Promoter choice affects the potency of HIV-1 specific RNA interference

Abstract

RNA interference (RNAi) is mediated by small interfering (si) RNAs that target and degrade mRNA in a sequence-specific manner. Cellular expression of siRNA can be achieved by the use of expression cassettes driven by RNA polymerase III (pol III) promoters. Here, we demonstrate that a modified tRNA(met)-derived (MTD) promoter effectively drives the cellular expression of HIV-1-specific siRNA. We observed up to 56% greater inhibition of virus production when the MTD promoter was used to drive the expression of short hairpin (sh) RNA targeting the HIV-1 transactivator protein tat compared to cassettes containing other pol III promoters such as H1, U6+1 and U6+27. We conclude that the MTD promoter is ideally suited to drive intracellular expression of HIV-1 specific siRNA and may serve as an important component of future RNAi vector delivery systems.

Figure 1

Schematic representation of the various shRNA expression units. Unique restriction sites in the AAV-DNA vector allowed the exchange of different promoters. Tat siRNA is expressed in the form of a hairpin transcript consisting of a 21-nt sense and 21-nt antisense sequence separated by a hexaloop. The five thymidine stretches serve as a RNA pol III termination signal whereas the pA_BGH polyadenylation sequence terminates RNA pol II transcription. Additional components of the recombinant AAV-DNA vector include inverted terminal repeats (ITRs) and a neomycin selection cassette.

Figure 2

Inhibition of HIV-1 expression by tat siRNA. Synthetic tat siRNA was co-transfected with HIV-1_NL4.3 in 293T cells and p24 production was assayed 48 h post transfection. Single-stranded sense and antisense tat RNA were used as controls. Values represent averages of three independent experiments, with the range indicated.

Figure 3

Expression levels of tat shRNA driven by different promoters were analyzed by northern blotting. Total cellular RNA was extracted from 293T cells 48 h after transfection with the indicated DNA constructs. Non-transfected 293T cells served as a negative control. Hybridization was performed using a ³²P-labeled DNA oligonucleotide probe complimentary to the sense tat transcript.

Figure 4

Inhibition of HIV-1 virus production by various tat shRNA expression constructs. 293T cells were co-transfected with HIV-1_NL4.3 and the indicated DNA vectors. A vector expressing luciferase specific shRNA (MTD-luc) served as a negative control. HIV-1 p24 antigen level in culture supernatant was quantified by ELISA 48 h after transfection. The antiviral effect of the MTD promoter was adjusted to 100% and the potency of the remaining pol III promoters is presented relative to the antiviral effect of the MTD promoter. Values represent the average of duplicate independent experiments, with the range indicated.

Figure 5

Reduction of HIV-1 p24 antigen in cell-free supernatant and cell lysate determined by western blot analysis. 293T cells were co-transfected with HIV-1_NL4.3 and the different shRNA expression vectors. Non- transfected 293T cells served as a negative control. The control plasmid MTD-luc expresses a luciferase shRNA. Tubulin served as a loading control.

Figure 6

Promoter dependent effects of tat shRNA on levels of spliced and unspliced intracellular HIV-1 RNA as quantified by real-time PCR. Variation of RNA input was normalized by GAPDH real-time PCR. To convert threshold cycles to copy number an external standard curve was created with known numbers of HIV-1 in vitro tat transcripts. A vector expressing luciferase specific shRNA (MTD-luc) served as a negative control. The greatest antiviral effect was observed in experiments using the MTD promoter, which was adjusted to 100% to allow comparisons with other promoters. Values represent averages of two independent experiments, with the range indicated.

Figure 7

Degradation of full length HIV-1 RNA by different tat shRNA expression constructs. Northern blot analysis of total unspliced HIV-1 RNA from 293T cells co-transfected with HIV-1_NL4.3 and various shRNA expression vectors 48 h post-transfection. Tubulin expression was used as a loading control.