Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5

Qin, F. Xiao-Feng; An, Dong Sung; Chen, Irvin S. Y.; Baltimore, David

doi:10.1073/pnas.232688199

Cited by 646 publications

(545 citation statements)

References 62 publications

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“…Of importance and paradoxically, previous studies of pharmacological drugs or small molecule inhibitors that target HIV-1 entry have shown them to be dramatically more potent at inhibiting cell-free viruses, as compared to cell:cell fusion. 70 Of note, inhibition of CCR5-directed HIV-1 cell-to-cell fusion by siRNAs was also not analyzed in a recent study, 53 as only effects on infection with cell-free viruses were reported and their results were comparable to our findings. Although, in the present study, full downregulation of CXCR4 was not demonstrated using transfection approaches of siRNAs, certain modifications are possible in further model systems to increase efficiency.…”

Section: Discussionsupporting

confidence: 85%

“…52 A very recent study suggests that RNAi targeting CCR5 mRNA may be useful in inhibiting HIV-1 strains found relatively early in clinical disease (ie about 65% decrease in HIV-1 p24 antigen expression in these infectivity studies). 53 Finally, both structural protein (gag) and regulatory protein (eg tat and rev) viral mRNAs have also been approached using siRNA inhibitors in relevant model systems in vitro. [47][48][49][50] Of note, one report has demonstrated the potentially very important finding that siRNAs may gain entrance to the preintegration complex of HIV-1, leading to cleavage of the 'in-coming' viral genome.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2004

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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.

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Section: Discussionsupporting

confidence: 85%

Section: Discussionmentioning

confidence: 99%

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

et al. 2004

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“…To measure lentiviral titration and transduction efficiency, EGFP encoding particles were coproduced utilizing pFG12. 28 In all, 2-10 EGFP-viral units per cell were added to 2 Â 10 3 HEK 293 FT cells. Transduction efficiency was assessed by EGFP expression analysis by flow cytometry (FC500, Beckman Coulter, Villepinte, France) and luciferase activity measurement, as previously described.…”

Section: Rnai Moleculesmentioning

confidence: 99%

Allele-specific Col1a1 silencing reduces mutant collagen in fibroblasts from Brtl mouse, a model for classical osteogenesis imperfecta

et al. 2013

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Gene silencing approaches have the potential to become a powerful curative tool for a variety of monogenic diseases caused by gain-of-function mutations. Classical osteogenesis imperfecta (OI), a dominantly inherited bone dysplasia, is characterized in its more severe forms by synthesis of structurally abnormal type I collagen, which exerts a negative effect on extracellular matrix. Specific suppression of the mutant (Mut) allele would convert severe OI forms to the mild type caused by a quantitative defect in normal collagen. Here, we describe the in vitro and ex vivo investigation of a small interfering RNA (siRNA) approach to allele-specific gene silencing using Mut Col1a1 from the Brtl mouse, a well-characterized model for classical human OI. A human embryonic kidney cell line, which expresses the firefly luciferase gene, combined with either wild-type or Mut Brtl Col1a1 exon 23 sequences, was used for the first screening. The siRNAs selected based on their specificity and the corresponding short hairpin RNAs (shRNAs) subcloned in a lentiviral vector were evaluated ex vivo in Brtl fibroblasts for their effect on collagen transcripts and protein. A preferential reduction of the Mut allele of up to 52% was associated with about 40% decrease of the Mut protein, with no alteration of cell proliferation. Interestingly, a downregulation of HSP47, a specific collagen chaperone known to be upregulated in some OI cases, was detected. Our data support further testing of shRNAs and their delivery by lentivirus as a strategy to specifically suppress the Mut allele in mesenchymal stem cells of OI patients for autologous transplantation.

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“…X. Qin and D. Baltimore (California Institute of Technology, Pasadena, CA) [18]. The FG12 vector construct contains a 5 0 long terminal repeat (LTR), multiple cloning sites, and a ubiquitin C promoter-driven enhanced green fluorescent protein, followed by a 3 0 LTR.…”

Section: Gene-kd Vector Preparationmentioning

confidence: 99%

The Actin Polymerization Regulator WAVE2 Is Required for Early Bone Marrow Repopulation by Hematopoietic Stem Cells

et al. 2009

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The Rho GTPase family members play essential roles in hematopoiesis. Of these, Rac1 is thought to be required for the appropriate spatial localization of hematopoietic stem and/or progenitor cells (HSPCs) within the bone marrow (BM), whereas Rac2 likely plays a role in BM retention of HSPCs. To elucidate the molecular mechanisms underlying Rac-mediated functions in hematopoietic stem cells (HSCs), we studied Wiskott-Aldrich syndrome protein family verprolin-homologous proteins (WAVEs), the specific effectors downstream of the Rac GTPases in actin polymerization. We here showed that CD34 2/low cKit 1 Sca-1 1 lineage 2 HSCs (CD34 2 KSL HSCs) express WAVE2 but neither WAVE1 nor WAVE3. Because WAVE2 knockout mice are embryonic-lethal, we utilized HSCs in which the expression of WAVE2 was reduced by small interfering RNA. We found that knockdown (KD) of WAVE2 in HSCs affected neither in vitro colony formation nor cell proliferation but did impair in vivo long-term reconstitution. Interestingly, WAVE2 KD HSCs exhibited unaltered homing but showed poor BM repopulation detected as early as day 5 after transplantation. The mechanistic studies on WAVE2 KD HSCs revealed modest but significant impairment in both cobblestone-like area-forming on stromal layers and actin polymerization upon integrin ligation by fibronectin. These results suggested that WAVE2-mediated actin polymerization, potentially downstream of Rac1, plays an important role in intramarrow mobilization and proliferation of HSCs, which are believed to be crucial steps for long-term marrow reconstitution after transplantation.

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Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5

Cited by 646 publications

References 62 publications

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

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

Allele-specific Col1a1 silencing reduces mutant collagen in fibroblasts from Brtl mouse, a model for classical osteogenesis imperfecta

The Actin Polymerization Regulator WAVE2 Is Required for Early Bone Marrow Repopulation by Hematopoietic Stem Cells

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