Akt Phosphorylation of Hepatitis C Virus NS5B Regulates Polymerase Activity and Hepatitis C Virus Infection

Hepatitis C virus (HCV) is a single-stranded RNA virus of positive polarity [ssRNA(+)] that replicates its genome through the activity of one of its proteins, called NS5B. This viral protein is responsible for copying the positive-polarity RNA genome into a negative-polarity RNA strand, which will be the template for new positive-polarity RNA genomes. The NS5B protein is phosphorylated by cellular kinases, including Akt. In this work, we have identified several amino acids of NS5B that are phosphorylated by Akt, with positions S27, T53, T267, and S282 giving the most robust results. Site-directed mutagenesis of these residues to mimic (Glu mutants) or prevent (Ala mutants) their phosphorylation resulted in a reduced NS5B in vitro RNA polymerase activity, except for the T267E mutant, the only non-conserved position of all those that are phosphorylated. In addition, in vitro transcribed RNAs derived from HCV complete infectious clones carrying mutations T53E/A and S282E/A were transfected in Huh-7.5 permissive cells, and supernatant viral titers were measured at 6 and 15 days post-transfection. No virus was rescued from the mutants except for T53A at 15 days post-transfection whose viral titer was statistically lower as compared to the wild type. Therefore, phosphorylation of NS5B by cellular kinases is a mechanism of viral polymerase inactivation. Whether this inactivation is a consequence of interaction with cellular kinases or a way to generate inactive NS5B that may have other functions are questions that need further experimental work.

Roles of N-linked and O-linked glycosylation sites in the activity of equine chorionic gonadotropin in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor

Background Equine chorionic gonadotropin (eCG), which comprises highly glycosylated α-subunit and β-subunit, is a unique member of the glycoprotein hormone family as it elicits both follicle-stimulating hormone (FSH)-like and luteinizing hormone (LH)-like responses in non-equid species. To examine the biological function of glycosylated sites in eCG, the following glycosylation site mutants were constructed: eCGβ/αΔ56, substitution of Asn56 of α-subunit with Gln; eCGβ-D/α, deletion of the O-linked glycosylation site at the carboxyl-terminal peptide (CTP) region of the β-subunit; eCGβ-D/αΔ56, double mutant. The recombinant eCG (rec-eCG) mutants were expressed in Chinese hamster ovary suspension (CHO-S) cells. The FSH-like and LH-like activities of the mutants were examined using CHO-K1 cells expressing rat lutropin/CG receptor (rLH/CGR) and rat FSH receptor (rFSHR). Results Both rec-eCGβ/α and rec-eCGβ/αΔ56 were efficiently secreted into the CHO-S cell culture medium on day 1 post-transfection. However, the secretion of eCGβ-D/α and eCGβ-D/αΔ56, which lack approximately 12 O-linked glycosylation sites, was slightly delayed. The expression levels of all mutants were similar (200–250 mIU/mL) from days 3 to 7 post-transfection. The molecular weight of rec-eCGβ/α, rec-eCGβ/αΔ56 and rec-eCG β-D/α were in the ranges of 40–45, 37–42, and 34–36 kDa, respectively. Treatment with peptide-N-glycanase F markedly decreased the molecular weight to approximately 5–10 kDa. Rec-eCGβ/αΔ56 exhibited markedly downregulated LH-like activity. The signal transduction activity of both double mutants was completely impaired. This indicated that the glycosylation site at Asn56 of the α-subunit plays a pivotal role in the LH-like activity of eCG. Similarly, the FSH-like activity of the mutants was markedly downregulated. eCGβ-D/α exhibited markedly downregulated LH-like and FSH-like activities. Conclusions Rec-eCGβ/α exhibits potent biological activity in cells expressing rLH/CGR and rFSHR. The findings of this study suggest that the LH-like and FSH-like activities of eCG are regulated by the N-linked glycosylation site at Asn56 of the eCG α-subunit and/or by the O-linked glycosylation sites of the eCG β-subunit. These findings improved our understanding of the mechanisms underlying both LH-like and FSH-like activities of eCG.

CRISPR/Cas9 Ribonucleoprotein-Based Genome Editing Methodology in the Marine Protozoan Parasite Perkinsus marinus

Perkinsus marinus (Perkinsozoa), a close relative of apicomplexans, is an osmotrophic facultative intracellular marine protozoan parasite responsible for “Dermo” disease in oysters and clams. Although there is no clinical evidence of this parasite infecting humans, HLA-DR40 transgenic mice studies strongly suggest the parasite as a natural adjuvant in oral vaccines. P. marinus is being developed as a heterologous gene expression platform for pathogens of medical and veterinary relevance and a novel platform for delivering vaccines. We previously reported the transient expression of two rodent malaria genes Plasmodium berghei HAP2 and MSP8. In this study, we optimized the original electroporation-based protocol to establish a stable heterologous expression method. Using 20 μg of pPmMOE[MOE1]:GFP and 25.0 × 106P. marinus cells resulted in 98% GFP-positive cells. Furthermore, using the optimized protocol, we report for the first time the successful knock-in of GFP at the C-terminus of the PmMOE1 using ribonucleoprotein (RNP)-based CRISPR/Cas9 gene editing methodology. The GFP was expressed 18 h post-transfection, and expression was observed for 8 months post-transfection, making it a robust and stable knock-in system.

Hepatitis B Virus-Like Particle: Targeted Delivery of Plasmid Expressing Short Hairpin RNA for Silencing the Bcl-2 Gene in Cervical Cancer Cells

Gene therapy research has advanced to clinical trials, but it is hampered by unstable nucleic acids packaged inside carriers and there is a lack of specificity towards targeted sites in the body. This study aims to address gene therapy limitations by encapsidating a plasmid synthesizing a short hairpin RNA (shRNA) that targets the anti-apoptotic Bcl-2 gene using truncated hepatitis B core antigen (tHBcAg) virus-like particle (VLP). A shRNA sequence targeting anti-apoptotic Bcl-2 was synthesized and cloned into the pSilencer 2.0-U6 vector. The recombinant plasmid, namely PshRNA, was encapsidated inside tHBcAg VLP and conjugated with folic acid (FA) to produce FA-tHBcAg-PshRNA VLP. Electron microscopy revealed that the FA-tHBcAg-PshRNA VLP has an icosahedral structure that is similar to the unmodified tHBcAg VLP. Delivery of FA-tHBcAg-PshRNA VLP into HeLa cells overexpressing the folate receptor significantly downregulated the expression of anti-apoptotic Bcl-2 at 48 and 72 h post-transfection. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay demonstrated that the cells’ viability was significantly reduced from 89.46% at 24 h to 64.52% and 60.63%, respectively, at 48 and 72 h post-transfection. As a conclusion, tHBcAg VLP can be used as a carrier for a receptor-mediated targeted delivery of a therapeutic plasmid encoding shRNA for gene silencing in cancer cells.

Improving the Intercellular Uptake and Osteogenic Potency of Calcium Phosphate via Nanocomplexation with the RALA Peptide

Calcium phosphate-base materials (e.g., alpha tri-calcium phosphate (α–TCP)) have been shown to promote osteogenic differentiation of stem/progenitor cells, enhance osteoblast osteogenic activity and mediate in vivo bone tissue formation. However, variable particle size and hydrophilicity of the calcium phosphate result in an extremely low bioavailability. Therefore, an effective delivery system is required that can encapsulate the calcium phosphate, improve cellular entry and, consequently, elicit a potent osteogenic response in osteoblasts. In this study, collagenous matrix deposition and extracellular matrix mineralization of osteoblast lineage cells were assessed to investigate osteogenesis following intracellular delivery of α-TCP nanoparticles. The nanoparticles were formed via condensation with a novel, cationic 30 mer amphipathic peptide (RALA). Nanoparticles prepared at a mass ratio of 5:1 demonstrated an average particle size of 43 nm with a zeta potential of +26 mV. The average particle size and zeta potential remained stable for up to 28 days at room temperature and across a range of temperatures (4–37 °C). Cell viability decreased 24 h post-transfection following RALA/α-TCP nanoparticle treatment; however, recovery ensued by Day 7. Immunocytochemistry staining for Type I collagen up to Day 21 post-transfection with RALA/α-TCP nanoparticles (NPs) in MG-63 cells exhibited a significant enhancement in collagen expression and deposition compared to an untreated control. Furthermore, in porcine mesenchymal stem cells (pMSCs), there was enhanced mineralization compared to α–TCP alone. Taken together these data demonstrate that internalization of RALA/α-TCP NPs elicits a potent osteogenic response in both MG-63 and pMSCs.

Influenza D pseudotyped lentiviruses: production, neutralisation assay and serological surveillance

Influenza D virus (IDV) has been reported in many animal species and potentially humans worldwide. Cattle are considered the major reservoir. There are currently three main lineages based on the haemagglutinin-esterase (HEF) gene: D/OK, D/660 and D/Japan. We performed pilot surveillance for IDV by using pseudotyped lentivirus (PVs) to generate a cell-based test to identify prior-exposure to IDV in animals. The expression plasmids of the HEF genes, D/swine/Italy/2015, D/bovine/France/2014, and D/bovine/Ibaraki/2016, were constructed. The HEF plasmid was co-transfected with lentiviral vector plasmid expressing luciferase, lentiviral Gag-Pol plasmid, and HAT protease plasmid in producer cells (HEK293T/17). Three days post-transfection, supernatants were collected and used for titration on various cell lines and in micro-neutralisation tests. Sera from pigs vaccinated with D/swine/Italy/2015 and D/swine/Oklahoma/2011 were used to undertake a preliminary validation of the micro-neutralisation assay. All pig sera have neutralising activity to influenza D (Italy) pseudotyped lentiviruses. Cow and sheep sera, 145 and 114 specimens, respectively, collected from UK farms were screened using the micro-neutralisation test. We found 97 bovine sera (66.9%) were influenza D antibody positive. Collectively, pseudotyped lentivirus technology opens up opportunities for serological surveillance of influenza D viruses.

MALAT1 inhibits the Wnt/β-catenin signaling pathway in colon cancer cells and affects cell proliferation and apoptosis

Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is a highly conserved long noncoding RNA, which has been related to various pathological processes, including cancer. The role and mechanism of MALAT1 in colon cancer are not clear. We investigated MALAT1 expression in colon cancer tissues, the effect of MALAT1 on proliferation and apoptosis of SW480 cells, and the signaling pathway involved in the MALAT1 effects. MALAT1 expression was determined in 60 colon cancer and para-carcinoma tissues using reverse transcription polymerase chain reaction (RT-PCR). Knockdown of MALAT1 in SW480 cells was induced by small interfering RNA (siRNA), and the cells were divided into three groups: untreated control, nonsense siRNA-treated control, and MALAT1 siRNA-treated group. SW480 cell apoptosis was assessed using TUNEL assay and flow cytometry. Apoptosis-related and Wnt/β-catenin signaling pathway-related proteins were detected by Western blotting in SW480 cells. SW480 cell proliferation was assessed by CCK-8 assay. MALAT1 expression was significantly higher in colon cancer vs. para-carcinoma tissues. Knockdown of MALAT1 by siRNA increased the number of apoptotic cells and the apoptosis rate at 24 h post-transfection in SW480 cells. Bcl2 associated X protein (Bax) expression was increased, B-cell lymphoma 2 (Bcl-2) expression was decreased, and the ratio of cleaved caspase-3 to truncated caspase-3 was increased in MALAT1 siRNA-transfected SW480 cells. MALAT1 knockdown decreased the proliferation of SW480 cells at 24 h, 48 h, and 72 h post-transfection. Wnt and β-catenin expression was inhibited in MALAT1 siRNA-transfected SW480 cells. Inhibition of MALAT1 expression in colon cancer may promote apoptosis and hinder cell proliferation by suppressing the activation of Wnt/β-catenin signaling pathway.

LncRNA UCA1/miR-143 miR-216b/HK2/MAPK signaling pathway is involved in the regulation of endothelial cell proliferation via the modulation of glycolysis in melanoma

Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs/miRs) are noncoding RNAs that function as regulators of tumor suppressors and oncogenes. The aim of the present study was to investigate the potential mechanism associated with the involvement of urothelial cancer associated 1 (UCA1) in melanoma. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed in order to determine the expression levels of UCA1, miR-143, miR-216b, and hexokinase 2 (HK2) in the melanoma and control groups, as well as the influence of UCA1, miR-143, and miR-216b on the expression of HK2, and the effect of lactate and UCA1 on the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK). Bioinformatics algorithm analysis and a luciferase assay were performed in order to predict miRNA targets. In addition, an MTT assay was performed in order to determine the effect of lactate and UCA1 expression on cell proliferation. A total of 39 participants, consisting of 18 patients with melanoma and 21 healthy control subjects, were included in the present study. The present study demonstrated that the expression levels of UCA1 mRNA, and HK2 mRNA and protein were enhanced in patients with melanoma compared with healthy controls; whereas the expression levels of miR-143 and miR-216b mRNA were suppressed in patients with melanoma compared with healthy controls. Furthermore, it was revealed that UCA1 negatively modulated the expression of miR-143 and miR-216b, and that miR-143 and miR-216b directly targeted the HK2 protein by binding to the HK2 3′ untranslated region (UTR). In addition, it was demonstrated that miR-143 and miR-216 suppressed the luciferase activity exhibited by wild-type HK2 3′-UTR. Furthermore, it was revealed that transfection with UCA1 small interfering RNA, and miR-143 and miR-216b mimics markedly suppressed HK2 mRNA and protein expression levels as well as lactate levels in human umbilical vein endothelial cells; however, O2 consumption was revealed to be enhanced post transfection. By contrast, transfection with UCA1 enhanced HK2 mRNA and protein expression levels as well as lactate production; however, O2 consumption was revealed to be suppressed post transfection. Lactate-induced phosphorylation of p38 MAPK was revealed to occur in a concentration-dependent manner, and UCA1 enhanced the phosphorylation level of p38 MAPK via the inhibition of miR-143 and miR-216b expression. Lactate and UCA1 were demonstrated to enhance cell proliferation. In conclusion, the present study demonstrated that the lncRNA UCA1/miR-143 miR-216b/HK2/lactic acid/MAPK axis may be involved in the pathogenesis of melanoma via the modulation of endothelial cells, and thus, lncRNA UCA1 may serve as a potential therapeutic target for melanoma treatment.

Enhanced transfection of a macromolecular lignin-based DNA complex with low cellular toxicity

Cationic polymers remain attractive tools for non-viral gene transfer. The effectiveness of these vectors rely on the ability to deliver plasmid DNA (pDNA) into the nucleus of cells. While we have previously demonstrated the potential of Lignin-PGEA-PEGMA as a non-viral gene delivery vector, alterations of cellular phenotype and cytotoxicity were observed post transfection. The present study aims to explore transfection conditions for high efficiency and low toxicity of the Lignin-PGEA-PEGMA based gene delivery system. Cellular toxicity was significantly reduced by using the centrifugation protocol, which enables rapid deposition of DNA complexes. Replacement of media post centrifugation resulted in minimal exposure of cells to excess polymers, which were toxic to cells. At an optimized DNA amount (500–750 ng) and molar ratios of nitrogen (N) in polymer to phosphate (P) in pDNA (N/P = 30–40), with the use of a novel transfection enhancer that facilitates endosomal escape and nuclear trafficking, the efficiency of gene delivery was increased significantly 24 h post transfection. The present study demonstrated an appropriately optimized protocol that enabled the utility of a novel cationic polymer blend with a mixture of fusogenic lipids and a histone deacetylate inhibitor in non-viral transfection, thereby providing an attractive alternative to costly commercial gene carriers.

Abstract 17202: Synthetic Modified RNA Driven Delivery of Insulin-Like Growth Factor-1 Promotes Early Cardiomyocyte Survival Post-Acute Myocardial Infarction

To extend the temporal window for cytoprotection in cardiomyocytes undergoing apoptosis after hypoxia and myocardial infarction (MI), a synthetic modified RNA (modRNA) was used to drive delivery of insulin-like growth factor-1 (IGF1) within the area at risk in a murine model of MI. Transient transfection of synthetic GFP modRNA as an tracking indicator, with polyethylenimine (PEI)-based nanoparticle, showed efficient delivery of modRNA derived protein and minimum cytotoxicity in HL-1 cardiomyocytes (CM; 44±5%). ModRNA-IGF1 protein expression and secreted levels increased 3.5 fold ( p <0.05) at 24 hours and peaked at 48 hours post-transfection. The expression efficiency of modRNA was further enhanced (~2 fold at 24 hours post-transfection; p <0.05) under hypoxia-induced apoptosis conditions. ModRNA augmented secreted and cell associated IGF1 promoting CM survival and abrogating cell apoptosis (71±5% in controls to 37±7%, p <0.05). Translation of modRNA-IGF1 was sufficient to induce downstream increases in Akt and Erk phosphorylation (3 fold and 2 fold, respectively; p <0.01). Downregulation of IGF1 specific miRNA-1 and -133 (52% and 56%, respectively; p <0.01) but not miR-145 expression, was also confirmed. As proof of concept, intramyocardial delivery of modRNA-IGF1 but not control modRNA-GFP significantly decreased in TUNEL-positive cells within the infarct border zone (BZ) at 24 hours (22±3% versus 53±8% in controls, p <0.01). Akt phosphorylation was augmented while caspase-9 activity and cleavage was downregulated in the infarct BZ compared to controls ( p <0.05). These findings demonstrate extended in vivo cytoprotective effect of IGF1 24 hours post-MI driven by synthetic modRNA delivery. This provides a novel strategy to reduce ischemic injury by controlled release of IGF1 using a controllable bioactive nanoparticle for ‘short burst’ IGF1 cytoprotective therapy.