Formamide buffer, Lane 2: TEN buffer without PNA oligomer, Lane 3–7: TEN buffer with the increasing concentration of PNA (in molar ratio) as indicated on top of each gel, Lane 8: Dimerization buffer. F – Formamide buffer, D – Dimerization buffer.

Figure 12. PNA effect on the equilibrium between the LDI and BMH conformations of HIV-1 leader RNA.

The RNA samples from conformer assays were analyzed on the non-denaturing gels and the intensity of bands corresponding to the LDI and BMH conformers on each gel was quantified. The relative shift from LDI to BMH conformation was calculated as e/E × 100%, where ‘e’ is the percentage intensity of LDI conformer in TEN buffer with different concentration of PNA oligomer and ‘E’ is the percentage intensity of LDI conformer in TEN buffer without PNA oligomers. Error bars indicate standard deviation of triplicate runs.