59 research outputs found
DNA fragments binding CTCF in vitro and in vivo are capable of blocking enhancer activity
<p>Abstract</p> <p>Background</p> <p>Earlier we identified ten 100-300-bp long CTCF-binding DNA fragments selected earlier from a 1-Mb human chromosome 19 region. Here the positive-negative selection technique was used to check the ability of CTCF-binding human genomic fragments to block enhancer-promoter interaction when inserted into the genome.</p> <p>Results</p> <p>Ten CTCF-binding DNA fragments were inserted between the CMV enhancer and CMV minimal promoter driving the herpes simplex virus thymidine kinase (HSV<it>-tk</it>) gene in a vector expressing also the <it>neo</it><sup>R </sup>gene under a separate promoter. The constructs were then integrated into the genome of CHO cells, and the cells resistant to neomycin and ganciclovir (positive-negative selection) were picked up, and their DNAs were PCR analyzed to confirm the presence of the fragments between the enhancer and promoter in both orientations.</p> <p>Conclusions</p> <p>We demonstrated that all sequences identified by their CTCF binding both <it>in vitro </it>and <it>in vivo </it>had enhancer-blocking activity when inserted between the CMV minimal promoter and enhancer in stably transfected CHO cells.</p
Divergent Effects of Human Cytomegalovirus and Herpes Simplex Virus-1 on Cellular Metabolism
Viruses rely on the metabolic network of the host cell to provide energy and macromolecular precursors to fuel viral replication. Here we used mass spectrometry to examine the impact of two related herpesviruses, human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1), on the metabolism of fibroblast and epithelial host cells. Each virus triggered strong metabolic changes that were conserved across different host cell types. The metabolic effects of the two viruses were, however, largely distinct. HCMV but not HSV-1 increased glycolytic flux. HCMV profoundly increased TCA compound levels and flow of two carbon units required for TCA cycle turning and fatty acid synthesis. HSV-1 increased anapleurotic influx to the TCA cycle through pyruvate carboxylase, feeding pyrimidine biosynthesis. Thus, these two related herpesviruses drive diverse host cells to execute distinct, virus-specific metabolic programs. Current drugs target nucleotide metabolism for treatment of both viruses. Although our results confirm that this is a robust target for HSV-1, therapeutic interventions at other points in metabolism might prove more effective for treatment of HCMV
Comparative Structural Analysis of Human DEAD-Box RNA Helicases
DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members
Human Cytomegalovirus IE1 Protein Elicits a Type II Interferon-Like Host Cell Response That Depends on Activated STAT1 but Not Interferon-Ξ³
Human cytomegalovirus (hCMV) is a highly prevalent pathogen that, upon primary
infection, establishes life-long persistence in all infected individuals. Acute
hCMV infections cause a variety of diseases in humans with developmental or
acquired immune deficits. In addition, persistent hCMV infection may contribute
to various chronic disease conditions even in immunologically normal people. The
pathogenesis of hCMV disease has been frequently linked to inflammatory host
immune responses triggered by virus-infected cells. Moreover, hCMV infection
activates numerous host genes many of which encode pro-inflammatory proteins.
However, little is known about the relative contributions of individual viral
gene products to these changes in cellular transcription. We systematically
analyzed the effects of the hCMV 72-kDa immediate-early 1 (IE1) protein, a major
transcriptional activator and antagonist of type I interferon (IFN) signaling,
on the human transcriptome. Following expression under conditions closely
mimicking the situation during productive infection, IE1 elicits a global type
II IFN-like host cell response. This response is dominated by the selective
up-regulation of immune stimulatory genes normally controlled by IFN-Ξ³ and
includes the synthesis and secretion of pro-inflammatory chemokines.
IE1-mediated induction of IFN-stimulated genes strictly depends on
tyrosine-phosphorylated signal transducer and activator of transcription 1
(STAT1) and correlates with the nuclear accumulation and sequence-specific
binding of STAT1 to IFN-Ξ³-responsive promoters. However, neither synthesis
nor secretion of IFN-Ξ³ or other IFNs seems to be required for the
IE1-dependent effects on cellular gene expression. Our results demonstrate that
a single hCMV protein can trigger a pro-inflammatory host transcriptional
response via an unexpected STAT1-dependent but IFN-independent mechanism and
identify IE1 as a candidate determinant of hCMV pathogenicity
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