Poly-ICLC, a TLR3 Agonist, Induces Transient Innate Immune Responses in Patients With Treated HIV-Infection: A Randomized Double-Blinded Placebo Controlled Trial

Objective: Toll-like receptor-3 agonist Poly-ICLC has been known to activate immune cells and induce HIV replication in pre-clinical experiments. In this study we investigated if Poly-ICLC could be used for disrupting HIV latency while simultaneously enhancing innate immune responses.Design: This was a randomized, placebo-controlled, double-blinded trial in aviremic, cART-treated HIV-infected subjects. Participants (n = 15) were randomized 3:1 to receive two consecutive daily doses of Poly-ICLC (1.4 mg subcutaneously) vs. placebo. Subjects were observed for adverse events, immune activation, and viral replication.Methods: Besides primary outcomes of safety and tolerability, several longitudinal immune parameters were evaluated including immune cell phenotype and function via flowcytometry, ELISA, and transcriptional profiling. PCR assays for plasma HIV-1 RNA, CD4+ T cell-associated HIV-1 RNA, and proviral DNA were performed to measure HIV reservoirs and latency.Results: Poly-ICLC was overall safe and well-tolerated. Poly-ICLC-related adverse events were Grade 1/2, with the exception of one Grade 3 neutropenia which was short-lived. Mild Injection site reactions were observed in nearly all participants in the Poly-ICLC arm. Transcriptional analyses revealed upregulation of innate immune pathways in PBMCs following Poly-ICLC treatment, including strong interferon signaling accompanied by transient increases in circulating IP-10 (CXCL10) levels. These responses generally peaked by 24–48 h after the first injection and returned to baseline by day 8. CD4+ T cell number and phenotype were unchanged, plasma viral control was maintained and no significant effect on HIV reservoirs was observed.Conclusions: These finding suggest that Poly-ICLC could be safely used for inducing transient innate immune responses in treated HIV+ subjects indicating promise as an adjuvant for HIV therapeutic vaccines.Trial Registration:www.ClinicalTrials.gov, identifier: NCT02071095

Hsa-mir183/EGR1-mediated regulation of E2F1 is required for CML stem/progenitor cell survival

Chronic myeloid leukemia (CML) stem/progenitor cells (SPC) express a transcriptional program characteristic of proliferation, yet can achieve and maintain quiescence. Understanding the mechanisms by which leukemic SPC maintain quiescence will help to clarify how they persist during long-term targeted treatment. We have identified a novel BCR-ABL1 protein kinase dependent pathway mediated by the up-regulation of hsa-mir183, the down-regulation of its direct target EGR1 and, as a consequence, up-regulation of E2F1. We show here that inhibition of hsa-mir183 reduced proliferation and impaired colony formation of CML SPC. Downstream of this, inhibition of E2F1 also reduced proliferation of CML SPC, leading to p53-mediated apoptosis. In addition, we demonstrate that E2F1 plays a pivotal role in regulating CML SPC proliferation status. Thus, for the first time, we highlight the mechanism of hsa-mir183/EGR1-mediated E2F1 regulation and demonstrate this axis as a novel, critical factor for CML SPC survival, offering new insights into leukemic stem cell eradication

The replication initiator protein of a geminivirus interacts with host monoubiquitination machinery and stimulates transcription of the viral genome

<div><p>Geminiviruses constitute a group of plant viruses, with a ssDNA genome, whose replication in the nucleus of an infected cell requires the function of geminivirus-encoded replication initiator protein (Rep). Our results suggest that monoubiquitinated histone 2B (H2B-ub) promotes tri-methylation of histone 3 at lysine 4 (H3-K4me3) on the promoter of Chilli leaf curl virus (ChiLCV). We isolated homologues of two major components of the monoubiquitination machinery: UBIQUITIN-CONJUGATING ENZYME2 (<i>NbUBC2)</i> and HISTONE MONOUBIQUITINATION1 (<i>NbHUB1</i>) from <i>N</i>. <i>benthamiana</i>. ChiLCV failed to cause disease in <i>NbUBC2</i>-, and <i>NbHUB1</i>-silenced plants, at the same time, H2B-ub and H3-K4me3 modifications were decreased, and the occupancy of RNA polymerase II on the viral promoter was reduced as well. In further investigations, Rep protein of ChiLCV was found to re-localize NbUBC2 from the cytoplasm to the nucleoplasm, like NbHUB1, the cognate partner of NbUBC2. Rep was observed to interact and co-localize with NbHUB1 and NbUBC2 in the nuclei of the infected cells. In summary, the current study reveals that the ChiLCV Rep protein binds the viral genome and interacts with NbUBC2 and NbHUB1 for the monoubiquitination of histone 2B that subsequently promotes trimethylation of histone 3 at lysine 4 on ChiLCV mini-chromosomes and enhances transcription of the viral genes.</p></div

Sub-cellular localization of Rep-EGFP in (A) pTRV-<i>NbHUB1</i> and (B) pTRV-<i>NbUBC2</i> silenced plants.

<p>DAPI is used as nuclear marker. Scale bar = 20μm.</p

Deposition of H2B-ub and H3-K4me3 on the ChiLCV genome.

<p><b>(A)</b> An <i>N</i>. <i>benthamiana</i> plant inoculated with the vector pCAMBIA2300. (B) Phenotype of a representative <i>N</i>. <i>benthamiana</i> plant inoculated with ChiLCV showing typical symptoms of leaf curl disease at 21 days post inoculation (dpi). (C) qPCR of viral DNA accumulation at different dpi. (D) Immunoblot analysis of global cellular H2B, H2B-ub, H3-K4me3 and H3-K27me3 levels in mock-, and virus-inoculated <i>N</i>. <i>benthamiana</i> plants at 7, 14 and 21 dpi. Immunoblotting was performed using anti-H3-K4me3, anti-H2B-ub, anti-H2B and anti-H3-K27me3 specific antibodies following standard protocol. (E) Schematic diagram of the circular genome of ChiLCV (2750 nt) indicating the relative positions of the viral ORFs and the position of the primers (red arrows) used for chromatin immunoprecipitation in the study. (F) Detection of occupancy of H3-K4me3, H2B-ub and H3-K27me3 on the <i>Actin</i> genic region by ChIP-PCR serves as control. (G-H) Detection of H3-K4me3, H2B-ub, H3-K27me3 and H2B on the promoter and C3 region of ChiLCV by ChIP-PCR at different time points following infection using anti-H2B, anti-H2B-ub, anti-H3-K4me3 and H3-K27me3 antibodies and primers specific to the ChiLCV promoter (2684–46 nt) and the C3 region (1020–1120 nt).</p

Sub-cellular localization and colocalization of Rep, NbHUB1, and NbUBC2 fluorescent fusion proteins in epidermal cells of <i>N</i>. <i>benthamiana</i> leaves.

<p>Sub- cellular localization of (A) Rep-DsRed, (B) NbHUB1-GFP (C) NbUBC2-DsRed, (D) Colocalization of Rep-DsRed and NbHUB1-GFP, (E) Colocalization of NbUBC2-DsRed and NbHUB1-GFP, (F) Colocalization of Rep-EGFPand NbUBC2-DsRed. DAPI is used as nuclear marker. Scale bar = 20μm (A-C, E), 200μm (D), and 50 μm (F). (G) Pearson’s correlation for colocalization was calculated using the NIS-Element 4.0 software. <a href="https://www.statisticssolutions.com/pearsons-correlation-coefficient/" target="_blank">Pearson's correlation coefficient</a> of NbUBC2-DsRed and Rep-EGFP (0.89), NbUBC2-DsRed and NbHUB1-GFP (0.80), Rep-DsRed and NbHUB1-GFP (0.96) are mentioned. (H) Graphical representation of percentage of cells showing NbUBC2-DsRed localization alone (n = 150) and in the presence of either NbHUB1-GFP (n = 124) or Rep-EGFP (n = 90). ‘n’ denotes total number of cells observed.</p

Silencing of <i>NbUBC2</i> and <i>NbHUB1</i> affected ChiLCV pathogenesis and deposition of H2B-ub and H3-K4me3 on the viral genome.

<p>(A-G) Phenotype of <i>N</i>. <i>benthamiana</i> plants infiltrated with pTRV-<i>NtPDS</i>, empty vector (pTRV: 00), with or without ChiLCV inoculated, pTRV-<i>NbUBC2</i>, pTRV-<i>NbHUB1</i> plants at 21 dpi. (H) Detection of <i>NbHUB1</i> specific siRNAs in three independent silenced plants (P1, P2, P3) of <i>N</i>. <i>benthamiana</i>. (I) Detection of <i>NbUBC2</i> specific siRNA in three different silenced plants of <i>N</i>. <i>benthamiana</i>. (J) Symptom severity graph showing disease development of ChiLCV on pTRV:00, <i>NbUBC2-</i>, and <i>NbHUB1</i>-silenced <i>N</i>. <i>benthamiana</i> plants. (K-L) Relative level of ChiLCV DNA accumulation in either mock-, or virus-inoculated silenced plants by Southern blot and qPCR assays. Total plant DNA loaded is indicated below the blot (K). The statistical significance between the mean values was calculated by <i>t</i>-test (***p<0.001, **p<0.01). (M) Expression analysis of C2 transcripts level in pTRV:00, <i>NBUBC2</i>-and<i>NbHUB1</i>-silenced plants inoculated with ChiLCV (21 dpi). The statistical significance of the differences between the mean values were calculated by performing <i>t</i>-test (***p<0.001, **p<0.01). (N) An analysis of the viral titer in the absence (WT) or in the presence of pTRV infiltrated <i>N</i>. <i>benthamiana</i> plants inoculated with ChiLCV at 21 dpi. (O) Detection of H3-K4me3 and H2B-ub in <i>NbUBC2</i>, and <i>NbHUB1</i> silenced <i>N</i>. <i>benthamiana</i> plants by immunoblotting assays.</p

Rep protein neither gets ubiquitinated nor influences global histone modifications.

<p>(A) Detection of Rep-EGFP ubiquitinationin vivo. Immunoprecipitation was carried out from the total nuclear proteins using anti-GFP specific antibody at 7 dpi followed by immunoblotting with an anti-ubiquitin specific-antibody. Immunoblotting with anti-GFP specific antibody serves as input. (B) In vivo ubiquitination assay of H2B. Immunoprecipitation of ubiquitinated H2B using anti-ubiquitin specific antibody and immunoblotting was carried out with anti-H2B specific antibody. Immunoblotting of total nuclear proteins with H2B served as input control whereas human H2B served as control of unmodified H2B. (C) The effect of Rep protein on the total cellular histone modifications. Immunoblotting was performed using anti-H2B, anti-H2B-ub, and anti-H3-K4me3 specific antibodies from the total histone-enriched nuclear proteins isolated from EGFP-, and Rep-EGFP-infiltrated leaves. (D) Quantitative graphical representation of total cellular histone modifications. (E) Rep protein binds to ChiLCV DNA unlike host DNA. ChIP-PCR assays were carried out using the anti-GFP specific antibody and viral promoter specific primers. pCAMBIA2300, Rep-EGFP, EGFP with ChiLCV, and Rep-EGFP with ChiLCV-infiltrated leaves were subjected to ChIP-PCR analysis. The sample processed without antibody was considered as a control. Actin is considered as negative control.</p

ChiLCV induces formation of punctate bodies consisting of Rep in the nucleus.

<p>(A-F) Colocalization of Rep-DsRed and NbHUB1-GFP in presence of ChiLCV at 7 dpi. Scale bar = 2μm. (G) Magnified view of the nucleus. Closer examination of punctate bodies indicated “beads-on-string” like organization and the number of “beads” varied from two to seven. (H) Enlarged picture of one of the representative punctate bodies consisting of seven beads organized in a circular manner. The punctate bodies were organized as “beads-on-string”-like structures that measured as approximately 400 nm in diameter and the average size of the beads was calculated to be ~100 nm. The brightness and contrast of the image were adjusted by using Adobe Photoshop (<a href="http://www.adobe.com/in/products/photoshop" target="_blank">www.adobe.com/in/products/photoshop</a>) in order to improve image clarity. (I) Rep-EGFP with Rep-DsRed, (J) Rep-EGFP and Rep-DsRed organized as punctate structures in presence of ChiLCV, (K) Rep_1-120-DsRed failed to form punctate bodies in presence of ChiLCV and (L) Rep_1-180-DsRed derived punctate bodies in presence of ChiLCV.</p

Rep interacts with NbHUB1 and NbUBC2 in vivo.

<p>In planta bimolecular fluorescence complementation assays were performed in the lower epidermis of <i>N</i>. <i>benthamiana</i> leaves (at 5 dpi). NbHUB1 and NbUBC2 were expressed as in-frame fusion with the N-terminal of the YFP protein using the pSPYNE vector. The Rep protein was expressed with C-terminal region of the YFP protein using pSPYCE vector. BiFC assay of interaction of (A) pSPYNE-NbHUB1 and pSPYCE-Rep, (B) pSPYNE-NbUBC2 and pSPYCE-Rep, (C) pSPYNE-NbHUB1 and pSPYCE-NbH2B. (D) pSPYNE-NbHUB1 / pSPYCE, (E) pSPYNE-NbUBC2 / pSPYCE, (F) pSPYCE-Rep / pSPYNE and (G) pSPYNE / pSPYCE serve as control. Scale bar = 100μm. (H) The protein–protein interaction was also monitored by FRET microscopy in the epidermal cells of <i>N</i>. <i>benthamiana</i> leaves coexpressing EGFP and DsRed, Rep-EGFP and Rep-DsRed, NbHUB1-GFP and Rep-DsRed, Rep-EGFP and NbUBC2-DsRed. Representative acceptor photobleaching images show EGFP (donor) and DsRed (acceptor) channels before and after bleaching. After bleaching, DsRed fluorescence decreases in the bleached are as indicated by rectangles/circles. The FRET profiles showed the proximity between the donor and acceptor molecules and the efficiency of FRET. (I) Graph represents the quantification of FRET efficiency (E_F) of three independent experiments. (J) Yeast two-hybrid assay of Rep and NbHUB1, Rep and NbUBC2 on non selective media (-Leu / -Trp) and selective media (-His / -Leu / -Trp with 2.5 mM 3-AT). β galactosidase activity were checked for each combination and corresponding negative controls._P53 and TAg served as positive control. (K) Schematic diagram of deletion mutants of Rep protein used in yeast two-hybrid assays. (L) Yeast two-hybrid and β-galactosidase assays of in vivo interaction between deletion mutants of Rep protein and TrAP with NbHUB1 and NbUBC2.</p