Gene silencing of HIV chemokine receptors using ribozymes and single-stranded antisense RNA

Abstract

The chemokine receptors CXCR4 and CCR5 are required for HIV-1 to enter cells, and the progression of HIV-1 infection to AIDS involves a switch in the co-receptor usage of the virus from CCR5 to CXCR4. These receptors therefore make attractive candidates for therapeutic intervention, and we have investigated the silencing of their genes by using ribozymes and single-stranded antisense RNAs. In the present study, we demonstrate using ribozymes that a depletion of CXCR4 and CCR5 mRNAs can be achieved simultaneously in human PBMCs (peripheral blood mononuclear cells), cells commonly used by the virus for infection and replication. Ribozyme activity leads to an inhibition of the cell-surface expression of both CCR5 and CXCR4, resulting in a significant inhibition of HIV-1 replication when PBMCs are challenged with the virus. In addition, we show that small single-stranded antisense RNAs can also be used to silence CCR5 and CXCR4 genes when delivered to PBMCs. This silencing is caused by selective degradation of receptor mRNAs.

Figure 1. Schematic illustration of CXCR4 and CCR5 ribozymes and antisense molecules binding to their respective targets

(A) Computer-generated structure of the CXCR4 ribozyme binding to its target mRNA. (B) Computer-generated structure of the CCR5 ribozyme binding to its target mRNA. (C) Computer-predicted structure of the CXCR4 antisense molecule binding to its target site. (D) Computer-predicted structure of the CCR5 antisense molecule binding to its target site. In each case, the ribozyme or antisense molecule is drawn as a thick line with the target as a thin line. The target for CXCR4 is from position 609 to 621 in the sequence (GenBank® accession number M99293) and that for CCR5 is from position 256 to 268 (GenBank® accession number X91492).

Figure 2. DNA cassettes used for producing ribozyme and antisense molecules

The arrangement of each cassette is shown, and below is a model of the corresponding transcript formed. In each model, the self-cleaving cis-acting hammerhead ribozyme is drawn as a thin line, and the thick line represents the trans-acting hammerhead molecule. The conformation of the active species produced after self-cleaving is indicated on the right of each Figure. (A) CXCR4 ribozyme cassette. (B) CCR5 ribozyme cassette. (C) CXCR4 antisense cassette. (D) CCR5 antisense cassette.

Figure 3. Ribozyme in vitro activity and specificity assay

Values on the left of the Figures represent size markers as number of bases. (A) Self-cleavage for ribozyme and antisense molecules. Lane a (control) shows the complete CCR5 ribozyme-generating transcript (93 bases) after incubation for 4 h in the absence of Mg²⁺ ions. Lane b shows a similar transcript in the presence of Mg²⁺ ions demonstrating cleavage and producing a 45 base cis-acting ribozyme and a 48 base trans-acting ribozyme that are indistinguishable in size on this gel. Lane c shows the complete CCR5 antisense-generating transcript (73 bases), after incubating in the absence of Mg²⁺ ions. Lane d shows a similar transcript after incubating with Mg²⁺ ions where it is self-cleaved into two fragments: a 27 base trans-acting molecule and a 46 base cis-acting ribozyme. Lane e shows the complete CXCR4 ribozyme-generating transcript (99 bases) after incubating in the absence of Mg²⁺ ions. Lane f shows a similar transcript in the presence of Mg²⁺, where it is self-cleaved into trans-acting (50 bases) and cis-acting (49 bases) products. Lane g shows the complete CXCR4 antisense-generating transcript (79 bases) in the absence of Mg⁺². Lane h shows a similar transcript in the presence of Mg²⁺, where it self-cleaved to generate two RNA fragments: one of 29 bases, the CXCR4 trans-acting antisense molecule, and another of 50 bases, the cis-acting ribozyme. (B) Ribozyme in vitro assays. Lane a (control) shows the CCR5 mRNA (1199 bases) after incubation with Mg²⁺ for 4 h at 37 °C, and lane b shows effects of incubating the CCR5 ribozyme with this mRNA at a molar ratio of 1:1 for 4 h. Lane c (control) shows the CXCR4 mRNA (1175 bases) after incubation, and lane d shows the effects of incubation of the CXCR4 ribozyme with this mRNA at a molar ratio of 1:1 for 4 h. (C) Ribozyme specificity studies. Lanes a and b show the effect of incubating CCR5 mRNA with CXCR4 ribozyme (lane a, control stopped at zero time; lane b, after overnight incubation in 24 mM Mg²⁺ at 37 °C). Lanes c and d show the effect of incubating CXCR4 mRNA with CCR5 ribozyme (lane c, control stopped at zero time; lane d, after incubating overnight in 24 mM Mg²⁺ at 37 °C).

Figure 4. CXCR4 and CCR5 ribozyme and antisense activity in human PBMCs

(A) Agarose gel results of RT–PCR using primers specific for mRNAs of actin, CCR5 and CXCR4. Lane a, control cells transfected with pCS2P+pZEQ expression systems; lane b, cells containing pCS2P+pCXCR4/CCR5Rz; lane c, cells containing pCS2P+pZEQ+pCXCR4/CCR5Rz; lane d, cells containing pCS2P+pCCR5As; lane d', cells containing pCS2P+pCCCR5As; lane e, cells containing pCS2P+pZEQ+CCR5 antisense system; lane e', cells containing pCS2P+pZEQ+pCXCR4As. (B) Quantification of the CCR5 and CXCR4 RT–PCR products produced in (A) as a percentage of the control. The same lettering is used as in (A) to identify the samples. Similar values were obtained from Lightcycler data after normalizing with actin as a control. (C) Results of FACS scan for CCR5 and CXCR4: (a) control cells, (b) cells transfected with pCS2P+pZEQ+pCCR5As/pCXCR4As, and (c) cells transfected with pCS2P+pZEQ+pCXCR4/CCR5Rz. Quantification shows that the cells transfected with pCS2P+pZEQ+pCXCR4/CCR5Rz have CCR5 levels depleted by up to 92% and CXCR4 levels depleted by up to 70%. The pCCR5As- or pCXCR4As-transfected cells have CCR5 levels lowered by 78% and CXCR4 levels lowered by 55% respectively.