Replication of Oral BK Virus in Human Salivary Gland Cells

BK polyomavirus (BKPyV) is the most common viral pathogen among allograft patients. Increasing evidence links BKPyV to the human oral compartment and to HIV-associated salivary gland disease (HIVSGD). To date, few studies have analyzed orally derived BKPyV. This study aimed to characterize BKPyV isolated from throat wash (TW) samples from HIVSGD patients. The replication potential of HIVSGD-derived clinical isolates HIVSGD-1 and HIVSGD-2, both containing the noncoding control region (NCCR) architecture OPQPQQS, were assessed and compared to urine-derived virus. The BKPyV isolates displayed significant variation in replication potential. Whole-genome alignment of the two isolates revealed three nucleotide differences that were analyzed for a potential effect on the viral life cycle. Analysis revealed a negligible difference in NCCR promoter activity despite sequence variation and emphasized the importance of functional T antigen (Tag) for efficient replication. HIVSGD-1 encoded full-length Tag, underwent productive infection in both human salivary gland cells and kidney cells, and expressed viral DNA and Tag protein. Additionally, HIVSGD-1 generated DNase-resistant particles and by far surpassed the replication potential of the kidney-derived isolate in HSG cells. HIVSGD-2 encoded a truncated form of Tag and replicated much less efficiently. Quantitation of infectious virus, via the fluorescent forming unit assay, suggested that HIVSGD BKPyV had preferential tropism for salivary gland cells over kidney cells. Similarly, the results suggested that kidney-derived virus had preferential tropism for kidney cells over salivary gland cells. Evidence of HIVSGD-derived BKPyV oral tropism and adept viral replication in human salivary gland cells corroborated the potential link between HIVSGD pathogenesis and BKPyV.     

Transformation of Human Embryonic Kidney Cells by a Viable Deletion Mutant of BK Virus

Infection by a viable deletion mutant of BK virus (pm-522) of human embryonic kidney (HEK) cultures originating from different fetuses generated cell lines, designated pmHEK, resistant to superinfection by the virus. However, infection of HEK cultures by a cloned wild-type BK virus (wt-501) did not generate a cell line. In medium with 10% calf serum all pmHEK cells reached saturation densities significantly higher than those of HEK cells and could grow in medium containing 1% serum. They did not form colonies in soft-agar medium, and had limited life-spans greatly extended beyond that of HEK cells. These results suggest that pmHEK cells are partially transformed. T antigen was uniformly expressed by all pmHEK cells, while V antigen was present in only a small minority of the cells. Thirty to 5,000 copies of the viral DNA per cell were detected, primarily in a nonintegrated form, in all pmHEK cell lines and the clones isolated from one of them. Since at least in two pmHEK lines, the actual quantities of free viral DNA per cell were sig-nificantly greater than those estimated on the basis of the assumption that free viral DNA was produced only by the V-antigen-positive cells, it can be concluded that free viral DNA is also present in the V-antigen-negative cells predominant in the cell populations. Although the existence of a few copies of integrated viral sequences in pmHEK cells was not ruled out, the large amount of free viral DNA present in each cell probably plays a prominent role in the production of T antigen required for maintenance of transformation.     

Stage-Specific Regulation of Reprogramming to Induced Pluripotent Stem Cells by Wnt Signaling and T Cell Factor Proteins

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转录因子HBP1抑制骨肉瘤细胞增殖

转录因子HBP1(HMG-box containing protein 1, HBP1)属HMG家族,是1个含有513个氨基酸残基的多肽,可与RB蛋白结合,抑制许多癌基因的表达,从而抑制细胞的增殖. 本工作构建了HBP1慢病毒表达载体,病毒包装后感染骨肉瘤细胞U2OS细胞.Western印迹结果显示,感染细胞cyclin D1、c-Myc蛋白质水平降低 ,而p53蛋白质水平增加;Real-time PCR检测cyclin D1、c-Myc及p53 mRNA水平与蛋白质检测结果一致. HBP1表达载体（pEFBOS-HBP1）和报告基因载体（含 cyclin D1、c-Myc或p53 promoter）共转染U2OS细胞后的荧光素酶分析发现,HBP1可抑制cyclin D1、c-Myc启动子转录激活,促进p53启动子转录激活,并且这种抑制或激活作用具有HBP1剂量依赖性. 以细胞代龄群体倍增值PD （population doubling）为指标测试细胞生长结合软琼脂集落形成实验证明,HBP1慢病毒感染的U2OS细胞生长速度减缓、集落形成能力下降. 上述结果提示,HBP1可能通过调控细胞增殖相关基因的表达抑制骨肉瘤细胞的增殖.     

Genome-wide Approaches Reveal Functional Vascular Endothelial Growth Factor (VEGF)-inducible Nuclear Factor of Activated T Cells (NFAT) c1 Binding to Angiogenesis-related Genes in the Endothelium

VEGF is a key regulator of endothelial cell migration, proliferation, and inflammation, which leads to activation of several signaling cascades, including the calcineurin-nuclear factor of activated T cells (NFAT) pathway. NFAT is not only important for immune responses but also for cardiovascular development and the pathogenesis of Down syndrome. By using Down syndrome model mice and clinical patient samples, we showed recently that the VEGF-calcineurin-NFAT signaling axis regulates tumor angiogenesis and tumor metastasis. However, the connection between genome-wide views of NFAT-mediated gene regulation and downstream gene function in the endothelium has not been studied extensively. Here we performed comprehensive mapping of genome-wide NFATc1 binding in VEGF-stimulated primary cultured endothelial cells and elucidated the functional consequences of VEGF-NFATc1-mediated phenotypic changes. A comparison of the NFATc1 ChIP sequence profile and epigenetic histone marks revealed that predominant NFATc1-occupied peaks overlapped with promoter-associated histone marks. Moreover, we identified two novel NFATc1 regulated genes, CXCR7 and RND1. CXCR7 knockdown abrogated SDF-1- and VEGF-mediated cell migration and tube formation. siRNA treatment of RND1 impaired vascular barrier function, caused RhoA hyperactivation, and further stimulated VEGF-mediated vascular outgrowth from aortic rings. Taken together, these findings suggest that dynamic NFATc1 binding to target genes is critical for VEGF-mediated endothelial cell activation. CXCR7 and RND1 are NFATc1 target genes with multiple functions, including regulation of cell migration, tube formation, and barrier formation in endothelial cells.