Dataset Information

Binding interactions between the encephalomyocarditis virus leader and protein 2A.

ABSTRACT: The leader (L) and 2A proteins of cardioviruses are the primary antihost agents produced during infection. For encephalomyocarditis virus (EMCV), the prototype of the genus Cardiovirus, these proteins interact independently with key cellular partners to bring about inhibition of active nucleocytoplasmic trafficking and cap-dependent translation, respectively. L and 2A also bind each other and require this cooperation to achieve their effects during infection. Recombinant L and 2A interact with 1:1 stoichiometry at a KD (equilibrium dissociation constant) of 1.5 ?M. The mapped contact domains include the amino-proximal third of 2A (first 50 amino acids) and the central hinge region of L. This contact partially overlaps the L segment that makes subsequent contact with Ran GTPase in the nucleus, and Ran can displace 2A from L. The equivalent proteins from Theiler's murine encephalomyelitis virus (TMEV; BeAn) and Saffold virus interact similarly in any subtype combination, with various affinities. The data suggest a mechanism whereby L takes advantage of the nuclear localization signal in the COOH region of 2A to enhance its trafficking to the nucleus. Once there, it exchanges partners in favor of Ran. This required cooperation during infection explains many observed codependent phenotypes of L and 2A mutations.Cardiovirus pathogenesis phenotypes vary dramatically, from asymptomatic, to mild gastrointestinal (GI) distress, to persistent demyelination and even encephalitic death. Leader and 2A are the primary viral determinants of pathogenesis, so understanding how these proteins cooperate to induce such a wide variety of outcomes for the host is of great important and interest to the field of virology, especially to those who use TMEV as a murine model for multiple sclerosis.

SUBMITTER: Petty RV

PROVIDER: S-EPMC4249092 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Binding interactions between the encephalomyocarditis virus leader and protein 2A.

Petty Ryan V RV Basta Holly A HA Bacot-Davis Valjean R VR Brown Bradley A BA Palmenberg Ann C AC

Journal of virology 20140910 22

<h4>Unlabelled</h4>The leader (L) and 2A proteins of cardioviruses are the primary antihost agents produced during infection. For encephalomyocarditis virus (EMCV), the prototype of the genus Cardiovirus, these proteins interact independently with key cellular partners to bring about inhibition of active nucleocytoplasmic trafficking and cap-dependent translation, respectively. L and 2A also bind each other and require this cooperation to achieve their effects during infection. Recombinant L and ...[more]

PMID: 25210192

Similar Datasets

Project description:Encephalomyocarditis virus and Theilovirus are species in the Cardiovirus genus of the Picornaviridae family. For all cardioviruses, the viral polyprotein is initiated with a short Leader (L) protein unique to this genus. The nuclear magnetic resonance (NMR) structure of LE from encephalomyocarditis virus (EMCV) has been determined. The protein has an NH2-proximal CHCC zinc finger, a central linker, and a contiguous, highly acidic motif. The theiloviruses encode the same domains, with one or two additional, COOH-proximal domains, characteristic of the human Saffold viruses (SafV) and Theiler's murine encephalomyelitis viruses (TMEV), respectively. The expression of a cardiovirus L, in recombinant form, or during infection/transfection, triggers an extensive, cell-dependent, antihost phosphorylation cascade, targeting nucleoporins (Nups) that form the hydrophobic core of nuclear pore complexes (NPC). The consequent inhibition of active nucleocytoplasmic trafficking is potent and prevents the host from mounting an effective antiviral response. For this inhibition, the L proteins themselves must be phosphorylated. In cells (extracts or recombinant form), LE was shown to be phosphorylated at Thr47 and Tyr41. The first reaction (Thr47), catalyzed by casein kinase 2 (CK2), is an obligatory precedent to the second event (Tyr41), catalyzed by spleen tyrosine kinase (Syk). Site mutations in LE, or kinase-specific inhibitors, prevented LE phosphorylation and subsequent Nup phosphorylation. Parallel experiments with LS (SafV-2) and LT (TMEV BeAn) proteins confirmed the general cardiovirus requirement for L phosphorylation, but CK2 was not the culpable kinase. It is likely that LS and LT are both activated by alternative kinases in different cell types, probably reactive within the Theilo-specific domains. IMPORTANCE An understanding of the diverse methods used by viruses to interfere with cellular processes is important because they can teach us how to control virus infections. This report shows how viruses in the same genus use different cellular enzymes to phosphorylate their proteins. If these processes are interfered with, the viruses are severely disabled.

Project description:Type I interferon (IFN)-mediated antiviral responses are critical for modulating host-virus responses, and indeed, viruses have evolved strategies to antagonize this pathway. Encephalomyocarditis virus (EMCV) is an important zoonotic pathogen, which causes myocarditis, encephalitis, neurological disease, reproductive disorders, and diabetes in pigs. This study aims to understand how EMCV interacts with the IFN pathway. EMCV circumvents the type I IFN response by expressing proteins that antagonize cellular innate immunity. Here, we show that EMCV VP2 is a negative regulator of the IFN-β pathway. This occurs via the degradation of the MDA5-mediated cytoplasmic double-stranded RNA (dsRNA) antiviral sensing RIG-I-like receptor (RLR) pathway. We show that structural protein VP2 of EMCV interacts with MDA5, MAVS, and TBK1 through its C terminus. In addition, we found that EMCV VP2 could significantly degrade RLRs by the proteasomal and lysosomal pathways. For the first time, EMCV VP2 was shown to play an important role in EMCV evasion of the type I IFN signaling pathway. This study expands our understanding that EMCV utilizes its capsid protein VP2 to evade the host antiviral response.IMPORTANCE Encephalomyocarditis virus is an important pathogen that can cause encephalitis, myocarditis, neurological diseases, and reproductive disorders. It also causes huge economic losses for the swine industry worldwide. Innate immunity plays an important role in defending the host from pathogen infection. Understanding pathogen microorganisms evading the host immune system is of great importance. Currently, whether EMCV evades cytosolic RNA sensing and signaling is still poorly understood. In the present study, we found that viral protein VP2 antagonized the RLR signaling pathway by degrading MDA5, MAVS, and TBK1 protein expression to facilitate viral replication in HEK293 cells. The findings in this study identify a new mechanism for EMCV evading the host's innate immune response, which provide new insights into the virus-host interaction and help develop new antiviral approaches against EMCV.

Project description:Epstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B-cell immortalization. EBNALP is thought to function primarily by coactivating EBNA2-mediated transcription. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) studies highlight that EBNALP frequently cooccupies DNA sites with host cell transcription factors (TFs), in particular, EP300, implicating a broader role in transcription regulation. In this study, we investigated the mechanisms of EBNALP transcription coactivation through EP300. EBNALP greatly enhanced EP300 transcription activation when EP300 was tethered to a promoter. EBNALP coimmunoprecipitated endogenous EP300 from lymphoblastoid cell lines (LCLs). EBNALP W repeat serine residues 34, 36, and 63 were required for EP300 association and coactivation. Deletion of the EP300 histone acetyltransferase (HAT) domain greatly reduced EBNALP coactivation and abolished the EBNALP association. An EP300 bromodomain inhibitor also abolished EBNALP coactivation and blocked the EP300 association with EBNALP. EBNALP sites cooccupied by EP300 had significantly higher ChIP-seq signals for sequence-specific TFs, including SPI1, RelA, EBF1, IRF4, BATF, and PAX5. EBNALP- and EP300-cooccurring sites also had much higher H3K4me1 and H3K27ac signals, indicative of activated enhancers. EBNALP-only sites had much higher signals for DNA looping factors, including CTCF and RAD21. EBNALP coactivated reporters under the control of NF-κB or SPI1. EP300 inhibition abolished EBNALP coactivation of these reporters. Clustered regularly interspaced short palindromic repeat interference targeting of EBNALP enhancer sites significantly reduced target gene expression, including that of EP300 itself. These data suggest a previously unrecognized mechanism by which EBNALP coactivates transcription through subverting of EP300 and thus affects the expression of LCL genes regulated by a broad range of host TFs.IMPORTANCE Epstein-Barr virus was the first human DNA tumor virus discovered over 50 years ago. EBV is causally linked to ∼200,000 human malignancies annually. These cancers include endemic Burkitt lymphoma, Hodgkin lymphoma, lymphoma/lymphoproliferative disease in transplant recipients or HIV-infected people, nasopharyngeal carcinoma, and ∼10% of gastric carcinoma cases. EBV-immortalized human B cells faithfully model key aspects of EBV lymphoproliferative diseases and are useful models of EBV oncogenesis. EBNALP is essential for EBV to transform B cells and transcriptionally coactivates EBNA2 by removing repressors from EBNA2-bound DNA sites. Here, we found that EBNALP can also modulate the activity of the key transcription activator EP300, an acetyltransferase that activates a broad range of transcription factors. Our data suggest that EBNALP regulates a much broader range of host genes than was previously appreciated. A small-molecule inhibitor of EP300 abolished EBNALP coactivation of multiple target genes. These findings suggest novel therapeutic approaches to control EBV-associated lymphoproliferative diseases.

Dataset Information

Binding interactions between the encephalomyocarditis virus leader and protein 2A.

Publications

Binding interactions between the encephalomyocarditis virus leader and protein 2A.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets