Antiviral effects of IFIT1 in human cytomegalovirus-infected fetal astrocytes.

The prominent feature of human cytomegalovirus (HCMV) is cell tropism specificity for human fetal nervous system, which leads to severe fetal nervous system damage especially in first-trimester gestation. In this study, human astrocytes isolated from fetal brain were infected with HCMV AD169 and whole genome transcriptome profile was performed. The results showed that the gene expression of interferon stimulated genes (ISGs), chemokine and chemokine receptors were significantly up-regulated (P < 0.01). The antiviral replication effects of IFIT1 (Interferon-induced protein with tetratricopeptide repeats 1, Fc = 148.17) was investigated. Lentivirus with IFIT1 overexpression or knockdown was transduced into astrocytes, respectively. The viral mRNA, protein expression and HCMV titers were determined. The results showed that IE1, IE2, pp65, and viral titers were significantly decreased in IFIT1 overexpression group and enhanced in the knockdown group compared with control one (P < 0.01). Taken together, this study revealed IFIT1 played an important antiviral role in HCMV infected fetal astrocytes. The prominent feature of human cytomegalovirus (HCMV) is cellular tropism specificity for human fetal brain nervous system leading to severe fetal nervous damage especially in first-trimester gestation. In this study, human astrocytes isolated from first-trimester fetal brain were infected with HCMV AD169 and IFIT1 was studied for its antiviral replication effects. The results provided insights into the function of IFIT1 as a key factor in antiviral defense contributing to development of targeted therapeutics to fetal brain with HCMV infection. J. Med. Virol. 89:672-684, 2017. © 2016 Wiley Periodicals, Inc.

DNA methylation level of promoter region of activating transcription factor 5 in glioma.

Transcription factors, which represent an important class of proteins that play key roles in controlling cellular proliferation and cell cycle modulation, are attractive targets for cancer therapy. Previous researches have shown that the expression level of activating transcription factor 5 (ATF5) was frequently increased in glioma and its acetylation level was related to glioma. The purposes of this study were to explore the methylation level of ATF5 in clinical glioma tissues and to explore the effect of ATF5 methylation on the expression of ATF5 in glioma. Methylation of the promoter region of ATF5 was assayed by bisulfite-specific polymerase chain reaction (PCR) sequencing analysis in 35 cases of glioma and 5 normal tissues. Quantitative real-time PCR (qRT-PCR) was also performed to detect ATF5 mRNA expression in 35 cases of glioma and 5 normal tissues. Clinical data were collected from the patients and analyzed. The percentages of methylation of the ATF5 gene in the promoter region in healthy control, patients with well-differentiated glioma, and those with poorly differentiated glioma were 87.78%, 73.89%, and 47.70%, respectively. Analysis of the methylation status of the promoter region of the ATF5 gene showed a gradually decreased methylation level in poorly differentiated glioma, well-differentiated glioma, and normal tissues (P<0.05). There was also a significant difference between well-differentiated glioma and poorly differentiated glioma (P<0.05). ATF5 mRNA expression in glioma was significantly higher than that in the normal tissues (P<0.05). This study provides the first evidence that the methylation level of ATF5 decreased, and its mRNA expression was evidently up-regulated in glioma.

HCMV induces dysregulation of glutamate uptake and transporter expression in human fetal astrocytes.

Human cytomegalovirus (HCMV) infections are the leading cause of viral induced birth defects, affecting the central nervous system (CNS) primarily. Fetal CNS is especially vulnerable to CMV induced injury. As HCMV permissive cells, astrocytes are responsible for major glutamate transport and regulate extracellular levels of glutamate avoiding its accumulation which is implicated in neurodegenerative disorders. In this study, highly purified astrocytes isolated from human first trimester aborted fetal brain were infected with HCMV AD169, glutamate uptake function was detected by (3)H labeling technic, and the expression level alterations of glutamate transporters (GLAST, GLT-1), glutamine synthetase (GS) and its activity were also investigated. Protein kinases C (PKC) inhibitor treatment was to identify whether PKC signalling involved in regulating glutamate uptake, protein expression of GLAST, GLT-1, GS and GS activity. Results indicated HCMV AD169 infection could modulate glutamate uptake, expression levels of GLAST, GLT-1, GS and it activity through PKC signalling, suggesting a great susceptibility of human fetal astrocytes to HCMV infection, which significantly alters the uptake and metabolism of an important excitatory amino acid, glutamate, may be a potential mechanism for HCMV associated neurological disease, and an effective therapeutic target in neural diseases.

Human cytomegalovirus infection modulates thrombospondins 1 and 2 in primary fetal astrocytes.

Transmission of human cytomegalovirus (HCMV) to the fetus is the most common type of intrauterine infection; the mechanism of HCMV pathogenesis in the developing central nervous system remains unclear. Thrombospondins 1 and 2 (TSP1, TSP2) produced by immature astrocytes are critical for fetal synaptogenesis. To examine the effect of HCMV on fetal astrocytes, human fetal astrocytes were isolated and cultured with HCMV AD169. Cells were harvested at different time points. Protein and mRNA expressions of TSP1 and TSP2 were determined using RT-qPCR, western blotting analysis, and enzyme-linked immunosorbent assay. The results showed that HCMV infection induced time-dependent decreases in mRNA and protein expressions of both TSP1 and TSP2 in astrocytes. Flow cytometry was used to detect apoptosis of HCMV-infected astrocytes, and the result indicated that there was no linkage between cell apoptosis and the decrease in TSP1 and TSP2 expressions induced by HCMV infection. When ganciclovir treatment was performed on HCMV-infected astrocytes, results showed that ganciclovir treatment inhibited the reduction of TSP1 and TSP2 expression in astrocytes. In the further study, pEGFP-N3-IE1 was transfected into astrocytes to identify that it was not IE1 but active viral replication that was essential in the continuous decrease of TSP1 and TSP2 expressions in HCMV-infected astrocytes.

Inhibitory effects of Ginsenoside Rb1 on apoptosis caused by HSV-1 in human glioma cells.

To investigate the inhibitory effects of Ginsenoside Rb1 (GRb1) on apoptosis caused by Herpes Simplex Virus-1(HSV-1) in Human Glioma Cells (U251), U251 cells were infected by HSV-1 at a multiplicity of infection of 5 and GRb1, GRb1+HSV-1, HSV-1 and control groups. MTT and cell apoptosis assays were used to detect the inhibitory effects of GRb1 on the apoptosis of U251 cells that caused by HSV-1 infection for various concentrations of drug and virus treatments by MTT assay. We found that in the 400 µg/mL GRb1 and 400 µg/mL GRb1+HSV-1 groups, MTT values were higher than control group at all times (P<0. 05). Moreover, the apoptosis rate in the 400 µg/mL GRb1+HSV-1 group was lower than the HSV-1 group (P<0. 05). These results confirmed that, at appropriate concentrations, GRb1 could inhibit nerve cell apoptosis in HSV-1 infections.

[Effect of HSV-1 infection on NGF and its receptor expression in human glioma cells].

Nerve growth factor (NGF) is mainly secreted by the neuroglia cells, which can exert biological effect through its receptors on the specific target cell surface. NGF is closely related to neurocyte growth, differentiation and apoptosis. As a neurotropic virus, HSV-1 an easily lead to neurocyte, neuroglia cells death or apoptosis. In this study, the U251 human glioma cells were chosen as target cells to study the change of NGF and its receptors in the apoptosis process of HSV-1 infection. Our results showed that U251 cells were permissive to HSV-1 replication. In the apoptosis process of HSV-1 infected U251 cells, the expression of both NGF and P75NTR increased and then decreased, while the expression of TrkA decreased gradually. These result indicated that HSV-1 was able to induce the abnormal expression of NGF and its receptors in U251 cells.

Effect of human cytomegalovirus infection on nerve growth factor expression in human glioma U251 cells.

OBJECTIVE: To explore the change of endogenic nerve growth factor (NGF) expression in human glioma cells infected with human cytomegalovirus (HCMV). METHODS: U251 cells were cultured in RPMI 1640 culture medium and infected with HCMV AD169 strain in vitro to establish a cell model of viral infection. Morphologic changes of U251 cells were observed under inverted microscope before and after infection with HCMV. Expression of NGF gene and protein of cells was detected by RT-PCR and Western blotting before and after infection with HCMV. RESULTS: The cytopathic effects of HCMV-infected cells appeared on day 5 after infection. However, differential NGF expression was evident on day 7. NGF expression was decreased significantly in U251 cells on day 7 after infection in comparison with control group (P < 0.05). CONCLUSION: HCMV can down-regulate endogenous NGF levels in human glioma cell line U251.

[The expression and anti-apoptotic function of HCMV IE2 protein controlled by Tet-On system].

During the infection of host cells, IE2 protein is one of the first and most abundantly expressed products of HCMV genome, which plays an important role in the controlling of cell cycle and apoptosis. But the correlation between expression level and anti-apoptotic activity of IE2 protein is still not clear. In this study, we successfully established a HCMV IE2 protein expression cell line that was controlled by Tet-On system. The effect of IE2 protein on cell apoptosis and the expression of p53 was detected under different condition of induction. Our results showed that the IE2 protein could inhibit cell apoptosis induced by TNF-alpha. Additionally, the anti-apoptotic activity of IE2 protein seemed to be relevant to its expression level. However, we failed to detect any difference of p53 expression between the IE2 protein expression and non-expression cells. These data indicated that the IE2 protein might inhibit cell apoptosis through regulating different signal pathways.

Isolation of a cDNA encoding a protease from Perinereis aibuhitensis Grube.

The cDNA encoding a protease of Perinereis aibuhitensis Grube (PPA) was cloned. The deduced amino acid sequence analysis showed that the protein had 49% identity to the C-terminal amino acid 169-246 of serine protease of Heterodera glycines. Northern blotting analysis indicated that the cDNA could hybridize with mRNA of approximately 260 bases isolated from the marine earthworm. The cDNA was amplified by polymerase chain reaction and cloned into pMAL-p2 to construct expression vector pMAL-PPA. pMAL-PPA was introduced into Escherichia coli BL21(DE3) and overexpression of PPA fused with maltose binding protein was achieved by isopropyl-beta-D-thiogalactopyranoside induction. The fusion protein was purified by affinity chromatography on an amylose resin column and ion-exchange chromatography on a diethylaminoethyl-Sepharose 4B column. Rabbits were immunized with the purified protein and antiserum was prepared. The antibody could react with a protein of approximately 9 kDa extracted from the marine earthworm as shown by Western blotting analysis. The activity analysis of the recombinant PPA suggested that it was probably a plasminogen activator.