The reaction was performed using the Vii7A Real-Time PCR System (Applied Biosystems)

The reaction was performed using the Vii7A Real-Time PCR System (Applied Biosystems). to harvest genomic GDC-0068 (Ipatasertib, RG-7440) DNA at indicated time points. 1ug of DNA was used to run Taqman for HCMV pp65, pp71 and US28 genes. Values were normalized to Rab14. Each sample was run in triplicate and copy numbers were estimated based on the standard curves shown in panels ACC.(PDF) pone.0116178.s002.pdf (177K) GUID:?604D92A9-4B80-492A-AC6C-F94DA4095809 S3 Fig: IE1 protein expression in 387 GSC. Protein was extracted from uninfected 387 GSC and AD169-infected GSC at 9 and 11 wks p.i and probed for IE1 and actin.(PDF) pone.0116178.s003.pdf (96K) GUID:?8C159F06-8E4C-4D5E-B130-59B609FEFE31 S4 Fig: CMV gene expression in 3832 GSC sorted for CD133. Primary-derived 3832 GSC cells were sorted into CD133+ and CD133? fractions using the Miltenyi AutoMACS system with CD133 microbeads. cDNA from each fraction was used to determine viral gene expression using a SYBR Green array with custom CMV primers. Heatmap represents Ct values of viral genes normalized to housekeeping gene RPL13A. Red shows higher gene expression and green shows lower gene expression.(PDF) pone.0116178.s004.pdf (117K) GUID:?A742665A-C600-4CAB-AFCC-E12D1B959E48 S1 Table: Sequences for primers and TaqMan probes. UL111A sequence was obtained from Chang WL, Baumgarth N, Yu D, Barry PA (2004) Human cytomegalovirus-encoded interleukin-10 homolog inhibits maturation of dendritic cells and alters their functionality. J Virol 78: 8720C8731.(PDF) pone.0116178.s005.pdf (167K) GUID:?753CFE52-F3F8-476B-A3E4-B4451E859E36 S2 Table: List of primers used for SYBR Green RT-PCR. All primer sequences are displayed for each viral gene tested.(PDF) pone.0116178.s006.pdf (255K) GUID:?4EEE73F8-10DD-4891-B436-D07F964246BB Data Availability StatementThe authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the Supporting Information files. Abstract The most common adult primary brain tumor, glioblastoma (GBM), is characterized by fifteen months median patient survival and has no clear etiology. We and others have identified the presence of human cytomegalovirus (HCMV) gene products endogenously GDC-0068 (Ipatasertib, RG-7440) expressed in GBM tissue and primary cells, with a subset of viral genes being consistently expressed in most samples. Among these viral genes, several ITGA9 have important oncomodulatory properties, regulating tumor stemness, proliferation, immune evasion, invasion and GDC-0068 (Ipatasertib, RG-7440) angiogenesis. These findings lead us to hypothesize that a specific HCMV gene signature GDC-0068 (Ipatasertib, RG-7440) may be associated with GBM pathogenesis. To investigate this hypothesis, we used glioma cell lines and primary glioma stem-like cells (GSC) infected with clinical and laboratory HCMV strains and measured relative viral gene expression levels along several time points up to 15 weeks post-infection. While HCMV gene expression was detected in several infected glioma lines through week 5 post-infection, only HCMV-infected GSC expressed viral gene products 15 weeks post-infection. Efficiency of infection across time was higher in GSC compared to cell lines. Importantly, HCMV-infected GSC outlived their uninfected counterparts, and this extended survival was paralleled by increased tumorsphere frequency and upregulation of stemness regulators, such as SOX2, p-STAT3, and BMX (a novel HCMV target identified in this study). Interleukin 6 (IL-6) treatment significantly upregulated HCMV gene expression in long-term infected glioma cultures, suggesting that pro-inflammatory signaling in the tumor milieu may further augment HCMV gene expression and subsequent tumor progression driven by viral-induced cellular signaling. Together, our data support a critical role for long-term, low-level HCMV infection in promoting survival, stemness, and proliferation of GSC that could significantly contribute to GBM pathogenesis. Introduction Glioblastoma multiforme (GBM), a grade IV glioma, is the most aggressive and malignant type of brain tumor [1]. The cause of GBM remains unknown, and even with current treatments, the median survival for patients with GBM is 15 months [2]. Glioma stem-like cells (GSC) constitute a small subset of tumor cells characterized by expression of various stem cell markers and endowed with tumor initiating capabilities (reviewed in [3]). GSC are resistant to radiation and chemotherapy and are primarily responsible for GBM recurrence [4]. There is an increased interest in elucidating the role of human cytomegalovirus (HCMV) in cancer since it has been associated with GBM and several other malignancies (reviewed in [5]). Our laboratory was the first to report that HCMV is present in over 95% of malignant gliomas [6], and since then, several other groups have corroborated these findings [7]C[14]. While the exact role of HCMV in GBM is still under investigation, evidence from several studies suggests that it might act as an oncomodulator, altering proliferative signaling, cell growth, angiogenesis, cell death, immune detection, and chromosome stability (reviewed in [15]). HCMV is a herpes virus affecting 50C80% of the population. HCMV infection can persist for the lifetime of the host in adult stem cells, particularly hematopoietic stem cells in the bone marrow (reviewed in [16]). There are two types of persistent viral infections: (1) latent, where no new virus is produced and (2) chronic, where new virus is produced at low levels [17]. Since HCMV gene expression pattern in endogenously infected GBM tissues is reminiscent of a low-level chronic.