Publications by authors named "Daniel A Engel"

22 Publications

  • Page 1 of 1

Ebola Virus Inclusion Body Formation and RNA Synthesis Are Controlled by a Novel Domain of Nucleoprotein Interacting with VP35.

J Virol 2020 07 30;94(16). Epub 2020 Jul 30.

Department of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia, USA

Ebola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection, IBs display dynamic properties regarding their size and location. The contents of IBs also must transition prior to further viral maturation, assembly, and release, implying additional steps in IB function. Interestingly, the expression of the viral nucleoprotein (NP) alone is sufficient for the generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30, and the RNA polymerase L. We previously defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here, we show that NP-Ct is necessary for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for the production of infectious virus-like particles (VLPs), and that defective VLPs with NP-Ct deletions are significantly reduced in viral RNA content. Furthermore, coexpression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins, only VP35 is able to overcome the defect in IB formation caused by the deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself and that is mediated by a newly identified functional domain of NP, the central domain. Inclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative-sense RNA viruses, including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.
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http://dx.doi.org/10.1128/JVI.02100-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7394894PMC
July 2020

Identification, design and synthesis of novel pyrazolopyridine influenza virus nonstructural protein 1 antagonists.

Bioorg Med Chem Lett 2019 05 26;29(9):1113-1119. Epub 2019 Feb 26.

Division of Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, United States. Electronic address:

Nonstructural protein 1 (NS1) plays a crucial function in the replication, spread, and pathogenesis of influenza virus by inhibiting the host innate immune response. Here we report the discovery and optimization of novel pyrazolopyridine NS1 antagonists that can potently inhibit influenza A/PR/8/34 replication in MDCK cells, rescue MDCK cells from cytopathic effects of seasonal influenza A strains, reverse NS1-dependent inhibition of IFN-β gene expression, and suppress the slow growth phenotype in NS1-expressing yeast. These pyrazolopyridines will enable researchers to investigate NS1 function during infection and how antagonists can be utilized in the next generation of treatments for influenza infection.
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http://dx.doi.org/10.1016/j.bmcl.2019.02.027DOI Listing
May 2019

The structure of the C-terminal domain of the nucleoprotein from the Bundibugyo strain of the Ebola virus in complex with a pan-specific synthetic Fab.

Acta Crystallogr D Struct Biol 2018 07 27;74(Pt 7):681-689. Epub 2018 Jun 27.

Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

The vast majority of platforms for the detection of viral or bacterial antigens rely on immunoassays, typically ELISA or sandwich ELISA, that are contingent on the availability of suitable monoclonal antibodies (mAbs). This is a major bottleneck, since the generation and production of mAbs is time-consuming and expensive. Synthetic antibody fragments (sFabs) generated by phage-display selection offer an alternative with many advantages over Fabs obtained from natural antibodies using hybridoma technology. Unlike mAbs, sFabs are generated using phage display, allowing selection for binding to specific strains or for pan-specificity, for identification of structural epitopes or unique protein conformations and even for complexes. Further, they can easily be produced in Escherichia coli in large quantities and engineered for purposes of detection technologies and other applications. Here, the use of phage-display selection to generate a pan-specific Fab (MJ20), based on a Herceptin Fab scaffold, with the ability to bind selectively and with high affinity to the C-terminal domains of the nucleoproteins (NPs) from all five known strains of the Ebola virus is reported. The high-resolution crystal structure of the complex of MJ20 with the antigen from the Bundibugyo strain of the Ebola virus reveals the basis for pan-specificity and illustrates how the phage-display technology can be used to manufacture suitable Fabs for use in diagnostic or therapeutic applications.
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http://dx.doi.org/10.1107/S2059798318007878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038385PMC
July 2018

Crystal structures of the methyltransferase and helicase from the ZIKA 1947 MR766 Uganda strain.

Acta Crystallogr D Struct Biol 2017 Sep 15;73(Pt 9):767-774. Epub 2017 Aug 15.

Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908-0736, USA.

Two nonstructural proteins encoded by Zika virus strain MR766 RNA, a methyltransferase and a helicase, were crystallized and their structures were solved and refined at 2.10 and 2.01 Å resolution, respectively. The NS5 methyltransferase contains a bound S-adenosyl-L-methionine (SAM) co-substrate. The NS3 helicase is in the apo form. Comparison with published crystal structures of the helicase in the apo, nucleotide-bound and single-stranded RNA (ssRNA)-bound states suggests that binding of ssRNA to the helicase may occur through conformational selection rather than induced fit.
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http://dx.doi.org/10.1107/S2059798317010737DOI Listing
September 2017

Molecular architecture of the nucleoprotein C-terminal domain from the Ebola and Marburg viruses.

Acta Crystallogr D Struct Biol 2016 Jan 1;72(Pt 1):49-58. Epub 2016 Jan 1.

Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908-0736, USA.

The Filoviridae family of negative-sense, single-stranded RNA (ssRNA) viruses is comprised of two species of Marburgvirus (MARV and RAVV) and five species of Ebolavirus, i.e. Zaire (EBOV), Reston (RESTV), Sudan (SUDV), Taï Forest (TAFV) and Bundibugyo (BDBV). In each of these viruses the ssRNA encodes seven distinct proteins. One of them, the nucleoprotein (NP), is the most abundant viral protein in the infected cell and within the viral nucleocapsid. It is tightly associated with the viral RNA in the nucleocapsid, and during the lifecycle of the virus is essential for transcription, RNA replication, genome packaging and nucleocapsid assembly prior to membrane encapsulation. The structure of the unique C-terminal globular domain of the NP from EBOV has recently been determined and shown to be structurally unrelated to any other known protein [Dziubańska et al. (2014), Acta Cryst. D70, 2420-2429]. In this paper, a study of the C-terminal domains from the NP from the remaining four species of Ebolavirus, as well as from the MARV strain of Marburgvirus, is reported. As expected, the crystal structures of the BDBV and TAFV proteins show high structural similarity to that from EBOV, while the MARV protein behaves like a molten globule with a core residual structure that is significantly different from that of the EBOV protein.
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http://dx.doi.org/10.1107/S2059798315021439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4905509PMC
January 2016

The structure of the C-terminal domain of the Zaire ebolavirus nucleoprotein.

Acta Crystallogr D Biol Crystallogr 2014 Sep 29;70(Pt 9):2420-9. Epub 2014 Aug 29.

Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908-0736, USA.

Ebolavirus (EBOV) causes severe hemorrhagic fever with a mortality rate of up to 90%. EBOV is a member of the order Mononegavirales and, like other viruses in this taxonomic group, contains a negative-sense single-stranded (ss) RNA. The EBOV ssRNA encodes seven distinct proteins. One of them, the nucleoprotein (NP), is the most abundant viral protein in the infected cell and within the viral nucleocapsid. Like other EBOV proteins, NP is multifunctional. It is tightly associated with the viral genome and is essential for viral transcription, RNA replication, genome packaging and nucleocapsid assembly prior to membrane encapsulation. NP is unusual among the Mononegavirales in that it contains two distinct regions, or putative domains, the C-terminal of which shows no homology to any known proteins and is purported to be a hub for protein-protein interactions within the nucleocapsid. The atomic structure of NP remains unknown. Here, the boundaries of the N- and C-terminal domains of NP from Zaire EBOV are defined, it is shown that they can be expressed as highly stable recombinant proteins in Escherichia coli, and the atomic structure of the C-terminal domain (residues 641-739) derived from analysis of two distinct crystal forms at 1.98 and 1.75 Å resolution is described. The structure reveals a novel tertiary fold that is distantly reminiscent of the β-grasp architecture.
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http://dx.doi.org/10.1107/S1399004714014710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157450PMC
September 2014

A direct and versatile assay measuring membrane penetration of adenovirus in single cells.

J Virol 2013 Nov 11;87(22):12367-79. Epub 2013 Sep 11.

Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.

Endocytosis is the most prevalent entry port for viruses into cells, but viruses must escape from the lumen of endosomes to ensure that viral genomes reach a site for replication and progeny formation. Endosomal escape also helps viruses bypass endolysosomal degradation and presentation to certain Toll-like intrinsic immunity receptors. The mechanisms for cytosolic delivery of nonenveloped viruses or nucleocapsids from enveloped viruses are poorly understood, in part because no quantitative assays are readily available which directly measure the penetration of viruses into the cytosol. Following uptake by clathrin-mediated endocytosis or macropinocytosis, the nonenveloped adenoviruses penetrate from endosomes to the cytosol, and they traffic with cellular motors on microtubules to the nucleus for replication. In this report, we present a novel single-cell imaging assay which quantitatively measures individual cytosolic viruses and distinguishes them from endosomal viruses or viruses at the plasma membrane. Using this assay, we showed that the penetration of human adenoviruses of the species C and B occurs rapidly after virus uptake. Efficient penetration does not require acidic pH in endosomes. This assay is versatile and can be adapted to other adenoviruses and members of other nonenveloped and enveloped virus families.
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http://dx.doi.org/10.1128/JVI.01833-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807902PMC
November 2013

The influenza virus NS1 protein as a therapeutic target.

Authors:
Daniel A Engel

Antiviral Res 2013 Sep 21;99(3):409-16. Epub 2013 Jun 21.

Department of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA 22908, United States. Electronic address:

Nonstructural protein 1 (NS1) of influenza A virus plays a central role in virus replication and blockade of the host innate immune response, and is therefore being considered as a potential therapeutic target. The primary function of NS1 is to dampen the host interferon (IFN) response through several distinct molecular mechanisms that are triggered by interactions with dsRNA or specific cellular proteins. Sequestration of dsRNA by NS1 results in inhibition of the 2'-5' oligoadenylate synthetase/RNase L antiviral pathway, and also inhibition of dsRNA-dependent signaling required for new IFN production. Binding of NS1 to the E3 ubiquitin ligase TRIM25 prevents activation of RIG-I signaling and subsequent IFN induction. Cellular RNA processing is also targeted by NS1, through recognition of cleavage and polyadenylation specificity factor 30 (CPSF30), leading to inhibition of IFN-β mRNA processing as well as that of other cellular mRNAs. In addition NS1 binds to and inhibits cellular protein kinase R (PKR), thus blocking an important arm of the IFN system. Many additional proteins have been reported to interact with NS1, either directly or indirectly, which may serve its anti-IFN and additional functions, including the regulation of viral and host gene expression, signaling pathways and viral pathogenesis. Many of these interactions are potential targets for small-molecule intervention. Structural, biochemical and functional studies have resulted in hypotheses for drug discovery approaches that are beginning to bear experimental fruit, such as targeting the dsRNA-NS1 interaction, which could lead to restoration of innate immune function and inhibition of virus replication. This review describes biochemical, cell-based and nucleic acid-based approaches to identifying NS1 antagonists.
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http://dx.doi.org/10.1016/j.antiviral.2013.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4373342PMC
September 2013

Yeast based small molecule screen for inhibitors of SARS-CoV.

PLoS One 2011 2;6(12):e28479. Epub 2011 Dec 2.

Department of Microbiology and Immunology, University of Maryland, Baltimore, Maryland, United States of America.

Severe acute respiratory coronavirus (SARS-CoV) emerged in 2002, resulting in roughly 8000 cases worldwide and 10% mortality. The animal reservoirs for SARS-CoV precursors still exist and the likelihood of future outbreaks in the human population is high. The SARS-CoV papain-like protease (PLP) is an attractive target for pharmaceutical development because it is essential for virus replication and is conserved among human coronaviruses. A yeast-based assay was established for PLP activity that relies on the ability of PLP to induce a pronounced slow-growth phenotype when expressed in S. cerevisiae. Induction of the slow-growth phenotype was shown to take place over a 60-hour time course, providing the basis for conducting a screen for small molecules that restore growth by inhibiting the function of PLP. Five chemical suppressors of the slow-growth phenotype were identified from the 2000 member NIH Diversity Set library. One of these, NSC158362, potently inhibited SARS-CoV replication in cell culture without toxic effects on cells, and it specifically inhibited SARS-CoV replication but not influenza virus replication. The effect of NSC158362 on PLP protease, deubiquitinase and anti-interferon activities was investigated but the compound did not alter these activities. Another suppressor, NSC158011, demonstrated the ability to inhibit PLP protease activity in a cell-based assay. The identification of these inhibitors demonstrated a strong functional connection between the PLP-based yeast assay, the inhibitory compounds, and SARS-CoV biology. Furthermore the data with NSC158362 suggest a novel mechanism for inhibition of SARS-CoV replication that may involve an unknown activity of PLP, or alternatively a direct effect on a cellular target that modifies or bypasses PLP function in yeast and mammalian cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028479PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3229576PMC
July 2012

Design, synthesis, and evaluation of novel small molecule inhibitors of the influenza virus protein NS1.

Bioorg Med Chem 2012 Jan 19;20(1):487-97. Epub 2011 Oct 19.

University of Virginia, Department of Chemistry, Charlottesville, VA 22904, USA.

Influenza is a continuing world-wide public health problem that causes significant morbidity and mortality during seasonal epidemics and sporadic pandemics. The existing vaccination program is variably effective from year to year, and drug resistance to available antivirals is a growing problem, making the development of additional antivirals an important challenge. Influenza virus non-structural protein 1 (NS1) is the centerpiece of the viral response to the host interferon (IFN) system. NS1 was demonstrated previously to be a potential therapeutic target for antiviral therapy by the identification of specific small-molecule inhibitors. One inhibitory compound, NSC125044, was subjected to chemical evaluation. Initial synthetic work comprised simplifying the core structure by removing unwanted functionality and determination of key features important for activity. Several subclasses of molecules were designed and synthesized to further probe activity and develop the basis for a structure-activity relationship. Apparent potency, as judged by activity in virus replication assays, increased dramatically for some analogs, without cytotoxicity. Results suggest that the target binding site tolerates hydrophobic bulk as well as having a preference for weakly basic substituents.
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http://dx.doi.org/10.1016/j.bmc.2011.10.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4373408PMC
January 2012

Insertion of CTCF-binding sites into a first-generation adenovirus vector reduces the innate inflammatory response and prolongs transgene expression.

Virology 2011 Mar 26;412(1):136-45. Epub 2011 Jan 26.

Department of Microbiology, University of Colorado Medical School, Aurora, CO 80045, USA.

We have made improvements to E1-deleted adenovirus (Ad) transducing vectors that both substantially reduce the innate inflammatory response provoked by the virus in BALB/c mouse ears and increase the duration of expression of the GFP transgene in BALB/c mouse liver. These improvements result from testing the hypothesis that induction of strong innate responses is primarily a result of the powerful enhancer contained within the strong CMV promoter activating expression of Ad genes retained within the vector. A DNA fragment containing four CTCF-binding sites, which was expected to act as a chromatin insulator, was introduced 5', 3', or both 5' and 3' of a CMV-GFP cassette in an attempt to reduce activation of Ad gene expression by the enhancer. The presence of this sequence in any of the configurations led to reduction of the innate immune response, as assayed by mouse ear swelling, to the low level induced by a virus deleted for the E1 region and carrying no introduced sequence. In addition, the duration of GFP expression in the liver more than doubled. The prolonged GFP expression indicates that GFP does not play the limiting role in shutting down vector expression. The CTCF-binding sequence introduced appears to act as a chromatin insulator in Ad DNA, but position-independence of the elements in reducing the innate immune response indicate unanticipated complexities in the mechanism by which Ad vectors induce innate immune responses.
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http://dx.doi.org/10.1016/j.virol.2010.12.053DOI Listing
March 2011

An integrated, valveless system for microfluidic purification and reverse transcription-PCR amplification of RNA for detection of infectious agents.

Lab Chip 2011 Mar 8;11(5):957-61. Epub 2010 Dec 8.

Department of Chemistry, University of Virginia, McCormick Road, P.O. Box 400319, Charlottesville, VA 22904, USA.

We describe the first miniaturized device capable of the front-end sample preparation essential for detecting RNA-based infectious agents. The microfluidic device integrates sample purification and reverse transcription PCR (RT-PCR) amplification for the identification and detection of influenza A. The device incorporates a chitosan-based RNA binding phase for the completely aqueous isolation of nucleic acids, avoiding the PCR inhibitory effects of guanidine and isopropanol used in silica-based extraction methods. The purified nucleic acids and the reagents needed for single-step RT-PCR amplification are fluidically mobilized simultaneously to a PCR chamber. Utilizing infrared (IR)-mediated heating allowed for a > 5-fold decrease in RT-PCR analysis time compared to a standard thermal cycling protocol used in a conventional thermal cycler. Influenza A virus [A/PR/8/34 (H1N1)] was used as a simulant in this study for virus-based infectious and biowarfare agents with RNA genomes, and was successfully detected in a mock nasal swab sample at clinically relevant concentrations. Following on-chip purification, a fragment specific to the influenza A nucleoprotein gene was first amplified via RT-PCR amplification using IR-mediated heating to achieve more rapid heating and cooling rates. This was initially accomplished on a two-chip system to optimize the SPE and RT-PCR, and then translated to an integrated SPE-RT-PCR device.
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http://dx.doi.org/10.1039/c0lc00136hDOI Listing
March 2011

Novel inhibitor of influenza non-structural protein 1 blocks multi-cycle replication in an RNase L-dependent manner.

J Gen Virol 2011 Jan 29;92(Pt 1):60-70. Epub 2010 Sep 29.

Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

Influenza virus non-structural protein 1 (NS1) is the centrepiece of the viral response to the host interferon (IFN) system. NS1 has been demonstrated previously to be a potential therapeutic target for antiviral therapy by identification of specific small-molecule inhibitors. This study demonstrated the biological mechanism for a potent new NS1 antagonist. Compound JJ3297 inhibited virus replication by more than three orders of magnitude without affecting cell viability. Importantly, it efficiently reversed NS1-induced inhibition of IFN mRNA production. The hypothesis was tested that JJ3297 facilitates IFN production in infected cells, leading to protection of the surrounding uninfected cells. Accordingly, the compound efficiently prevented virus spread through a cell population during a 48 h multi-cycle infection initiated at a very low m.o.i. Consistent with the hypothesis, the compound had no detectable influence on a 6 h single-cycle infection initiated at a high m.o.i. The effect of JJ3297 on virus replication was not caused by inhibition of NS1 expression or its mislocalization in the cell. JJ3297 facilitated the induction of an IFN-like antiviral state, resulting in increased resistance to subsequent challenge with vesicular stomatitis virus. The activity of JJ3297 absolutely required the function of cellular RNase L, indicating that an intact IFN system is required for function of the compound. These results support a model in which inhibition of NS1 function results in restoration of the IFN-induced antiviral state and inhibition of virus replication and spread. This represents a new direction for anti-influenza virus drug development that exploits the IFN pathway to challenge virus replication.
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http://dx.doi.org/10.1099/vir.0.025015-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3052532PMC
January 2011

Accurate single-day titration of adenovirus vectors based on equivalence of protein VII nuclear dots and infectious particles.

J Virol Methods 2009 Aug 3;159(2):251-8. Epub 2009 May 3.

Department of Microbiology, University of Virginia Health System, P.O. Box 800734, Charlottesville, VA 22908-0734, USA.

Protein VII is an abundant component of adenovirus particles and is tightly associated with the viral DNA. It enters the nucleus along with the infecting viral genome and remains bound throughout early phase. Protein VII can be visualized by immunofluorescent staining as discrete dots in the infected cell nucleus. Comparison between protein VII staining and expression of the 72kDa DNA-binding protein revealed a one-to-one correspondence between protein VII dots and infectious viral genomes. A similar relationship was observed for a helper-dependent adenovirus vector expressing green fluorescent protein. This relationship allowed accurate titration of adenovirus preparations, including wild-type and helper-dependent vectors, using a 1-day immunofluorescence method. The method can be applied to any adenovirus vector and gives results equivalent to the standard plaque assay.
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http://dx.doi.org/10.1016/j.jviromet.2009.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774845PMC
August 2009

Novel influenza virus NS1 antagonists block replication and restore innate immune function.

J Virol 2009 Feb 3;83(4):1881-91. Epub 2008 Dec 3.

Department of Microbiology, University of Virginia School of Medicine, Charlottesville, 22908, USA.

The innate immune system guards against virus infection through a variety of mechanisms including mobilization of the host interferon system, which attacks viral products mainly at a posttranscriptional level. The influenza virus NS1 protein is a multifunctional facilitator of virus replication, one of whose actions is to antagonize the interferon response. Since NS1 is required for efficient virus replication, it was reasoned that chemical inhibitors of this protein could be used to further understand virus-host interactions and also serve as potential new antiviral agents. A yeast-based assay was developed to identify compounds that phenotypically suppress NS1 function. Several such compounds exhibited significant activity specifically against influenza A virus in cell culture but had no effect on the replication of another RNA virus, respiratory syncytial virus. Interestingly, cells lacking an interferon response were drug resistant, suggesting that the compounds block interactions between NS1 and the interferon system. Accordingly, the compounds reversed the inhibition of beta interferon mRNA induction during infection, which is known to be caused by NS1. In addition, the compounds blocked the ability of NS1 protein to inhibit double-stranded RNA-dependent activation of a transfected beta interferon promoter construct. The effects of the compounds were specific to NS1, because they had no effect on the ability of the severe acute respiratory syndrome coronavirus papainlike protease protein to block beta interferon promoter activation. These data demonstrate that the function of NS1 can be modulated by chemical inhibitors and that such inhibitors will be useful as probes of biological function and as starting points for clinical drug development.
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http://dx.doi.org/10.1128/JVI.01805-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2643796PMC
February 2009

Inhibition of cyclin D1 gene transcription by Brg-1.

Cell Cycle 2008 Mar 12;7(5):647-55. Epub 2007 Dec 12.

Kimmel Cancer Center, Department of Cancer Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

The evolutionarily conserved SWI-SNF chromatin remodeling complex regulates cellular proliferation. A catalytic subunit, BRG-1, is frequently down regulated, silenced or mutated in malignant cells, however, the mechanism by which BRG-1 may function as a tumor suppressor or block breast cancer cellular proliferation is not understood. The cyclin D1 gene is a collaborative oncogene overexpressed in greater than 50% of human breast cancers. Herein, BRG-1 inhibited DNA synthesis and cyclin D1 expression in human MCF-7 breast cancer epithelial cells. The cyclin D1 promoter AP-1 and CRE sites were required for repression by BRG-1 in promoter assays. BRG-1 deficient cells abolished and siRNA to BRG-1 reduced, formation of the BRG-1 chromatin complex. The endogenous cyclin D1 promoter AP-1 site bound BRG-1. Estradiol treatment of MCF-7 cells induced recruitment of BRG-1 to the endogenous hpS2 gene promoter. Estradiol, which induced cyclin D1 abundance, was associated with a reduction in recruitment of the co-repressors HP1alpha/HDAC1 to the endogenous cyclin D1 promoter AP-1/BRG-1 binding sites. These studies suggest the endogenous cyclin D1 promoter BRG-1 binding site functions as a molecular scaffold in the context of local chromatin upon which coactivators and corepressors are recruited to regulate cyclin D1.
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http://dx.doi.org/10.4161/cc.7.5.5446DOI Listing
March 2008

Transcription releases protein VII from adenovirus chromatin.

Virology 2007 Dec 20;369(2):411-22. Epub 2007 Sep 20.

Department of Microbiology, University of Virginia Health System, PO Box 800734, Charlottesville, VA 22908-0734, USA.

Adenovirus protein VII is the major protein component of the viral nucleoprotein core. It is a nonspecific DNA-binding protein that condenses viral DNA inside the capsid. Protein VII remains associated with viral chromatin throughout early phase, indicating its continuing role during infection. Here we characterize the release of protein VII from infectious genomes during a time period that corresponds to the late phase of infection. Interestingly, the early viral transactivator E1A, but not other early gene products, is responsible for releasing protein VII by a mechanism that requires ongoing transcription but not viral DNA replication. Moreover transcription per se, in the absence of E1A, is also sufficient to trigger release. Accordingly, a recombinant genome containing only non-coding "stuffer" DNA is unable to support release of protein VII. Our data support a model in which early gene transcription results in a change in the structure of the viral chromatin.
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http://dx.doi.org/10.1016/j.virol.2007.08.012DOI Listing
December 2007

Adenovirus protein VII functions throughout early phase and interacts with cellular proteins SET and pp32.

J Virol 2005 Feb;79(4):2474-83

Department of Microbiology, University of Virginia Health System, Charlottesville, VA 22908, USA.

Adenovirus protein VII is the major component of the viral nucleoprotein core. It is a highly basic nonspecific DNA-binding protein that condenses viral DNA inside the capsid. We have investigated the fate and function of protein VII during infection. "Input" protein VII persisted in the nucleus throughout early phase and the beginning of DNA replication. Chromatin immunoprecipitation revealed that input protein VII remained associated with viral DNA during this period. Two cellular proteins, SET and pp32, also associated with viral DNA during early phase. They are components of two multiprotein complexes, the SET and INHAT complexes, implicated in chromatin-related activities. Protein VII associated with SET and pp32 in vitro and distinct domains of protein VII were responsible for binding to the two proteins. Interestingly, protein VII was found in novel nuclear dot structures as visualized by immunofluorescence. The dots likely represent individual infectious genomes in association with protein VII. They appeared within 30 min after infection and localized in the nucleus with a peak of intensity between 4 and 10 h postinfection. After this, their intensity decreased and they disappeared between 16 and 24 h postinfection. Interestingly, disappearance of the dots required ongoing RNA synthesis but not DNA synthesis. Taken together these data indicate that protein VII has an ongoing role during early phase and the beginning of DNA replication.
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http://dx.doi.org/10.1128/JVI.79.4.2474-2483.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC546597PMC
February 2005

Adenovirus protein VII condenses DNA, represses transcription, and associates with transcriptional activator E1A.

J Virol 2004 Jun;78(12):6459-68

Department of Microbiology, University of Virginia Health System, P.O. Box 800734, Charlottesville, VA 22908, USA.

Adenovirus protein VII is the major protein component of the viral nucleoprotein core. It is highly basic, and an estimated 1070 copies associate with each viral genome, forming a tightly condensed DNA-protein complex. We have investigated DNA condensation, transcriptional repression, and specific protein binding by protein VII. Xenopus oocytes were microinjected with mRNA encoding HA-tagged protein VII and prepared for visualization of lampbrush chromosomes. Immunostaining revealed that protein VII associated in a uniform manner across entire chromosomes. Furthermore, the chromosomes were significantly condensed and transcriptionally silenced, as judged by the dramatic disappearance of transcription loops characteristic of lampbrush chromosomes. During infection, the protein VII-DNA complex may be the initial substrate for transcriptional activation by cellular factors and the viral E1A protein. To investigate this possibility, mRNAs encoding E1A and protein VII were comicroinjected into Xenopus oocytes. Interestingly, whereas E1A did not associate with chromosomes in the absence of protein VII, expression of both proteins together resulted in significant association of E1A with lampbrush chromosomes. Binding studies with proteins produced in bacteria or human cells or by in vitro translation showed that E1A and protein VII can interact in vitro. Structure-function analysis revealed that an N-terminal region of E1A is responsible for binding to protein VII. These studies define the in vivo functions of protein VII in DNA binding, condensation, and transcriptional repression and indicate a role in E1A-mediated transcriptional activation of viral genes.
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http://dx.doi.org/10.1128/JVI.78.12.6459-6468.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC416553PMC
June 2004

Adenovirus type 5 DNA-protein complexes from formaldehyde cross-linked cells early after infection.

Virology 2003 Jul;312(1):204-12

Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.

We report here the properties of viral DNA-protein complexes that purify with cellular chromatin following formaldehyde cross-linking of intact cells early after infection. The cross-linked viral DNA fractionated into shear-sensitive (S) and shear- resistant (R) components that were separable by sedimentation, which allowed independent characterization. The R component had the density and sedimentation properties expected for DNA-protein complexes and contained intact viral DNA. It accounted for about 50% of the viral DNA recovered at 1.5 h after infection but less than 20% by 4.5 h. The proportion of R component was independent of multiplicity of infection, even at less than one particle per cell. Viral hexon and protein VII, but not protein VI, were detected in the fractions containing the R component. These properties are consistent with those of partially uncoated virions associated with the nuclear envelope. A substantial proportion of the S component viral DNA had the same density as cellular chromatin. Protein VII was the most abundant viral protein present in gradient fractions that contained the S component. Complexes containing USF transcription factor cross-linked to the adenovirus major late promoter were detected by viral chromatin immunoprecipitation of the fractions containing S component. The S component probably contained uncoated nuclear viral DNA that assembles into early viral transcription complexes.
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http://dx.doi.org/10.1016/s0042-6822(03)00194-6DOI Listing
July 2003

Adenovirus E1A requires the yeast SAGA histone acetyltransferase complex and associates with SAGA components Gcn5 and Tra1.

Oncogene 2002 Feb;21(9):1411-22

Department of Microbiology and Cancer Center, University of Virginia School of Medicine, Charlottesville, Virginia, VA 22908, USA.

The budding yeast Saccharomyces cerevisiae was used as a model system to study the function of the adenovirus E1A oncoprotein. Previously we demonstrated that expression of the N-terminal 82 amino acids of E1A in yeast causes pronounced growth inhibition and specifically interferes with SWI/SNF-dependent transcriptional activation. Further genetic analysis identified the yeast transcription factor Adr1 as a high copy suppressor of E1A function. Transcriptional activation by Adr1 requires interaction with co-activator proteins Ada2 and Gcn5, components of histone acetyltransferase complexes including ADA and SAGA. Analysis of mutant alleles revealed that several components of the SAGA complex, including proteins from the Ada, Spt, and Taf classes were required for E1A-induced growth inhibition. Growth inhibition also depended on the Gcn5 histone acetyltransferase, and point mutations within the Gcn5 HAT domain rendered cells E1A-resistant. Also required was SAGA component Tra1, a homologue of the mammalian TRRAP protein which is required for c-myc and E1A induced cellular transformation. Additionally, Gcn5 protein could associate with E1A in vitro in a manner that depended on the N-terminal domain of E1A, and Tra1 protein was co-immunoprecipitated with E1A in vivo. These results indicate a strong requirement for intact SAGA complex for E1A to function in yeast, and suggest a role for SAGA-like complexes in mammalian cell transformation.
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http://dx.doi.org/10.1038/sj.onc.1205201DOI Listing
February 2002