Pharmacological suppression of glycogen synthase kinase-3 reactivates HIV-1 from latency via activating Wnt/β-catenin/TCF1 axis in CD4+ T cells

ABSTRACT
 HIV-1 latency posts a major obstacle for HIV-1 eradication. Currently, no desirable latency reversing agents (LRAs) have been implicated in the “Shock and Kill” strategy to mobilize the latently infected cells to be susceptible for clearance by immune responses. Identification of key cellular pathways that modulate HIV-1 latency helps to develop efficient LRAs. In this study, we demonstrate that the Wnt downstream β-catenin/TCF1 pathway is a crucial modulator for HIV-1 latency. The pharmacological activation of the β-catenin/TCF1 pathway with glycogen synthase kinase-3 (GSK3) inhibitors promoted transcription of HIV-1 proviral DNA and reactivated latency in CD4+ T cells; the GSK3 kinase inhibitor 6-bromoindirubin-3′-oxime (6-BIO)-induced HIV-1 reactivation was subsequently confirmed in resting CD4+ T cells from cART-suppressed patients and SIV-infected rhesus macaques. These findings advance our understanding of the mechanisms responsible for viral latency, and provide the potent LRA that can be further used in conjunction of immunotherapies to eradicate viral reservoirs.


Introduction
The combination antiretroviral therapy (cART) has changed AIDS from a fatal disease into a chronic disease. However, the persistence of latent viral reservoirs posts an obstacle for cure [1][2][3]. The latent reservoir is described as a hidden sanctuary of the HIV-1 that is not recognized by the immune system, and the lifelong cART is necessary to prevent viral rebound [1][2][3]. The "Shock and Kill" paradigm represents a potential therapeutic approach to purge HIV-1 reservoirs, in which HIV-1 provirus expression in latently infected cells was reactivated with latency reversing agents (LRAs) ("Shock"), followed by immune responses or ART that clear the activated cells ("Kill") [1][2][3]. Although dozens of LRAs with distinct mechanistic classes have been tested in clinical trials, no LRA that fully induce viral reactivation has been identified, this is partly due to the heterogeneity of cells and tissues that form the HIV-1 reservoirs and the complexity of molecular mechanisms that regulate viral latency [4][5][6][7][8]. The low inducibility of latent proviruses is a major problem for the "Shock and Kill" strategy for curing HIV-1 infection [9]. Recently, the administration of compound AZD5582 for activating the non-canonical NF-κB pathway or the interleukin-15 superagonist N-803 in conjunction with CD8 + lymphocyte depletion has shown the potent reactivation of HIV or SIV in infected humanized mice and rhesus macaques, respectively [10,11]. Identification of the key host factors/pathways that modulate viral latency helps develop new antiretroviral therapies.
Glycogen synthase kinase-3 (GSK3) is a ubiquitously expressed, highly conserved serine/threonine protein kinase found in all eukaryotes [12]. GSK3 is constitutively active in most cells and serves as a regulator for multiple biological processes, ranging from glycogen metabolism, cell development, gene transcription, and protein translation to cytoskeletal organization, cell cycle regulation, proliferation, and apoptosis [12]. Accordingly, the aberrancy in the GSK-3 activity has been implicated in a wide variety of diseases; thus, targeting GSK3 as a therapy has been tested clinically in human diseases. These include GSK3 inhibitors such as Tideglusib, LY2090314, Enzastaurin, and LiCl [13][14][15], though the potential off-target pharmacological and toxicological effects necessitate extensive evaluation for their usage [16,17].
GSK3 is a key negative regulator of canonical Wnt signalling, and in the turned-off status of Wnt signalling, GSK3 functionally associates with axin and adenomatous polyposis coli (APC) protein to phosphorylate β-catenin for the sequential proteasomal degradation; while the activation of Wnt signalling prevents GSK3mediated β-catenin phosphorylation and stimulates the subsequent translocation of β-catenin to nucleus for regulating gene transcription [18][19][20]. The nuclear-located β-catenin binds factors of the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) family, enabling the recognition of Wnt target genes through TCF/LEF-specific sequence motifs [18][19][20].
In this study, we found that pharmacological suppression of GSK3 by small compound 6-BIO reactivated HIV-1 from latently infected CD4 + T cells, and 6-BIO-induced HIV-1 reactivation was evaluated in SIV-infected rhesus macaques and Peripheral blood mononuclear cells (PBMCs) isolated from cART-suppressed patients.

Ethics statement
The usages of human samples and rhesus macaques (Macaca mulatta) and the related methods and experimental protocols have been approved by the Medical Ethics Review Committee of Institut Pasteur of Shanghai, Chinese Academy of Sciences (CAS), the Medical Ethics Review Committee of Zhengzhou Sixth People's Hospital, and the Institutional Animal Care and Use Committee of Guangzhou Institutes of Biomedicine and Health, CAS. Informed consent has been signed by HIV-1 patients. All experiments were performed in accordance with relevant national guidelines and regulations.

Cells and virus
The HIV-1 latently infected CD4 + CEM cell ACH2 and HIV-1 chronically infected U937 cells (U1) were provided by Dr Shibo Jiang and Dr Lu Lu (Fudan University, Shanghai, China). HeLa-cell-derived TZM-bl indicator cells which contain LTR-driven luciferase reporter were a gift from Dr Paul Zhou (Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China). The HEK293T cells were kindly provided by Dr Li Wu (The Ohio State University, USA). Cells were cultured in RPMI 1640 medium (Gibco) or Dulbecco's Modified Eagle Medium (DMEM) (Hyclone) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 μg/ml of streptomycin (Invitrogen) at 37°C under 5% CO 2 .

shRNAs
The targeted sequences of shRNAs were listed in Supplementary Table 1. shRNA was cloned into the PLKO.1-puro shRNA expression vector. The packaging of shRNA lentiviruses was performed according to the PLKO.1 protocol (Addgene). Calcium phosphate-mediated transfection of HEK293T cells was used to generate shRNA lentiviruses as previously described [22].

Real-time qPCR
Total cellular RNA was extracted by TRIzol reagent (Invitrogen), and reverse transcribed into cDNA using the ReverTra Ace qPCR reverse transcription master mix with gDNA (genomic DNA) Remover (Toyobo). Real-time polymerase chain reaction (PCR) was performed using the Thunderbird SYBR qPCR Mix (Toyobo) with pre-denaturation at 95°C for 2 min, amplification with 40 cycles of denaturation (95°C, 15 s), and annealing (60°C, 30 s), on the ABI 7900HT Real-Time PCR system. The data were analysed by a SYBR green-based system (Toyobo), semiquantified and normalized with glyceraldehyde-3phosphate dehydrogenase (GAPDH). The primers were listed in Supplementary Table 1
For immunoblotting, cells were lysed for 1 h at 4°C in ice-cold lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.5 mM EGTA, 1% protease inhibitor cocktail [Sigma], 1 mM sodium orthovanadate, 1 mM NaF, 1% [vol/vol] Triton X-100, and 10% [vol/ vol] glycerol). After centrifugation for 10 min at 12,000g, the supernatant was boiled in reducing SDS sample loading buffer and subjected by SDS-PAGE. The indicated specific primary antibodies were used, followed by horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (Sigma) as the secondary antibody. Nuclear and cytoplasmic protein fractions were purified using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to the manufacturer's introduction.

Chromatin immunoprecipitation
ChIP experiments were performed according to the protocol provided by EZ-ChIP Chromatin Immunoprecipitation kit (Millipore) as previously described [22]. Cells were cross-linked with 1% formaldehyde for 10 min at room temperature and quenched with 0.125 M glycine for 5 min. After lysis, chromatin was sheared by use of a sonicator (Bioruptor UCD-200; Diagenode) for a total of 12 min (2s on and 6s off) on ice to obtain DNA fragments of 200-500 base pairs. Five per cent of the total sheared chromatin DNA was used as the input sample. Other sheared chromatin was incubated overnight at 4°C with an antibody against TCF1, H3K4me3, H3K27ac, H3K9me3, H3K27me3, or rabbit IgG (Cell Signalling), followed by incubating with 40 μl Protein G/Alabelled Dynabeads at 4°C for 4 h for immunoprecipitation. After washing and reversing cross-link, the input and immunoprecipitated DNA was purified and analysed by real-time PCR using primers specifically targeting for nucleotide position of HIV-1 proviral DNA. The primers were listed in Supplementary Table 1.

Viral reactivation in resting CD4 + T cells from cART-suppressed HIV-1 infected patients
Peripheral blood from combination antiretroviral treatment (cART)-treated HIV-1 infected patients were collected from the cohort patients of Zhengzhou Sixth People's Hospital. Informed consent has been signed. These patients had undergone cART comprised of TDF (tenofovir disoproxil fumarate), 3TC (lamivudine), and EFV (efavirenz) for more than 3 years with an undetectable HIV-1 viral load in the plasma. CD4 + counts were analysed by flow cytometry (BD Biosciences). HIV-1 viral loads were quantified by PCR (Cobas Amplicor). HIV subtypes were determined by phylogenetic analysis of Gag-RT sequence with subtype reference (Supplementary Table 2).

Rhesus macaque performances
Chinese rhesus macaques (Macaca mulatta) were housed in the Experimental Animal Center of Guangzhou Institutes of Biomedicine and Health (Guangzhou, China). SIV mac239 (5, 000TCID50) intravenously challenged macaques were treated with a potent and long-lasting nucleoside reverse transcriptase inhibitor FNC (2'-deoxy-2'-β-fluoro-4'-azidocytidine) (0.4 mg/kg) we developed recently for viral suppression (Chang J et al., patents: ZL 200710137548.0, US 8835615 B2, EP2177527 B1) [23]. Animals were followed intravenously treated with 6-BIO (T1917; Topscience). Levels of plasma and cell-associated SIV gag mRNA were quantitated by real-time PCR as described previously [24]. Briefly, plasma viral RNA was isolated using the QIAamp Viral RNA Minikit (Qiagen) and cellular RNA was extracted using a Total RNA Extraction Kit (Promega). RNAs were reverse transcribed into complementary DNA (cDNA) using the ReverTra Ace qPCR reverse transcription master mix with gDNA (genomic DNA) Remover (Toyobo). Plasma viral RNA copy number was then determined with a SYBR green-based real-time quantitative PCR (Takara) using SIV-gag-specific primers (Supplementary Table 1). The copy number of viral RNA was calculated based on the standard curve of an in vitrotranscribed fragment of the SIVmac239 gag gene. The limitation for assay was 100 copies/mL plasma. The reverse transcribed cellular cDNAs were used as the template for PCR amplification by Phanta Max Super-Fidelity DNA Polymerase (Vazyme) with SIVgag-specific 1st pair of primers (Supplementary  Table 1); the diluted PCR products were further used as the qPCR template, and cell-associated gag mRNA number was determined by real-time qPCR with TB Green Premix Ex Taq II (TAKARA) with SIV-gag-specific 2nd pair of primers (Supplementary Table 1). The number of circulating CD3 + , CD4 + , and CD8 + T-lymphocytes were determined using BD TruCount tubes according to the manufacturer's instructions (BD Biosciences).

RNA sequencing and data analysis
Total RNAs from cells were extracted using Trizol (Invitrogen) according to the manufacturer's protocol, and ribosomal RNA removed using QIAseq FastSelect-rRNA HMR Kits (QIAGEN, Germany). Fragmented RNAs (average length approximately 200 bp) were subjected to first strand and second strand cDNA synthesis, followed by adaptor ligation and enrichment with a lowcycle according to the instructions of NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA). The purified library products were evaluated using the Agilent 2200 TapeStation and Qubit2.0 (Life Technologies, USA). The libraries were paired-end sequenced (PE150, Sequencing reads were 150 bp) using Illumina HiSeq 3000 platform.
Raw RNA sequencing (RNA-seq) reads were filtered using Trimmomatic v0. 36. The filtered reads were mapped to the human (hg38) reference genomes using HISAT v2.1 with corresponding gene annotations (GRCh38.p13) with default settings. Total counts per mapped gene were determined using featureCounts function in SubReads package v1.5.3 with default parameter. Next, counts matrix obtained from feature-Counts was used as input for differential expression gene analysis with the bioconductor package DESeq2 v1.26 in Rv4.0. Gene counts more than 5 reads in a single sample or more than 50 total reads across all samples were retained for further analysis. Filtered counts matrix was normalized using the DESeq2 method to remove the library-specific artefacts. Principal component analysis was based on global transcriptome data using the build-in function prcomp in R software. The genes with log2 fold change >1 or < −1 and adjusted p value <0.05 corrected for multiple testing using the Benjamini-Hochberg method were considered significant. Transcription-factor enrichment analysis and functional enrichment analysis were performed using Metascape server tool (https:// metascape.org/gp/index.html#/main/step1). Gene set enrichment analysis (GSEA) used the R package clus-terProfiler v3.18.1.

Statistical analysis
Graphpad Prism 8.0 was used for statistical analysis. For intra-group direct comparisons, Student's unpaired two-tailed t test was performed to analyse significant differences. For comparisons of multiple groups, one-way ANOVAs were performed. Significance levels are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001.

Data and code availability
All raw RNA-seq data used in this study are available under BioProject accession code PRJNA747246 at the NCBI database (https://www.ncbi.nlm.nih.gov/ bioproject/PRJNA747246).

Results
Suppression of GSK3 kinase activity by 6-BIO reactivates HIV-1 from multiple latently infected CD4 + T cells To investigate the role of GSK3 in modulating HIV-1 latency, GSK3 inhibitor 6-BIO was used to treat the HIV-1 latently infected CD4 + T cell ACH2, and viral reactivation was investigated. GSK3 exists as two isoforms encoded by separate genes: GSK3α (51 kDa) and GSK3β (47 kDa). The treatment of ACH2 cells with 6-BIO inhibited GSK3 kinase activity as demonstrated by the diminished phosphorylation of both α and β isoforms of GSK3 at Tyr216 and Tyr 279 (Figure 1(A)), and as a result, β-catenin phosphorylation was reduced and it became stable in the cytosol and efficiently translocated to the nucleus (Figure 1(A, B)). The 6-BIO treatment reactivated HIV-1 in ACH2 cells, as demonstrated by the significantly increased expressions of HIV-1 gag mRNA (Figure 1(C)). A dose-dependent manner for 6-BIO inactivating GSK3 and reactivating HIV-1 in ACH2 cells has been observed ( Figure 1(A-C)). When ACH2 cells were treated with 6-BIO for different time, we found that the expression of β-catenin was increased, and the expression kept stable after 24 h stimulation (Figure 1(D)). HIV-1 reactivation from the treated cells has been observed over time (Figure 1(F)).
To confirm the observation is not an artefact in a single cell line, we performed the same experiment in HIV-1 latently infected J-Lat cells 10.2 and A2 clones [21], and a dose-dependent manner for 6-BIO inactivating GSK3 and reactivating HIV-1 has been observed ( Figure 1(G, H)).
Taken together, these results demonstrate that the pharmacological inactivation of GSK3 kinase activity reactivates HIV-1 from latently infected CD4 + T cells.
Transcriptome analysis reveals 6-BIO-mediated activation of T cells and cellular β-catenin/TCF1 signalling Next, we performed transcriptome analysis to confirm 6-BIO-mediated activation of CD4 + T cells and cellular β-catenin/TCF1 signalling. ACH2 cells were treated with 6-BIO for 24 h, then the transcriptome was analysed using standard protocols. Data from three independent repeats were analysed. The volcano plot displayed a total of 567 up-regulated genes and 626 down-regulated genes after ACH2 cells were treated with 6-BIO (Figure 3(A)). Gene ontology (GO) functional enrichment analysis of differently expressed genes (DEGs) showed obvious upregulation of gene sets that regulate T cell activation, in contrast, the gene sets that regulate leukocytes differentiation, cell proliferation and development, and small GTPasemediated signal transduction were down-regulated (Figure 3(B)). The GSEA linked the up-regulated genes to the regulation of T cell activation and Wnt signalling pathway (Figure 3(C)). Transcription-factor enrichment analysis of DEGs showed that the upregulated transcription factors were mainly the signal transducer and transcription activators, such as STAT3, FOXP3, VDR, etc., and the down-regulated transcription factors were mainly linked to the regulations of cell proliferation and cell cycle, such as E2F1, TAL1, etc. (Figure 3(D)).

6-BIO treatment enhances TCF1's binding to 5 ′ ′ ′ ′ ′ -LTR to promote HIV-1 transcription
We next dissected the mechanism of 6-BIO-mediated activation of β-catenin/TCF1 signalling for reactivating HIV-1. The nuclear β-catenin binds to TCF1 and acts as a co-activator to enable the recognition of TCF1specific sequence motifs in promoters and enhancers of Wnt targeted genes [27]. HIV-1 latency is mainly characterized by a reversible silencing of 5 ′ -long-terminal repeat (LTR)-driven transcription of provirus. To explore whether 6-BIO treatment can promote TCF1 binding within the 5 ′ -LTR promoter of HIV-1 proviral DNA, we performed ChIP assay in ACH2 cells, and found that 6-BIO treatment significantly promoted TCF1 binding to the positioned nucleosomes (NUC0, NUC1, NUC2) and the intervening enhancer regions DHS (DNase hypersensitive site) of 5 ′ -LTR ( Figure 4  (A, B)). As the consequence, 6-BIO treatment significantly increased the initiation and elongation of HIV-1 5 ′ -LTR-driven transcription (Figure 4(C)). The initiation and elongation of HIV-1 5 ′ -LTR-driven transcription were investigated through monitoring (realtime PCR products with specific primers (Figure 4 (A)) (Supplementary Table 1) [28,29].
To further confirm the regulation of 6-BIO-triggered β-catenin/TCF1 signalling on HIV-1 transcription, Jurkat T cells were acutely infected with pseudotyped single-cycle infectious HIV-Luc/NL-3 (CXCR4 tropic) for 24 h. A dose-dependent manner of enhancement of viral infection was observed upon 6-BIO treatment, and 6-BIO treatment significantly increased the initiation and elongation of HIV-1 5 ′ -LTR-driven transcription (Figure 4(D)). Concurrently, 6-BIO treatment activated β-catenin/TCF1 signalling with a dose-dependent manner. GSK3 kinase activity in Jurkat T cells was inhibited by 6-BIO treatment as demonstrated by the diminished phosphorylation of both α and β isoforms of GSK3 at Tyr216 and Tyr 279 ( Figure  4(E)), and as a result, β-catenin phosphorylation was reduced and it became stable in the cytosol and efficiently translocated to the nucleus (Figure 4(E, F)), and a dose-dependent upregulation of TCF1 expression and increased nuclear location were observed (Figure 4(E,  F)). Taken together, these data demonstrated that 6-BIO treatment enhances β-catenin/TCF1 signalling and promotes HIV-1 transcription.

6-BIO treatment alters epigenetic modification of histones in HIV-1 5 ′ ′ ′ ′ ′ -LTR promoter
Besides the direct promotion of viral transcription by binding to HIV-1 5 ′ -LTR, TCF1 regulates gene expression can also be achieved through its ability to influence epigenetic modifications. The selective targeting of TCF1 in silent chromatin creates chromatin accessibility through epigenetically modifying histones by gaining active marks H3K27ac, H3K4me3, and erasing repressive marks H3K9me3, H3K27me3, and the ability of TCF1-mediated epigenetic modifications are known to regulate T cell development [30]. Notably, the above-mentioned repressive and activate epigenetic marks have also been found to guide chromatin-remodelling complex of HIV-1 5 ′ -LTR promoter to modulate transcriptional activity of HIV-1 latency [4,31,32].

6-BIO-triggered β-catenin/TCF1 signalling reactivates HIV-1 in cART-suppressed patient cells
To confirm the role of 6-BIO-triggered activation of βcatenin/TCF1 pathway in reactivating HIV-1 observed using cell lines, we then tested 6-BIO treatment in primary PBMCs isolated from cART-suppressed patients (Supplementary Table 2). Phytohemagglutinin-P (PHA-P) treatment was used as the positive stimulation control. Viral reactivation was detected with flow cytometry to analyse the HIV-1 Env expression on CD4 + T cells (Figure 6(A)). Results showed that 6-BIO treatment significantly reactivated HIV-1 in patient CD4 + T cells (Figure 6(B)), and this was also confirmed by significantly increased quantity of cellassociated HIV-1 gag and tat-rev mRNA (Figure 6 (C)). 6-BIO-mediated suppression of GSK3 kinase activity and the followed activation of β-catenin/ TCF1 signalling were also confirmed in PBMCs from 2 cART-suppressed patients. The inhibition on GSK3 kinase activity was demonstrated by reduced phosphorylation of Tyr216 and Tyr 279 (Figure 6(D)), increased cytosolic stabilization and greater nuclear translocation of β-catenin ( Figure 6(D, E)), and increased TCF1 nuclear location ( Figure 6(E)). These results demonstrate that 6-BIO-induced suppression of GSK3 kinase activity and the followed activation of β-catenin/TCF1 axis reactivates HIV-1 in PBMCs isolated from cART-suppressed patients.

6-BIO reverses SIV latency in rhesus macaques
To further investigate the role of 6-BIO in a more controlled setting, we next evaluated the latency reversal activity of 6-BIO in four Chinese-origin rhesus macaques. These macaques were challenged with 5000TCID 50 of SIV mac239 , and then were treated with a novel nucleoside reverse transcriptase inhibitor   (Figure 7(B), upper panels). Intravenous infusions of 0.4 mg/kg 6-BIO were administered every two days. Viral reactivation, defined as the re-production of both plasma (Figure 7(B), upper panels) and cell-associated gag mRNAs ( Figure  7(B), lower panels), was observed after the first dose and kept increase along with the further 6-BIO administration in all macaques. Non-specific activation of host cells can provide more susceptible target cells for HIV-1 infection [33]. The administration of 6-BIO treatment did not induce the non-specific activation of CD4 + T, CD8 + T, and CD14 + monocytes, as shown by no increased surface expressions of CD25 and/or HLD-DR (Figure 7(C)); moreover, 6-BIO did not lead to decline of the absolute number of CD4 + and CD8 + T cells over the period of treatment (Figure 7(D)). Taken together, these results demonstrate that 6-BIO efficiently induces SIV latency reversal in rhesus macaques.

Discussion
The reversible repression of HIV-1 5 ′ -LTR-mediated transcription represents the main mechanism for HIV-1 to maintain latency [1][2][3]6,8,9]. Identification of host factors/pathways that modulate LTR activity and viral latency helps develop new antiretroviral therapies. In this study, by targeting GSK3 for inhibition with small molecule agonists 6-BIO and LiCl, we provide evidence that activation of β-catenin/ TCF-1 pathway reactivates HIV-1 from latently infected CD4 + T cells. Given 6-BIO has been proposed PBMCs (1×10 7 ) isolated from cART-suppressed HIV-1 patients were treated with 6-BIO (1 μM) or PHA-P (5 μg/ml) for 5 days, viral reactivation was measured by quantifying the percentage of Env + CD4 + T cells (A, B), or by quantifying the production of intracellular gag or tat-rev mRNAs, and the enrichment fold for viral reactivation relative to medium treatment was calculated (C). (D, E) 6-BIO promotes β-catenin nuclear translocation in PBMCs from cART-suppressed HIV-1 patients. The PBMCs from 2 representative donors were harvested for subjecting immunoblotting, to detect the total levels and protein expression and phosphorylation (E), and to determine nuclear and cytoplasmic levels of β-catenin and TCF1. *P <0.05 and ****P <0.001 denote significant difference.
for treatments of a wide range of diseases [34][35][36][37], it provides a potent LRA candidate that can be used in combination with cell immunotherapies or antiretroviral drugs in the "Shock and Kill" strategy to eradicate HIV reservoirs.
The canonical Wnt/β-catenin pathway is evolutionarily conserved and plays important roles in stem cell proliferation and pluripotency, neurodevelopment, embryonic development, and tumorigenesis [38][39][40][41]. Dysregulation of Wnt/β-catenin signalling pathway has been associated with cancers and neurodegenerative diseases, and small molecules targeting Wnt/β-catenin pathway have been tested as potential drugs for treating these diseases [41][42][43][44]. Of note, Wnt/β-catenin signalling modulates the proliferation of CD4 + T cells that form longlived HIV-1 reservoir. Blockade of the interaction between β-catenin and co-activator CREB-binding protein, by a Wnt/β-catenin pathway small molecule inhibitor PRI-724, decreases the proliferation of Figure 7. 6-BIO reverses SIV latency in rhesus macaques. Four Chinese rhesus macaques were challenged with 5000TCID 50 amounts of SIV mac239 intravenously via saphenous vein, and animals then were treated with FNC (0.4 mg/kg), followed with intravenous infusions of 0.4 mg/kg 6-BIO every two days (A). (B) Viral reactivation was measured by defining the production of both plasma and cell-associated gag mRNAs. (C) Non-specific activation assay. PBMCs were isolated at the time point of pre-(day 0) and post-(day 8) treatment with 6-BIO, and cells were immunostaining and the surface expressions of CD25 and/or HLD-DR on the CD4 + T-lymphocytes, CD8 + T-lymphocytes, and CD14 + monocytes were analysed with flow cytometry. (D) The plasma cell numbers of CD4 + -and CD8 + -T-lymphocytes were longitudinally monitored using BD TruCount tubes. *P <0.05 denotes significant difference.
stem cell memory-and central memory-CD4 + T in ART-suppressed SIV mac251 -infected rhesus macaques, but unable to not significantly reduce the viral reservoir size [45].
Transcriptome data also confirm 6-BIO-mediated activation of β-catenin/TCF1 signalling in CD4 + T cells ACH2. The core DEGs related to the regulation of inflammation and cytokine signalling mainly included CCR1, IL-10, IL12RB2, IL18RAP, IL2RA, TNFSF8, etc. Non-specific activation of host cells can provide more susceptible target cells for HIV-1 infection [33]. It seems that 6-BIO does not induce the non-specific activation of these HIV-1 target cells, at least in our study.
Our study has some limitations. 6-BIO evaluation was performed in the limited number of 4 SIVinfected monkeys; the 6-BIO reactivation was started right after the SIV suppression in monkeys, and a longer duration for SIV suppression is more appropriate in reflecting HIV-1 latency in the long-term cARTsuppressed patients. Besides of the monkey/SIV models, the humanized mouse models provide alternative tools for fully evaluating 6-BIO activity.
Taken together, by using HIV-1 latently infected CD4 + T cell lines, resting CD4 + T cells from cART-suppressed patients and SIV-infected rhesus macaques, we demonstrate that the pharmacological suppression GSK3 kinase activity by 6-BIO reactivates HIV-1 from latently infected CD4 + T cells. These findings advance our understanding of the mechanisms responsible for viral latency, and provide the potent LRA that can be further used in conjunction of immunotherapies to eradicate viral reservoirs.