Endogenous expression of a high-affinity pseudoknot RNA aptamer suppresses replication of HIV-1

Abstract

Aptamers, small oligonucleotides derived from an in vitro evolution process called SELEX, are promising therapeutic and diagnostic agents. Although very effective in vitro, only a few examples are available showing their potential in vivo. We have analyzed the effect of a well characterized pseudoknot RNA aptamer selected for tight binding to human immunodeficiency virus (HIV) type 1 reverse transcriptase on HIV replication. Transient intracellular expression of a chimeric RNA consisting of the human initiator tRNA(Met) (tRNA(Meti))/aptamer sequence in human 293T cells showed inhibition of HIV particle release by >75% when the cells were co-transfected with proviral HIV-1 DNA. Subsequent virus production of human T-lymphoid C8166 cells, infected with viral particles derived from co-transfected 293T cells, was again reduced by >75% as compared with the control. As the observed effects are additive, in this model for virus spread, the total reduction of HIV particle formation by transient intracellular expression of the pseudoknot RNA aptamer amounts to >95%. Low-dose HIV infection of human T cells stably expressing the aptamer did not show any virus replication over a period of 35 days. This is the first example of an RNA aptamer selected against a viral enzyme target to show powerful antiviral activity in HIV-1-permissive human T-lymphoid cell lines.

Figure 1

Vector construction. (A) The original retroviral vector DCN2A contains a Δ3′-5 mutant tRNA^Meti gene and a polymerase III termination signal (TER) in the U3 region of the 3′ long terminal repeat. An oligonucleotide containing the pseudoknot sequence was cloned downstream of the tRNA gene and upstream of the polymerase III termination signal generating the DCT5T-pseudoknot vector. (B) Secondary structure prediction of the pseudoknot motif within the chimeric tRNA-pseudoknot RNA transcript. The prediction was calculated using the pseudoknot folding software PKNOTS. The pseudoknot part is shown in bold letters. The additional base at the 3′-end of the transcript arises from the termination signal.

Figure 2

Binding studies. Filter binding assay of radiolabeled pseudoknot RNA and tRNA-pseudoknot RNA with RT. 5′-³²P-labeled RNAs (200 pM) were titrated with increasing amounts of RT. The curves show best fits to a quadratic equation yielding a K_D of 37 (±14) and 86 (±17) pM for the pseudoknot (circles) and the tRNA-pseudoknot (squares), respectively.

Figure 3

Inhibition of RT activity in vitro. RNA-dependent DNA polymerase activity on poly(rA)/oligo(dT)_12–18 substrates was measured for 10 min at 37°C. 2.88 nM HIV-1 (circles) or HIV-2 (squares). RT was preincubated with increasing amounts of pseudoknot (open symbols) or tRNA-pseudoknot RNA (filled symbols).

Figure 4

Subcellular distribution of the tRNA-pseudoknot chimera. Total nuclear and cytoplasmic RNAs were prepared from 10⁷ cells (HS 68, 293T and Jurkat) transfected with the DCT5T-pseudoknot vector. Each lane was loaded with the same amount of RNA (20 µg) as determined by OD₂₆₀ measurements. (A) Pseudoknot RNA, (B) U6snRNA and (C) tRNA^Lys3. Lane 1, control (137 nt RNA chimera prepared by in vitro transcription). Lanes 2–4, nuclear RNA. Lanes 5–7, cytoplasmic RNA from transfected HS 68 (2,5), 293T (3,6) and Jurkat (4,7) cells.

Figure 5

Transient transfection and infection experiments. Each experiment was performed in triplicate and repeated twice. The error bars indicate the standard deviation error from the mean. (A) 293T cells were co-transfected with a HIV-1 proviral DNA and different retroviral constructs: the parental vector (DCT5T), an antisense RNA vector (DCT5T-αrev) and the tRNA^Meti-pseudoknot vector (DCT5T-pseudoknot). Virus production was measured 36 h post-transfection via ELISA analysis. (B) Infection of human T-lymphoid cell line (C8166) with viruses produced by the 293T cell line (see Fig. 7A). In each experiment the same amount of virus was applied. HIV-1 replication was measured 48 h post-infection by ELISA.

Figure 6

Infection of stably transfected Jurkat cells. Jurkat cell clones either stably transfected with the parental vector DCT5T (circles) or the DCT5T-pseudoknot vector (triangles) were infected with different amounts HIV-1 particles. (A) 190 pg p24 per 10⁵ cells and (B) 1700 pg p24 per 10⁵ cells. At the time points given, viral replication was measured by p24 ELISA.