Human cytomegalovirus infection downregulates the expression of glial fibrillary acidic protein in human glioblastoma U373MG cells: identification of viral genes and protein domains involved

Abstract

Human cytomegalovirus (HCMV) has tropism for glial cells, among many other cell types. It was reported previously that the stable expression of HCMV immediate-early protein 1 (IE1) could dramatically reduce the RNA level of glial fibrillary acidic protein (GFAP), an astroglial cell-specific intermediate filament protein, which is progressively lost with an increase in glioma malignancy. To understand this phenomenon in the context of virus infection, a human glioblastoma cell line, U373MG, was infected with HCMV (strain AD169 or Towne). The RNA level of GFAP was reduced by more than 10-fold at an m.o.i. of 3 at 48 h post-infection, whilst virus treated with neutralizing antibody C23 or with UV light had a much-reduced effect. Treatment of infected cells with ganciclovir did not prevent HCMV-mediated downregulation of GFAP. Although the expression of GFAP RNA is downregulated in IE1-expressing cells, a mutant HCMV strain lacking IE1 still suppressed GFAP, indicating that other IE proteins may be involved. IE2 is also proposed to be involved in GFAP downregulation, as an adenoviral vector expressing IE2 could also reduce the RNA level of GFAP. Data from the mutational analysis indicated that HCMV infection might affect the expression of this structural protein significantly, primarily through the C-terminal acidic region of the IE1 protein. INTRODUCTIONHuman cytomegalovirus (HCMV), a member of the subfamily Betaherpesvirinae, persistently infects the majority of the world's population. Endothelial, astroglial and neuronal cells in the human brain have been shown to be infected by HCMV in vitro (Plachter et al., 1996;Poland et al., 1990;Schmidbauer et al., 1989). Among the many different cell types that the virus infects in vivo, HCMV has tropism for brain cells and is associated with serious disorders of the central nervous system in congenitally infected infants and HCMV-infected AIDS patients (Cinque et al., 1997). Recently, the presence of the HCMV genome and gene products, including the immediate-early protein 1 (IE1), has been detected in a high percentage of human malignant glioma samples, suggesting that HCMV might contribute to the malignant phenotype of glioma (Cobbs et al., 2002; Mitchell et al., 2008;Sabatier et al., 2005;Scheurer et al., 2008).The major IE genes encode the first de novo virus-encoded proteins synthesized following infection, some of which function to regulate viral and cellular gene expression during virus replication. The 72 kDa IE1 and 86 kDa IE2 proteins are the most abundantly expressed IE gene products. RNA transcripts originating from the major IE gene region span five major exons that are alternatively spliced to produce IE1 (exons 1-4) and IE2 (exons 1-3 and 5) (Stenberg et al., 1984(Stenberg et al., , 1985. Thus, exon 5 is unique to IE2, whilst exon 4 is unique to IE1 (Mocarski, 2001). The translation of IE1 and IE2 initiates from the ATG codon in exon 2, and these two proteins share 85 identical residues at their N termini. Many data have indicated that both IE1 and IE2 stimu...

Human Cytomegalovirus Immediate-Early 1 Protein Rewires Upstream STAT3 to Downstream STAT1 Signaling Switching an IL6-Type to an IFNγ-Like Response

“…We predict that many more of the genes repressed by IE1 than those listed in Table 1 and marked as STAT3-responsive in Table 2 will turn out to be activated by signaling via pSTAT3. Although, to our knowledge, this is the first systematic genome-wide analysis of transcriptional repression by IE1, our results are consistent with earlier findings linking the viral protein to down-regulation of the genes encoding glial fibrillary acidic protein [ 77 , 78 ] and SOCS3 [ 30 ], both of which are activated by pSTAT3 [ 79 – 82 ]. Our results are also in line with previous reports on inhibition of IL6 signaling by hCMV, although hCMV-dependent activation of IL6 signaling has also been observed [ 30 , 83 ].…”

Human Cytomegalovirus IE1 Protein Elicits a Type II Interferon-Like Host Cell Response That Depends on Activated STAT1 but Not Interferon-γ

“…IE1 may accomplish transcriptional activation via interactions with a diverse set of cellular transcription regulatory proteins thereby acting through multiple DNA elements [50], [51], [52], [54], [55], [56], [57], [58], [59], [105], [106], [109], [110], [111], [112], [113], [117], [118], [119], [120], [121], [122], [123], [124], [125], [126] as well as epigenetic mechanisms including histone acetylation [53], [59], [127]. More recently, IE1 has also been implicated in transcriptional repression [31], [32], [33], [57], [62], [63], [64]. Our own work ([31] and this study, Figure 4 B) and a report by Huh et al (2008) has demonstrated that IE1 can inhibit the hCMV- or IFN-α/β-dependent activation of human ISGs including ISG54, MxA, PKR, and CXCL10.…”

“…Consequently, IE1 stimulates expression from a broad range of viral and cellular promoters in transient transfection assays. However, IE1-mediated activation or repression of merely a few single endogenous human genes has been demonstrated so far [58], [59], [60], [61], [62], [63], [64].…”

Human cytomegalovirus IE1 downregulates Hes1 in neural progenitor cells as a potential E3 ubiquitin ligase

“…For example, IE1 up-regulates the transcription of interleukin 6 (IL-6) and the genes activated by signal transducer and activator of transcription 1 (STAT1) [ 36 ]. Moreover, IE1 inhibits transactivation of p53-dependent downstream genes, disrupts transcription of STAT3-activated genes, and downregulates certain essential NPC markers such as the glial fibrillary acidic protein (GFAP) [ 37 – 40 ].…”