Inhibition of HIV-1 fusion with small interfering RNAs targeting the chemokine coreceptor CXCR4

Abstract

RNA interference (RNAi) is an evolutionarily conserved process by which plants and animals protect their genomes utilizing small, double-stranded RNAs to degrade target RNAs in a sequence-specific manner. Post-transcriptional gene silencing by these moieties can lead to degradation of both cellular and viral RNAs. It has recently been shown that double-stranded, small interfering RNAs (siRNAs) of 21-25 nucleotides can be transfected into relevant cells to target specific RNAs. This approach was utilized to inhibit human immunodeficiency virus type I (HIV-1) infection in human cells. siRNAs with homology to a motif in the mRNA that encodes for the HIV-1 chemokine coreceptor CXCR4 was utilized. Complementary studies via immunofluorescence microscopy and fluorescence-activated cell sorting demonstrated downregulation of CXCR4 from the surface of cells transfected with the specific siRNAs. As well, siRNAs without sequence homology to CXCR4 were used as controls and demonstrated no downregulation of CXCR4. siRNAs targeted to another chemokine coreceptor, APJ, showed specificity for downregulation of APJ but had no effects on CXCR4.Transfections with siRNAs targeting CXCR4 mRNA were shown to inhibit HIV-1 envelope fusion, which is relatively resistant to most viral inhibitors targeting chemokine coreceptors. The specificity of this effect was demonstrated by the inhibition of fusion by CXCR4-tropic and dual-tropic (CXCR4 and CCR5) envelope glycoproteins from HIV-1 on CXCR4+ indicator cells, but the lack of effects by siRNAs targeting CXCR4 mRNA on dual-tropic HIV-1 envelopes in CCR5+ indicator cells utilizing these fusion assays. Interestingly, siRNAs targeting CXCR4 selectively inhibited CXCR4-tropic cell-free virus infection of human cells but at only modest levels as compared to cell:cell fusion. siRNA may be a potential molecular therapeutic approach to alter a cellular cofactor critical for infection of human cells by relevant strains of HIV-1. The targeting of a cellular cofactor, rather than the HIV-1-specific mRNAs or genomic RNA, holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi directly against this virus.

Expression of Nef Downregulates CXCR4, the Major Coreceptor of Human Immunodeficiency Virus, from the Surfaces of Target Cells and Thereby Enhances Resistance to Superinfection

“…A recent study reported a marked inhibition of X4 HIV Env fusion efficiency (65 to 75% reduction) in HOS CD4/CXCR4 cells, in which CXCR4 levels had been decreased by 80% by delivery of small interfering RNA (siRNA) (76). Another study employing short hairpin RNA against CXCR4 and CCR5 in MAGI-CXCR4 and GHOST-CCR5 cells showed that a ϳ70% downregulation of the coreceptors resulted in an over 10-fold reduction in viral antigen levels in culture supernatants (3).…”

Antiviral Effects of Human Immunodeficiency Virus Type 1-Specific Small Interfering RNAs against Targets Conserved in Select Neurotropic Viral Strains

“…As noted above, the antiviral effects of HIV-1-specific siRNAs were demonstrated by several research groups (4,13,15,16,33,38,44,50,54,74). However, the targets that were selected in previous studies are not conserved among neurotropic strains of HIV-1.…”

“…Substantial protection of lymphocyte populations from CCR5-tropic HIV-1 infection was achieved (54). In another study, Zhou et al demonstrated selective inhibition of CXCR4-tropic cell-free virus infection of human cells by using siRNA targeting the CXCR4 coreceptor (74).…”

“…Both viral proteins (15,16,33,38,50) and the cellular coreceptors (4,13,44,54,74) used by HIV-1 have been targeted for down-regulation by siRNAs. There are certain perceived shortcomings to both approaches.…”

siRNA, miRNA and HIV: promises and challenges

“…In addition to targeting HIV genes (nef, tat, gag, vif, env) [26,30,31], some have also targeted cellular proteins important to the HIV-1 life cycle. Thus, siRNAs were designed and tested against different steps of virus replication: i) at the entry level to HIV coreceptors CCR5 and CXCR4 [32][33][34]; ii) at the pre-integration step to the Arp2/3 complex responsible for actin polymerization involved in the routing of HIV toward the nucleus [35], and to PARP-1 [poly (ADPribose) polymerase-1] an abundant nuclear enzyme required for HIV integration [36]; iii) to cyclin T1, CDK9 and SPT5 which function at the level of HIV transcription for elongation of RNA polymerase II on the HIV-LTR [37,38], and iv) to other key components such as cyclophilin A [39] and cdk2 [40]. All these targets were found to be efficiently inhibited by transfection of specific siRNAs, and their inhibition correlated with reduction in HIV replication in tissue culture [41].…”