scholarly journals Functional Interaction between the pp71 Protein of Human Cytomegalovirus and the PML-Interacting Protein Human Daxx

2002 ◽  
Vol 76 (11) ◽  
pp. 5769-5783 ◽  
Author(s):  
Heike Hofmann ◽  
Hilde Sindre ◽  
Thomas Stamminger
Keyword(s):  
Human Cytomegalovirus ◽  
Protein Function ◽  
Fluorescent Protein ◽  
De Novo ◽  
Human Fibroblasts ◽  
Specific Interaction ◽  
Immediate Early ◽  
Regulatory Function ◽  
Viral Protein Synthesis ◽  
Interacting Protein

ABSTRACT The tegument protein pp71 (UL82) of human cytomegalovirus (HCMV) has previously been shown to transactivate the major immediate-early enhancer-promoter of HCMV. Furthermore, this protein is able to enhance the infectivity of viral DNA and to accelerate the infection cycle, suggesting an important regulatory function during viral replication. To gain insight into the underlying mechanisms that are used by pp71 to exert these pleiotropic effects, we sought for cellular factors interacting with pp71 in a yeast two-hybrid screen. Here, we report the isolation of the human Daxx (hDaxx) protein as a specific interaction partner of HCMV pp71. hDaxx, which was initially described as an adapter protein involved in apoptosis regulation, has recently been identified as a nuclear protein that interacts and colocalizes with PML in the nuclear domain ND10. In order to assess whether pp71 can also be detected in ND10 structures, a vector expressing pp71 in fusion with the green fluorescent protein was used for transfection of human fibroblasts. This revealed a colocalization of pp71 with the ND10 proteins PML and Sp100. In addition, cotransfection of a hDaxx expression vector resulted in an enhanced recruitment of pp71 to ND10. Targeting of pp71 to nuclear dots could also be observed in infected human fibroblasts in the absence of de novo viral protein synthesis. Moreover, cotransfection experiments revealed that pp71-mediated transactivation of the major immediate-early enhancer-promoter was synergistically enhanced in the presence of hDaxx. These results suggest an important role of hDaxx for pp71 protein function.

scholarly journals A Strong Negative Transcriptional Regulatory Region between the Human Cytomegalovirus UL127 Gene and the Major Immediate-Early Enhancer

1999 ◽  
Vol 73 (11) ◽  
pp. 9039-9052 ◽  
Author(s):  
Christopher A. Lundquist ◽  
Jeffrey L. Meier ◽  
Mark F. Stinski
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Genome ◽  
De Novo ◽  
Human Fibroblasts ◽  
Regulatory Region ◽  
Fold Increase ◽  
Immediate Early ◽  
Wild Type ◽  
Viral Protein Synthesis ◽  
Unique Region

ABSTRACT The region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region. DNase I protection analysis with human cell nuclear extracts demonstrated multiple protein binding sites in this region of the viral genome (P. Ghazal, H. Lubon, C. Reynolds-Kohler, L. Hennighausen, and J. A. Nelson, Virology 174:18–25, 1990). However, the function of this region in the context of the viral genome is not known. In wild-type human CMV-infected human fibroblasts, cells permissive for viral replication, there is little to no transcription from UL127. We determined that the unique region prevented transcription from the UL127 promoter but had no effect on the divergent MIE promoter. In transient-transfection assays, the basal level of expression from the UL127 promoter increased significantly when the wild-type unique sequences were mutated. In recombinant viruses with similar mutations in the unique region, expression from the UL127 promoter occurred only after de novo viral protein synthesis, typical of an early viral promoter. A 111-bp deletion-substitution of the unique sequence caused approximately a 20-fold increase in the steady-state level of RNA from the UL127 promoter and a 245-fold increase in the expression of a downstream indicator gene. This viral negative regulatory region was also mutated at approximately 50-bp regions proximal and distal to the UL127 promoter. Although some repressive effects were detected in the distal region, mutations of the region proximal to the UL127 promoter had the most significant effects on transcription. Within the proximal and distal regions, there are potential cis sites for known eucaryotic transcriptional repressor proteins. This region may also bind unknown viral proteins. We propose that the unique region upstream of the UL127 promoter and the MIE enhancer negatively regulates the expression from the UL127 promoter in permissive human fibroblast cells. This region may be a regulatory boundary preventing the effects of the very strong MIE enhancer on this promoter.

scholarly journals Visualization of Intracellular Transport of Vesicular Stomatitis Virus Nucleocapsids in Living Cells

2006 ◽  
Vol 80 (13) ◽  
pp. 6368-6377 ◽  
Author(s):  
Subash C. Das ◽  
Debasis Nayak ◽  
You Zhou ◽  
Asit K. Pattnaik
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Protein Function ◽  
Intracellular Transport ◽  
Fluorescent Protein ◽  
De Novo ◽  
Virus Production ◽  
Hinge Region ◽  
Cell Periphery ◽  
P Protein ◽  
Vesicular Stomatitis

ABSTRACT The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the viral RNA polymerase. In previous studies, we demonstrated that insertion of 19 amino acids in the hinge region of the protein had no significant effect on P protein function. In the present study, we inserted full-length enhanced green fluorescent protein (eGFP) in frame into the hinge region of P and show that the fusion protein (PeGFP) is functional in viral genome transcription and replication, albeit with reduced activity. A recombinant vesicular stomatitis virus encoding PeGFP in place of the P protein (VSV-PeGFP), which possessed reduced growth kinetics compared to the wild-type VSV, was recovered. Using the recombinant VSV-PeGFP, we show that the viral replication proteins and the de novo-synthesized RNA colocalize to sites throughout the cytoplasm, indicating that replication and transcription are not confined to any particular region of the cytoplasm. Real-time imaging of the cells infected with the eGFP-tagged virus revealed that, following synthesis, the nucleocapsids are transported toward the cell periphery via a microtubule (MT)-mediated process, and the nucleocapsids were seen to be closely associated with mitochondria. Treatment of cells with nocodazole or Colcemid, drugs known to inhibit MT polymerization, resulted in accumulation of the nucleocapsids around the nucleus and also led to inhibition of infectious-virus production. These findings are compatible with a model in which the progeny viral nucleocapsids are transported toward the cell periphery by MT and the transport may be facilitated by mitochondria.

scholarly journals Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-β pathway

2010 ◽  
Vol 207 (11) ◽  
pp. 2331-2341 ◽  
Author(s):  
John R. Grainger ◽  
Katie A. Smith ◽  
James P. Hewitson ◽  
Henry J. McSorley ◽  
Yvonne Harcus ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Fluorescent Protein ◽  
De Novo ◽  
Foxp3 Expression ◽  
Dominant Negative ◽  
Regulatory Function ◽  
Intestinal Helminth ◽  
Helminth Parasites

Foxp3-expressing regulatory T (T reg) cells have been implicated in parasite-driven inhibition of host immunity during chronic infection. We addressed whether parasites can directly induce T reg cells. Foxp3 expression was stimulated in naive Foxp3− T cells in mice infected with the intestinal helminth Heligmosomoides polygyrus. In vitro, parasite-secreted proteins (termed H. polygyrus excretory-secretory antigen [HES]) induced de novo Foxp3 expression in fluorescence-sorted Foxp3− splenocytes from Foxp3–green fluorescent protein reporter mice. HES-induced T reg cells suppressed both in vitro effector cell proliferation and in vivo allergic airway inflammation. HES ligated the transforming growth factor (TGF) β receptor and promoted Smad2/3 phosphorylation. Foxp3 induction by HES was lost in dominant-negative TGF-βRII cells and was abolished by the TGF-β signaling inhibitor SB431542. This inhibitor also reduced worm burdens in H. polygyrus–infected mice. HES induced IL-17 in the presence of IL-6 but did not promote Th1 or Th2 development under any conditions. Importantly, antibody to mammalian TGF-β did not recognize HES, whereas antisera that inhibited HES did not affect TGF-β. Foxp3 was also induced by secreted products of Teladorsagia circumcincta, a related nematode which is widespread in ruminant animals. We have therefore identified a novel pathway through which helminth parasites may stimulate T reg cells, which is likely to be a key part of the parasite’s immunological relationship with the host.

scholarly journals Role of the Proximal Enhancer of the Major Immediate-Early Promoter in Human Cytomegalovirus Replication

2004 ◽  
Vol 78 (23) ◽  
pp. 12788-12799 ◽  
Author(s):  
Hiroki Isomura ◽  
Tatsuya Tsurumi ◽  
Mark F. Stinski
Keyword(s):  
Virus Replication ◽  
Human Cytomegalovirus ◽  
Human Fibroblast ◽  
Recombinant Virus ◽  
Immediate Early ◽  
Wild Type Virus ◽  
Fibroblast Cells ◽  
Viral Protein Synthesis ◽  
Enhancer Element ◽  
Human Fibroblast Cells

ABSTRACT The human cytomegalovirus (CMV) enhancer has a distal component (positions −550 to −300) and a proximal component (−300 to −39) relative to the transcription start site (+1) of the major immediate-early (MIE) promoter. Without the distal enhancer, human CMV replicates slower and has a small-plaque phenotype. We determined the sequence requirements of the proximal enhancer by making 5′-end deletions to positions −223, −173, −116, −67, and −39. Even though recombinant virus with the proximal enhancer deleted to −39 has the minimal TATA box-containing MIE promoter element, it cannot replicate independently in human fibroblast cells. Recombinant virus with a deletion to −67 has an Sp-1 transcription factor binding site which may represent a minimal enhancer element for recombinant virus replication in human fibroblast cells. Although recombinant virus with a deletion to −223 replicates to titers at least 100-fold less than that of the wild-type virus, it replicates to titers 8-fold higher than that of recombinant virus with a deletion to −173 and 20-fold higher than that of virus with a deletion to −67. Recombinant virus with a deletion to −173 replicates more efficiently than that with a deletion to −116. There was a direct correlation between the level of infectious virus replication and time after infection, amount of MIE gene transcription, MIE and early viral protein synthesis, and viral DNA synthesis. The extent of the proximal enhancer determines the efficiency of viral replication.

scholarly journals Recruitment of cdk9 to the Immediate-Early Viral Transcriptosomes during Human Cytomegalovirus Infection Requires Efficient Binding to Cyclin T1, a Threshold Level of IE2 86, and Active Transcription

2009 ◽  
Vol 83 (11) ◽  
pp. 5904-5917 ◽  
Author(s):  
Anokhi J. Kapasi ◽  
Charles L. Clark ◽  
Karen Tran ◽  
Deborah H. Spector
Keyword(s):  
Human Cytomegalovirus ◽  
De Novo ◽  
Threshold Level ◽  
Immediate Early ◽  
Cyclin T1 ◽  
Cellular Factors ◽  
Active Transcription ◽  
Exogenous Expression ◽  
Kinetics Of ◽  
De Novo Protein Synthesis

ABSTRACT Human cytomegalovirus (HCMV) infection results in the formation of nuclear viral transcriptosomes, which are sites dedicated to viral immediate-early (IE) transcription. At IE times of the infection, viral and cellular factors, including several components of transcription such as cyclin-dependent kinase 9 (cdk9), localize at these sites. To determine the mechanism and requirements of specific recruitment of cdk9 to the viral transcriptosomes, infection in the presence of inhibitor drugs and infection of cell lines expressing exogenous mutant cdk9 were performed. We found that cdk9 localization to the viral transcriptosomes requires de novo protein synthesis. In addition, active transcription is required for recruitment and maintenance of cdk9 at the viral transcriptosomes. In cells infected with a recombinant IE2 HCMV (IE2 86 ΔSX virus) in which IE2 gene expression is greatly reduced, cdk9 localization at the transcriptosome is delayed and corresponds to the kinetics of accumulation of the IE2 protein at these sites. Infection in the presence of the cdk9 inhibitors Flavopiridol and DRB (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole) allowed cdk9 localization to the viral transcriptosomes. A kinase-inactive cdk9 (D167N) expressed during the infection also localizes to the viral transcriptosomes, indicating that kinase activity of cdk9 is not a requirement for its localization to the sites of IE transcription. Exogenous expression of additional cdk9 mutants indicates that binding of Brd4 to the cdk9 complex is not required but that efficient binding to cyclin T1 is essential.

scholarly journals Resistance to Methylation de novo of the Human Cytomegalovirus Immediate Early Enhancer in a Model for Virus Latency and Reactivation in vitro

1987 ◽  
Vol 68 (11) ◽  
pp. 2839-2852 ◽  
Author(s):  
R. Boom ◽  
J. L. Geelen ◽  
C. J. Sol ◽  
R. P. Minnaar ◽  
J. Van Der Noordaa
Keyword(s):  
Human Cytomegalovirus ◽  
De Novo ◽  
Immediate Early ◽  
Virus Latency

scholarly journals DNA Microarrays of the Complex Human Cytomegalovirus Genome: Profiling Kinetic Class with Drug Sensitivity of Viral Gene Expression

1999 ◽  
Vol 73 (7) ◽  
pp. 5757-5766 ◽  
Author(s):  
James Chambers ◽  
Ana Angulo ◽  
Dhammika Amaratunga ◽  
Hongqing Guo ◽  
Ying Jiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Dna Sequences ◽  
De Novo ◽  
Expression Profiles ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Viral Dna ◽  
Viral Protein Synthesis ◽  
Entire Genome

ABSTRACT We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (α), early (β), early-late (γ1), and late (γ2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5′ noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for β, γ1, and γ2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.

Energetic basis of excited-state enzyme design and function

2021 ◽  
Author(s):  
Chi-Yun Lin ◽  
Matthew Romei ◽  
Irimpan Mathews ◽  
Steven Boxer
Keyword(s):  
Excited State ◽  
Protein Function ◽  
Fluorescent Protein ◽  
De Novo ◽  
Fitness Landscape ◽  
Quantitative Model ◽  
Efficient Solutions ◽  
Catalyst Design ◽  
Quantum Yields ◽  
Fluorescence Quantum Yields

The last decades have witnessed an explosion of de novo protein designs with a remarkable range of scaffolds. It remains challenging, however, to design catalytic functions that are competitive with naturally occurring counterparts as well as biomimetic or non-biological catalysts. Although directed evolution often offers efficient solutions, the fitness landscape remains opaque. Green fluorescent protein (GFP), which has revolutionized biological imaging and assays, is one of the most re-designed proteins. While not an enzyme in the conventional sense, GFPs feature competing excited-state decay pathways with the same steric and electrostatic origins as conventional ground-state catalysts, and they exert exquisite control over multiple reaction outcomes through the same principles. Thus, GFP is an “excited-state enzyme”. Herein we show that rationally designed mutants and hybrids that contain environmental mutations and substituted chromophores provide the basis for a quantitative model and prediction that describes the influence of sterics and electrostatics on excited-state catalysis of GFPs. As both perturbations can selectively bias photoisomerization pathways, GFPs with fluorescence quantum yields (FQYs) and photoswitching characteristics tailored for specific applications could be predicted and then demonstrated. The underlying energetic landscape, readily accessible via spectroscopy for GFPs, offers an important missing link in the design of protein function that is generalizable to catalyst design.

scholarly journals Reversal of Human Cytomegalovirus Major Immediate-Early Enhancer/Promoter Silencing in Quiescently Infected Cells via the Cyclic AMP Signaling Pathway

2007 ◽  
Vol 81 (12) ◽  
pp. 6669-6681 ◽  
Author(s):  
Michael J. Keller ◽  
Allen W. Wu ◽  
Janet I. Andrews ◽  
Patrick W. McGonagill ◽  
Eric E. Tibesar ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Signaling Pathway ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Lytic Cycle ◽  
Immediate Early ◽  
Infected Cells ◽  
Ifn Γ ◽  
Stimulation Of

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early (MIE) enhancer contains five functional cyclic AMP (cAMP) response elements (CRE). Because the CRE in their native context do not contribute appreciably to MIE enhancer/promoter activity in lytically infected human fibroblasts and NTera2 (NT2)-derived neurons, we postulated that they might have a role in MIE enhancer/promoter reactivation in quiescently infected cells. Here, we show that stimulation of the cAMP signaling pathway by treatment with forskolin (FSK), an adenylyl cyclase activator, greatly alleviates MIE enhancer/promoter silencing in quiescently infected NT2 neuronal precursors. The effect is immediate, independent of de novo protein synthesis, associated with the phosphorylation of ATF-1 serine 63 and CREB serine 133, dependent on protein kinase A (PKA) and the enhancer's CRE, and linked to viral-lytic-cycle advancement. Coupling of FSK treatment with the inhibition of either histone deacetylases or protein synthesis synergistically activates MIE gene expression in a manner suggesting that MIE enhancer/promoter silencing is optimally relieved by an interplay of multiple regulatory mechanisms. In contrast, MIE enhancer/promoter silence is not overcome by stimulation of the gamma interferon (IFN-γ) signaling pathway, despite the enhancer having two IFN-γ-activated-site-like elements. We conclude that stimulation of the cAMP/PKA signaling pathway drives CRE-dependent MIE enhancer/promoter activation in quiescently infected cells, thus exposing a potential mode of regulation in HCMV reactivation.

scholarly journals The Human Cytomegalovirus Major Immediate-Early Distal Enhancer Region Is Required for Efficient Viral Replication and Immediate-Early Gene Expression

2000 ◽  
Vol 74 (4) ◽  
pp. 1602-1613 ◽  
Author(s):  
Jeffery L. Meier ◽  
Jonathan A. Pruessner
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Early Gene ◽  
Immediate Early ◽  
Regulatory Function ◽  
Wild Type Virus ◽  
Cell Type ◽  
Distal Enhancer ◽  
Unique Region ◽  
Enhancer Region

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early (MIE) genes, encoding IE1 p72 and IE2 p86, are activated by a complex enhancer region (base positions -65 to -550) that operates in a cell type- and differentiation-dependent manner. The expression of MIE genes is required for HCMV replication. Previous studies analyzing functions of MIE promoter-enhancer segments suggest that the distal enhancer region variably modifies MIE promoter activity, depending on cell type, stimuli, or state of differentiation. To further understand the mechanism by which the MIE promoter is regulated, we constructed and analyzed several different recombinant HCMVs that lack the distal enhancer region (-300 to -582, -640, or -1108). In human fibroblasts, the HCMVs without the distal enhancer replicate normally at high multiplicity of infection (MOI) but replicate poorly at low MOI in comparison to wild-type virus (WT) or HCMVs that lack the neighboring upstream unique region and modulator (-582 or -640 to -1108). The growth aberrancy was normalized after restoring the distal enhancer in a virus lacking this region. For HCMVs without a distal enhancer, the impairment in replication at low MOI corresponds to a deficiency in production of MIE RNAs compared to WT or virus lacking the unique region and modulator. An underproduction of viral US3 RNA was also evident at low MOI. Whether lower production of IE1 p72 and IE2 p86 causes a reduction in expression of the immediate-early (IE) class US3 gene remains to be determined. We conclude that the MIE distal enhancer region possesses a mechanism for augmenting viral IE gene expression and genome replication at low MOI, but this regulatory function is unnecessary at high MOI.

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