HIV-1 LTR as a target for synthetic ribozyme-mediated inhibition of gene expression: site selection and inhibition in cell culture

Abstract

A library of three synthetic ribozymes with randomized arms, targeting NUX, GUX and NXG triplets, respectively, were used to identify ribozyme-accessible sites on the HIV-1 LTR transcript comprising positions -533 to 386. Three cleavable sites were identified at positions 109, 115 and 161. Ribozymes were designed against these sites, either unmodified or with 2'-modifications and phosphorothioate groups, and their cleavage activities of the transcript were determined. Their biological activities were assessed in cell culture, using a HIV-1 model assay system where the LTR is a promoter for the expression of the reporter gene luciferase in a transient expression system. Intracellular efficiency of the ribozymes were determined by cotransfection of ribozyme and plasmid DNA, expressing the target RNA. Modified ribozymes, directed against positions 115 and 161, lowered the level of LTR mRNA in the cell resulting in inhibition of expression of the LTR-driven reporter gene luciferase of 87 and 61%, respectively. In the presence of Tat the inhibitions were 43 and 25%. The inactive variants of these ribozymes exhibited a similar inhibitory effect. RNase protection revealed a reduction of RNA which was somewhat stronger for the active than the inactive ribozymes, particularly for ribozyme 115. Unmodified ribozymes showed no inhibition in the cell. The third ribozyme, targeting a GUG-triplet at position 109, possessed only low cleavage activity in vitro and no inhibitory effect in cell culture.

Figure 1

Plasmid pLTRLuc, expressing the reporter gene luciferase under control of the HIV-1 LTR promoter for cell culture experiments.

Figure 2

Sequences of hammerhead and hammerhead-like ribozymes with randomized substrate binding arms.

Figure 3

Detection of cleavage sites of the HIV-1 LTR mRNA by primer extension analysis. The in vitro transcript pOTH33, containing the HIV-1 LTR region, was incubated with three different synthetic ribozyme pools, containing randomized binding arms. Cleavage sites were detected by primer extension of the 3′-cleavage product. As a control LTR-transcript was incubated under the same conditions without ribozyme pool. Products were analyzed with 8% polyacrylamide–urea gel electrophoresis, aligned to the DNA sequencing ladder of pOTH33 and generated by using the same primer as described in Materials and Methods. Cleavage products are indicated by an arrow. Numbers are according to the cleavage positions in the LTR region.

Figure 4

Sequences and modifications of ribozymes targeted against the HIV-1 LTR mRNA at position 109, 115 and 161. 2′-Fluoro-2′-deoxypyrimidine nucleotides, circled; 2′-amino-2′-deoxyuridine nucleotides, boxed. S, phosphorothioate linkages.

Figure 5

Inhibition of HIV-1 LTR dependent luciferase expression by ribozymes. HeLa cells were cotransfected with plasmid and active ribozymes or their corresponding controls. After 18 h of growth luciferase activity was determined. Luciferase activity of cells, transfected with the inverted form of the ribozyme, was defined as 100% control. Results present an average of at least three experiments each done in duplicate. (A and B) Luciferase activity of cells transfected with ribozymes 161M and 115M, respectively, in the absence of Tat. (C and D) Activity in the presence of Tat.

Figure 6

RNase protection analysis of the LTR mRNA in cells transfected with ribozymes, their corresponding controls and plasmid pLTRLuc and pRSVTat. Total RNA of transfected cells was purified after 18 h of growth and probed with the antisense transcript of the LTR. LTR mRNA levels were normalized to the level of β-actin mRNA. As a marker dephosphorylated pUC19 Sau3AI digested DNA, labeled with [γ-³²P]ATP, was used.

Figure 7

Northern blot analysis of intracellular ribozymes. HeLa cells were transfected with ribozymes. After 18 h of growth total RNA was isolated and subjected to northern blot analysis as described. Upper panel, β-actin; lower panel, ribozymes.