Broadly active zinc finger protein-guided transcriptional activation of HIV-1

Human immunodeficiency virus type 1 (HIV-1) causes a persistent viral infection resulting in the demise of immune regulatory cells. Clearance HIV-1 results integration proviral DNA into genome host cells, which provides means for evasion and long-term persistence. A therapeutic compound that specifically targets sustainably activates latent provirus could be transformative is goal “shock-and-kill” approach to functional cure HIV-1. Substantial progress has been made toward development recombinant proteins target specific genomic loci gene activation, repression, or inactivation by directed mutations. However, most these modalities are too large complex efficient application. We describe here testing novel zinc finger protein transactivator, ZFP-362-VPR, potently enhances transcription both established latency models activity across different clades. Additionally, ZFP-362-VPR-activated reporter expression well-established primary human CD4+ T cell model off-target pathways were determined transcriptome analyses. This study clear proof concept application novel, therapeutically relevant, transactivator purge cellular reservoirs establishes remains hidden from system. infected cells was suggested take many decades.1Murray A.J. Kwon K.J. Farber D.L. Siliciano R.F. The Latent Reservoir HIV-1: How Immunologic Memory Clonal Expansion Contribute Persistence.J. Immunol. 2016; 197: 407-417Crossref PubMed Scopus (80) Google Scholar Activation purging macrophages, monocytes, with ∼1 per million an ongoing experimental struggle, being find reversing agents (LRAs) can used along antiretroviral therapy (ART). aims eliminate reservoir inducing activation death clearance presenting antigen.2Sadowski I. Hashemi F.B. Strategies eradicate HIV patients: elimination reservoirs.Cell. Mol. 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Next, tested breadth ability A–G assessed co-transfected containing subtype-specific LTRs driving luciferase 1E). subtypes A, B, D, F less C, E, noteworthy observation conserved F, while E G contain point mutations S1A). Subtype C triple site, aligns better between second third motifs mismatch deletion S1B). Overall, data support notion ZFP-362-VPR wide range subtypes. To clearly determine chromatin immunoprecipitation assay (ChIP)30Brena Chipman Minelli Akam trunk Hox genes centipede Strigamia maritima: sense anti-sense transcripts.Evol. 8: 252-265Crossref (30) performed dCas9-VPR+sgF2-362 treated localize 1F), enrichment pMo lacking S2). verify confirmed western blot S3). Collectively, suggest fusion construct dCas9+sgF2-362 variety models, whereas variable observed LRAs.7Saayman similarly lines clonally selected J-Lat “6.3,” “10.6,” “15.4,” similar potency 2). Furthermore, reliably activated commonly 3). These demonstrate consistently independent signaling pathways.Figure 3Comparison vitro modelsShow full captionThree (J-Lat 6.3, 10.6, 15.4) subjected either electroporation dCas-VPR+sgF2-362 various LRAs: TNF-α, CD3/CD28 beads, prostratin, PMA, PMA+ionomycin. DMSO untreated (mock) included negative controls. 72 hr after treatment FACS GFP. Fold percentage relative set 1. triplicate-treated cultures SEM. Statistically significant differences, one-way ANOVA Dunnett’s test (∗p < 0.05, ∗∗p 0.005, ∗∗∗∗p 0.0001) analyzed mock respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Three respectively. It if model. 17 days prior pNL4.3-Δenv-nluc replication-incompetent expressing nano-luciferase pseudotyped VSVG. As reduces expression, population uninfected separated productively day 17. transfected h post-separation, level measured readout There increase 4). Likewise, LTR, anti-CD3/CD28 stimulation, resulted high Although donor, donors pronounced S4). Nonetheless, major concern toxicity oncogenic same sgF2-362, expression. dCas9+sgF2-362, pcDNA3.1 bulk RNA-sequencing analysis. Principal component analysis showed tight clustering sample groups, indicating source variation S5A). plots show on-target reporter, components detected S5B S5C). 191 differentially expressed dCas9-VPR-sgF2-362 (Table S1) 219 ZFP-362-VPR-treated samples S2) (|log2FC| ³ 2, false rate [FDR] = 0.05). ZFN-362-VPR had profile 5A): 358 common treatments, 12 dCas9-VPR+sgF2-362-treated samples, 40 5B; Tables S3 looked enriched Ontology (GO) terms KEGG associated respect controls, neurotransmitter transport cardiomyocytes 5C). Similarly, GO suggesting profiles 5D). none oncogene proliferation. versatile modular. HIV-1, designed against SP1 LTR,31Kim Y.S. J.M. Jung Kang J.S. Seol Shin H.C. H.S. Van Lint al.Artificial fusions Sp1-binding trans-activator-responsive element HIV-1.J. Biol. 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