Figure 1.
Schematic representation of a natural substrate (ptRNA)(A), a small model substrate (EGS:mRNA) for ribonuclease P and M1 RNA from E. coli (B), and a complex formed between a M1GS RNA and its mRNA substrate (C).
A part of the EGS:mRNA and M1GS:mRNA complexes resembles the acceptor stem and T-stem domains of a ptRNA (shown in bold type). The site of cleavage by RNase P or M1 RNA is marked with a filled arrow. (D) Schematic representation of the substrate used in the study. The targeted sequences that bind to the guide sequences of the ribozymes are highlighted.
Table 1.
Overall cleavage rate [(kcat/Km)s] and binding affinity (Kd) in cleavage reactions of tat37 with RNase P ribozymes.
Figure 2.
Cleavage of substrate tat37 by M1GS RNA.
Substrate (20 nM) was incubated alone (lanes 1), with 1 nM of V38-TAT (lane 2), 5 nM of M1-TAT (lanes 3), or 5 nM of C-TAT ribozyme (lanes 4). Cleavage reactions were carried out for 30 min in buffer A (50 mM Tris.HCl, pH 7.5, 100 mM NH4Cl, 100 mM MgCl2) at 37°C. Cleavage products were separated in 15% polyacrylamide gels containing 8 M urea.
Figure 3.
Northern analyses of the expression of M1GS ribozymes from the RNA fractions isolated from 293T cells that were transfected with pHIVNL4-3 alone (-, lanes 4 and 8) or from cells that were co-transfected with pHIVNL4-3 and a M1GS DNA construct to express V38-TAT (lanes 1 and 5), M1-TAT (lines 2 and 6), and C-TAT (lanes 3 and 7).
Equal amounts of each RNA sample (25 µg) were separated on 2% agarose gels that contained formaldehyde, transferred to a nitrocellulose membrane, and hybridized to a [32P]-radiolabeled probe that contained the DNA sequence coding for M1 RNA (lanes 5–8) or H1 RNA (lanes 1–4), the RNA subunit of human RNase P [52]. The size of H1 RNA (∼380 nts) is very similar to that of M1GS RNA (∼450 nts) and therefore, can be used as a size marker. The expression of human H1 RNA was used as the internal loading control for the quantification of M1GS RNA expression. The hybridized products corresponding to the full-length retroviral transcripts (∼6 kb), transcribed from the LTR promoter, are at the top of the gel and are not shown.
Figure 4.
Expression of HIV p24 protein, as detected by Western blot analysis with a chemiluminescent substrate.
Total protein fractions were isolated from 293T cell cultures that were transfected with pHIVNL4-3 alone (-; lanes 1, 5, and 9) or from cells that were co-transfected with pHIVNL4-3 and a M1GS DNA construct to express M1-TAT (lanes 2, 6, and 10), C-TAT (lines 3, 7, and 11), and V38-TAT (lanes 4, 8, and 12). Cells and culture media were harvested at 48 hours postinfection, and protein samples from cells (cell lysate, A–B) and cell-free supernatants (supernatant, C) were loaded on the gels. Equal amounts of protein samples (40 µg) isolated from cells were separated in SDS-polyacrylamide gels. The membranes were stained with the antibodies against human actin (A) and HIV p24 (B–C). The expression of human actin was used as the internal loading control for the quantification of HIV p24 expression.
Figure 5.
Schematic representation of the levels of supernatant HIV-1 p24 and total (unspliced and spliced) intracellular HIV RNA in pHIVNL4-3-transfected 293 T cells that did not express a ribozyme [P(293T)] or expressed ribozyme C-TAT (C-TAT), M1-TAT (M1-TAT), and V38-TAT (V38-TAT).
The RNA and protein samples were isolated from cells and culture media at 48 hours postinfection, respectively. The levels of HIV RNA were determined using a real-time PCR assay while the levels of p24 protein were measured with a HIV p24 ELISA kit. The values shown are the averages from three independent experiments. The standard deviation is indicated by the error bars. Solid bars: HIV p24 protein; open bars: HIV RNA.
Figure 6.
Northern analyses of the expression of M1GS ribozymes from the RNA fractions isolated from parental H9 cells (-, lane 4 and 8) or different cloned cell lines that expressed V38-TAT (lanes 1 and 5), M1-TAT (lanes 2 and 6), and C-TAT (lanes 3 and 7).
Equal amounts of each RNA sample (25 µg) were separated on 2% agarose gels that contained formaldehyde, transferred to a nitrocellulose membrane, and hybridized to a [32P]-radiolabeled probe that contained the DNA sequence coding for H1 RNA (lanes 1–4) or M1 RNA (lanes 5–8). The expression of human H1 RNA was used as the internal loading control for the quantification of M1GS RNA expression.
Figure 7.
Schematic representation of the expression levels of supernatant HIV-1 p24 protein and total (unspliced and spliced) intracellular HIV RNA in viral infected H9 cells that did not express a ribozyme [P(H9)] or stably-expressed ribozyme C-TAT (C-TAT), M1-TAT (M1-TAT), and V38-TAT (V38-TAT).
The values shown are the averages from three independent experiments. The standard deviation is indicated by the error bars. Solid bars: HIV p24 protein; open bars: HIV RNA.
Figure 8.
Growth of HIV-1 in H9 cells and cell lines that expressed M1GS RNAs.
5×105 cells were infected with HIV-1 at a MOI of 0.02–0.1. Viral production was determined by a p24 antigen assay as a function of time postinfection. The parental H9 cells were used as a negative control. The values are the means from triplicate experiments. The standard deviation is indicated by the error bars.