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Ann C. Palmenberg - One of the best experts on this subject based on the ideXlab platform.
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three Cardiovirus leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways
Virology, 2015Co-Authors: Jessica J Ciomperlik, Holly Basta, Ann C. PalmenbergAbstract:Cardiovirus infections inhibit nucleocytoplasmic trafficking by Leader protein-induced phosphorylation of Phe/Gly-containing nucleoporins (Nups). Recombinant Leader from encephalomyocarditis virus, Theiler׳s murine encephalomyelitis virus and Saffold virus target the same subset of Nups, including Nup62 and Nup98, but not Nup50. Reporter cell lines with fluorescence mCherry markers for M9, RS and classical SV40 import pathways, as well as the Crm1-mediated export pathway, all responded to transfection with the full panel of Leader proteins, showing consequent cessation of path-specific active import/export. For this to happen, the Nups had to be presented in the context of intact nuclear pores and exposed to cytoplasmic extracts. The Leader phosphorylation cascade was not effective against recombinant Nup proteins. The findings support a model of Leader-dependent Nup phosphorylation with the purpose of disrupting Nup-transportin interactions.
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binding interactions between the encephalomyocarditis virus leader and protein 2a
Journal of Virology, 2014Co-Authors: Ryan V Petty, Bradley A. Brown, Holly A. Basta, Valjean R Bacotdavis, Ann C. PalmenbergAbstract: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 K D (equilibrium dissociation constant) of 1.5 μM. 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 Theiler9s 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. IMPORTANCE 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.
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encephalomyocarditis virus leader is phosphorylated by ck2 and syk as a requirement for subsequent phosphorylation of cellular nucleoporins
Journal of Virology, 2014Co-Authors: Holly A. Basta, Valjean R Bacotdavis, Jessica J Ciomperlik, Ann C. PalmenbergAbstract: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
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Binding interactions between the encephalomyocarditis virus leader and protein 2A. J.Virol
2014Co-Authors: Ryan V Petty, Bradley A. Brown, Holly A. Basta, Valjean R Bacot-davis, Ann C. PalmenbergAbstract: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
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encephalomyocarditis virus leader protein hinge domain is responsible for interactions with ran gtpase
Virology, 2013Co-Authors: Valjean R Bacotdavis, Ann C. PalmenbergAbstract:Encephalomyocarditis virus (EMCV), a Cardiovirus, initiates its polyprotein with a short 67 amino acid Leader (L) sequence. The protein acts as a unique pathogenicity factor, with anti-host activities which include the triggering of nuclear pore complex hyperphosphorylation and direct binding inhibition of the active cellular transport protein, Ran GTPase. Chemical modifications and protein mutagenesis now map the Ran binding domain to the L hinge-linker region, and in particular, to amino acids 35–40. Large deletions affecting this region were shown previously to diminish Ran binding. New point mutations, especially K35Q, D37A and W40A, preserve the intact L structure, abolish Ran binding and are deficient for nucleoporin (Nup) hyperphosphorylation. Ran itself morphs through multiple configurations, but reacts most effectively with L when in the GDP format, preferably with an empty nucleotide binding pocket. Therefore, L:Ran binding, mediated by the linker-hinge, is a required step in L-induced nuclear transport inhibition.
Frank J. M. Van Kuppeveld - One of the best experts on this subject based on the ideXlab platform.
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structure and genome release mechanism of the human Cardiovirus saffold virus 3
Journal of Virology, 2016Co-Authors: Edukondalu Mullapudi, Frank J. M. Van Kuppeveld, Jiři Novacek, Lenka Palkova, Pavel Kulich, Michael A Lindberg, Pavel PlevkaAbstract:In order to initiate an infection, viruses need to deliver their genomes into cells. This involves uncoating the genome and transporting it to the cytoplasm. The process of genome delivery is not well understood for nonenveloped viruses. We address this gap in our current knowledge by studying the uncoating of the nonenveloped human Cardiovirus Saffold virus 3 (SAFV-3) of the family Picornaviridae. SAFVs cause diseases ranging from gastrointestinal disorders to meningitis. We present a structure of a native SAFV-3 virion determined to 2.5 angstrom by X-ray crystallography and an 11-angstrom-resolution cryo-electron microscopy reconstruction of an "altered" particle that is primed for genome release. The altered particles are expanded relative to the native virus and contain pores in the capsid that might serve as channels for the release of VP4 subunits, N termini of VP1, and the RNA genome. Unlike in the related enteroviruses, pores in SAFV-3 are located roughly between the icosahedral 3- and 5-fold axes at an interface formed by two VP1 and one VP3 subunit. Furthermore, in native conditions many Cardioviruses contain a disulfide bond formed by cysteines that are separated by just one residue. The disulfide bond is located in a surface loop of VP3. We determined the structure of the SAFV-3 virion in which the disulfide bonds are reduced. Disruption of the bond had minimal effect on the structure of the loop, but it increased the stability and decreased the infectivity of the virus. Therefore, compounds specifically disrupting or binding to the disulfide bond might limit SAFV infection. IMPORTANCE A capsid assembled from viral proteins protects the virus genome during transmission from one cell to another. However, when a virus enters a cell the virus genome has to be released from the capsid in order to initiate infection. This process is not well understood for nonenveloped viruses. We address this gap in our current knowledge by studying the genome release of Human Saffold virus 3. Saffold viruses cause diseases ranging from gastrointestinal disorders to meningitis. We show that before the genome is released, the Saffold virus 3 particle expands, and holes form in the previously compact capsid. These holes serve as channels for the release of the genome and small capsid proteins VP4 that in related enteroviruses facilitate subsequent transport of the virus genome into the cell cytoplasm.
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PI4KA is essential for Cardiovirus replication.
2015Co-Authors: Cristina M. Dorobantu, Lucian Albulescu, Christian Harak, Qian Feng, Mirjam Van Kampen, Jeroen R. P. M. Strating, Alexander E. Gorbalenya, Volker Lohmann, Hilde M. Van Der Schaar, Frank J. M. Van KuppeveldAbstract:(A-D) Effects of PI4KA knockdown on EMCV infection. HeLa R19 cells were reverse transfected with siRNA against PI4K2A, PI4K2B, PI4KA, PI4KB or scrambled siRNA as a control (A) or siRNA against PI4KA, PI4KB or scrambled siRNA (B-D). At 48 h post transfection (p.t.), cells were infected with EMCV (A-C) or RLuc-EMCV (D) at an MOI of 0.1 or transfected with full-length infectious EMCV in vitro-transcribed RNA (C). After 8 h, cells were freeze-thawed to release intracellular virus particles and the total virus titers were determined by endpoint titration (A-C). Alternatively, cells were lysed and Renilla luciferase activity was determined as a measure of viral RNA replication (D). In parallel, a cell viability assay was performed to evaluate the cytotoxicity of the siRNA treatment (E). (F) Western blot analysis showing efficient knockdown of PI4KA and PI4KB. Actin was used as loading control. (G-I) AL-9 treatment inhibits Cardiovirus replication. HeLa R19 cells were infected with virus at an MOI of 0.1 (G and H) or 1 (I), followed by AL-9 treatment for 8 h, after which cells were lysed and virus replication was measured by endpoint titration (G and I) or by determining the Renilla luciferase activity (H). Cytotoxicity of AL-9 was determined in a cell viability assay run in parallel (J). Bars represent mean values of triplicates ± standard error of the means (SEM). Means were statistically compared using unpaired t tests. *P < 0.05, **P
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saffold Cardiovirus and multiple sclerosis no evidence for an association
Annals of clinical and translational neurology, 2014Co-Authors: Jochem M D Galama, Frank J. M. Van Kuppeveld, Jan Zoll, Kjerstin Lanke, Arjan S De Jong, Jeroen Melief, Inge Huitinga, Marcel M VerbeekAbstract:Saffold Cardiovirus, a newly discovered human Cardiovirus, has close similarity with Theiler's murine encephalomyelitis virus (TMEV) which can cause a chronic demyelinating encephalomyelitis in mice. In this study, we tested whether Saffold Cardiovirus infection of the brain is associated with multiple sclerosis (MS). Autopsy white matter samples from 19 MS and 9 normal brain donors were tested by polymerase chain reaction. All were negative. Paired cerebrospinal fluid and serum samples from 24 MS patients and 27 controls were tested for Saffold Cardiovirus-specific oligoclonal bands, two patients and two controls reacted positive. We conclude that an association between Saffold Cardiovirus and MS is highly improbable.
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the crystal structure of a Cardiovirus rna dependent rna polymerase reveals an unusual conformation of the polymerase active site
Journal of Virology, 2014Co-Authors: Laia Vivesadrian, Celia Lujan, Baldo Oliva, Lonneke Van Der Linden, Barbara Selisko, Bruno Coutard, Bruno Canard, Frank J. M. Van KuppeveldAbstract:Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-Å resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 Å from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-π interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the Cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A. IMPORTANCE The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first Cardiovirus RdRp, EMCV 3Dpol, we captured an altered conformation of a conserved motif in the polymerase active site (motif A) containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, which interferes with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting enzyme activity, and this type of modulation appears to be general to all picornaviruses.
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Conservation of Cardiovirus OriL SL-A element.
2013Co-Authors: Jan Zoll, Willem J. G. Melchers, Marc M. Hahn, Paul Gielen, Hans A. Heus, Frank J. M. Van KuppeveldAbstract:(A) Multiple alignment of SL-A of the Cardiovirus OriL. The upper part shows the alignment of the EMCV subgroup of the Cardiovirus genus. The lower part shows the alignment of the theiloviruses, including the human Theiler's like virus. The apical part of the stem-loop is shaded and includes the EMCV SL-A octaloop and the Theilovirus SL-A tetraloop with two closing base-pairs. (B) Secondary structures of OriL SL-A elements from Cardioviruses and parechovirus 5. Structures were calculated using MFold [14].
Bert L. Semler - One of the best experts on this subject based on the ideXlab platform.
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vpg unlinkase tdp2 in Cardiovirus infected cells re localization and proteolytic cleavage
Virology, 2018Co-Authors: Sonia Maciejewski, Wendy Ullmer, Bert L. SemlerAbstract:Cardioviruses cause diseases in many animals including, in rare cases, humans. Although they share common features with all picornaviruses, Cardioviruses have unique properties that distinguish them from other family members, including enteroviruses. One feature shared by all picornaviruses is the covalent attachment of VPg to the 5' end of genomic RNA via a phosphotyrosyl linkage. For enteroviruses, this linkage is cleaved by a host cell protein, TDP2. Since TDP2 is divergently required during enterovirus infections, we determined if TDP2 is necessary during infection by the prototype Cardiovirus, EMCV. We found that EMCV yields are reduced in the absence of TDP2. We observed a decrease in viral protein accumulation and viral RNA replication in the absence of TDP2. In contrast to enterovirus infections, we found that TDP2 is modified at peak times of EMCV infection. This finding suggests a unique mechanism for Cardioviruses to regulate TDP2 activity during infection.
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VPg unlinkase/TDP2 in Cardiovirus infected cells: Re-localization and proteolytic cleavage.
Virology, 2018Co-Authors: Sonia Maciejewski, Wendy Ullmer, Bert L. SemlerAbstract:Cardioviruses cause diseases in many animals including, in rare cases, humans. Although they share common features with all picornaviruses, Cardioviruses have unique properties that distinguish them from other family members, including enteroviruses. One feature shared by all picornaviruses is the covalent attachment of VPg to the 5' end of genomic RNA via a phosphotyrosyl linkage. For enteroviruses, this linkage is cleaved by a host cell protein, TDP2. Since TDP2 is divergently required during enterovirus infections, we determined if TDP2 is necessary during infection by the prototype Cardiovirus, EMCV. We found that EMCV yields are reduced in the absence of TDP2. We observed a decrease in viral protein accumulation and viral RNA replication in the absence of TDP2. In contrast to enterovirus infections, we found that TDP2 is modified at peak times of EMCV infection. This finding suggests a unique mechanism for Cardioviruses to regulate TDP2 activity during infection.
Valjean R Bacotdavis - One of the best experts on this subject based on the ideXlab platform.
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binding interactions between the encephalomyocarditis virus leader and protein 2a
Journal of Virology, 2014Co-Authors: Ryan V Petty, Bradley A. Brown, Holly A. Basta, Valjean R Bacotdavis, Ann C. PalmenbergAbstract: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 K D (equilibrium dissociation constant) of 1.5 μM. 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 Theiler9s 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. IMPORTANCE 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.
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encephalomyocarditis virus leader is phosphorylated by ck2 and syk as a requirement for subsequent phosphorylation of cellular nucleoporins
Journal of Virology, 2014Co-Authors: Holly A. Basta, Valjean R Bacotdavis, Jessica J Ciomperlik, Ann C. PalmenbergAbstract: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
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encephalomyocarditis virus leader protein hinge domain is responsible for interactions with ran gtpase
Virology, 2013Co-Authors: Valjean R Bacotdavis, Ann C. PalmenbergAbstract:Encephalomyocarditis virus (EMCV), a Cardiovirus, initiates its polyprotein with a short 67 amino acid Leader (L) sequence. The protein acts as a unique pathogenicity factor, with anti-host activities which include the triggering of nuclear pore complex hyperphosphorylation and direct binding inhibition of the active cellular transport protein, Ran GTPase. Chemical modifications and protein mutagenesis now map the Ran binding domain to the L hinge-linker region, and in particular, to amino acids 35–40. Large deletions affecting this region were shown previously to diminish Ran binding. New point mutations, especially K35Q, D37A and W40A, preserve the intact L structure, abolish Ran binding and are deficient for nucleoporin (Nup) hyperphosphorylation. Ran itself morphs through multiple configurations, but reacts most effectively with L when in the GDP format, preferably with an empty nucleotide binding pocket. Therefore, L:Ran binding, mediated by the linker-hinge, is a required step in L-induced nuclear transport inhibition.
Holly A. Basta - One of the best experts on this subject based on the ideXlab platform.
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binding interactions between the encephalomyocarditis virus leader and protein 2a
Journal of Virology, 2014Co-Authors: Ryan V Petty, Bradley A. Brown, Holly A. Basta, Valjean R Bacotdavis, Ann C. PalmenbergAbstract: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 K D (equilibrium dissociation constant) of 1.5 μM. 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 Theiler9s 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. IMPORTANCE 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.
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encephalomyocarditis virus leader is phosphorylated by ck2 and syk as a requirement for subsequent phosphorylation of cellular nucleoporins
Journal of Virology, 2014Co-Authors: Holly A. Basta, Valjean R Bacotdavis, Jessica J Ciomperlik, Ann C. PalmenbergAbstract: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
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Binding interactions between the encephalomyocarditis virus leader and protein 2A. J.Virol
2014Co-Authors: Ryan V Petty, Bradley A. Brown, Holly A. Basta, Valjean R Bacot-davis, Ann C. PalmenbergAbstract: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