Objectives: Duplexes of 21 base pair RNA, known as short-interfering RNA (siRNA), have been shown to inhibit gene expression by a sequence-specific RNA degradation mechanism termed RNA interference (RNAi). The objective of our study was to evaluate the effect of chemokine receptor gene suppression by RNAi on the entry and replication of HIV-1.
Methods: A flow cytometry and microscopy evaluation of HIV co-receptor expression of cells transfected with siRNA. An evaluation of the effect of siRNA on HIV entry and replication by intracellular p24 antigen detection, and virus production by infected cells, respectively.
Results: siRNA that target CXCR4 and CCR5 could effectively impede cell surface protein expression and their consequent function as HIV co-receptors. The inhibitory effect of RNAi directed to CXCR4 was detected 48 h after transfection of CXCR4+ U87-CD4+ cells. The expression of CXCR4 and CCR5 was blocked in 63 and 48% of positive cells by the corresponding siRNA. However, siRNA directed to CXCR4 or CCR5 did not have an effect on CD4 cells or green fluorescence protein expression. siRNA directed to CXCR4 did not suppress CCR5 expression or vice versa. The suppression of HIV-1 co-receptor expression effectively blocked the acute infection of CXCR4+ or CCR5+ U87-CD4+ cells by X4 (NL4-3) or R5 (BaL) HIV-1 strains. Inhibition of virus replication occurred regardless of the multiplicity of infection employed.
Conclusion: Our results demonstrate that RNAi may be used to block HIV entry and replication through the blockade of cellular gene expression. Gene silencing by siRNA may become a valid alternative for HIV intervention.
RNA interference (RNAi) is the latest strategy for attacking mRNA. Others include inhibition of translation via the use of single-stranded oligodeoxynucleotides and ribozymal RNA cleavage. RNAi occurs through the activity of small interfering RNA molecules (siRNA) that are homologous to the gene to be silenced. Upon entry to the cell, siRNA can mediate post-transcriptional sequence-specific degradation of mRNA. This mechanism protects against viral infection by silencing key cellular genes required for viral entry, as in this article, or by silencing essential viral genes. siRNAs act without inducing alpha-interferon signalling pathways or the activation of double-stranded RNA-dependent protein kinase R that also mediates antiviral host defences.
Martinez and his colleagues, whose study is highlighted in this issue, have used RNAi as a tool to specifically suppress expression of the HIV co-receptors CXCR4 and CCR5 and thus block entry and subsequent replication of HIV-1. The investigators used chemically synthesized single-stranded, 21-nucleotide siRNA molecules targeting the coding regions of CXCR4 and of CCR5. These purified siRNAs were transfected into CD4-expressing monocytoid cells also stably expressing either CXCR4 or CCR5, and which were then challenged with X4 or R5 strains of HIV-1. The specific siRNAs reduced CXCR4 and CCR5 expression by 48-80% without altering CD4 expression. This reduction in cell co-receptor expression caused a proportionate reduction in HIV entry and replication. They also found that siRNAs directed against HIV nef and pol genes likewise blocked HIV production without inhibition of co-receptor expression. Other investigators have previously demonstrated suppression of HIV genes.[2,3] Thus a two-pronged approach, suppressing both viral and relevant cellular genes, may be feasible.
As the authors point out, these findings provide evidence that RNAi is not just a tool for examining the function of cellular genes, it is also a potentially powerful therapeutic tool. The effect of the siRNAs was not as dramatic in inhibiting HIV replication as that achieved in the same system using CXCR4 and CCR5 receptor antagonists. However, similar to anti-sense technology, where low affinity of potential oligodeoxynucleotides towards their target nucleotide molecules has required development of modified nucleotides with higher affinity, strategies with RNAi must address inefficient transfection of target cells and ensure durability. DNA vectors for stable expression of siRNAs and stable gene suppression are being developed. Furthermore, interference of these genes in primary cells must be demonstrated -- a further challenge. The ability to target both viral and cellular genes provides a very attractive therapeutic approach, provided that the cellular functions of the host genes are not essential and that HIV does not escape from this interference. The real proof will be when this new tool is shown to work in humans.
Suppression of CXCR4 by short-interfering RNA. HeLa-green fluorescent protein (GFP) cells were mock transfected (control) or transfected with a short-interfering RNA (siRNA) directed to CXCR4 (RNAX42i). After 48 h, cells were labelled with anti-CXCR4 monoclonal antibody (12G5) and a secondary rhodamine-conjugated antibody and visualized in a Nikon uorescence microscope. CXCR4 and GFP expression were quantified by ow cytometry analysis. Mean uorescence intensity (MFI) values are shown.
Inhibition of HIV replication by short-interfering RNA. U87-CD4 cells expressing CXCR4 or CCR5 were mock transfected or transfected with the appropriate short-interfering RNA (siRNA) or an irrelevant siRNA (GFPi). After 48 h, cells were infected with HIV-1 NL4-3, multiplicity of infection (m.o.i.) 0.006 (a) or BaL, m.o.i. 0.12 (b), further incubated for 24 h and stained for intracellular p24 antigen and ow cytometry analysis (white bars). At 48 h post-transfection, supernatant from infected cells was assayed for p24 antigen using enzyme-linked immunoassay (black bars). The chemokine receptor antagonists AMD3100 and TAK-779 are known inhibitors of HIV entry and replication.[11,12] Values represent the mean SD of at least two experiments.
AIDS. 2003;17(18s) © 2003 Lippincott Williams & Wilkins
Lippincott Williams & Wilkins
Cite this: Suppression of Chemokine Receptor Expression by RNA Interference Allows for Inhibition of HIV-1 Replication - Medscape - Dec 01, 2003.