Ann C Palmenberg

University of Wisconsin–Madison, Madison, Wisconsin, United States

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Publications (114)639.57 Total impact

  • Jessica J Ciomperlik, Holly A Basta, Ann C Palmenberg
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    ABSTRACT: 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. Copyright © 2015 Elsevier Inc. All rights reserved.
    Virology 06/2015; 484:194-202. DOI:10.1016/j.virol.2015.06.004 · 3.28 Impact Factor
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    ABSTRACT: Members of rhinovirus C (RV-C) species are more likely to cause wheezing illnesses and asthma exacerbations compared with other rhinoviruses. The cellular receptor for these viruses was heretofore unknown. We report here that expression of human cadherin-related family member 3 (CDHR3) enables the cells normally unsusceptible to RV-C infection to support both virus binding and replication. A coding single nucleotide polymorphism (rs6967330, C529Y) was previously linked to greater cell-surface expression of CDHR3 protein, and an increased risk of wheezing illnesses and hospitalizations for childhood asthma. Compared with wild-type CDHR3, cells transfected with the CDHR3-Y529 variant had about 10-fold increases in RV-C binding and progeny yields. We developed a transduced HeLa cell line (HeLa-E8) stably expressing CDHR3-Y529 that supports RV-C propagation in vitro. Modeling of CDHR3 structure identified potential binding sites that could impact the virus surface in regions that are highly conserved among all RV-C types. Our findings identify that the asthma susceptibility gene product CDHR3 mediates RV-C entry into host cells, and suggest that rs6967330 mutation could be a risk factor for RV-C wheezing illnesses.
    Proceedings of the National Academy of Sciences 04/2015; 112(17). DOI:10.1073/pnas.1421178112 · 9.81 Impact Factor
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    ABSTRACT: The Rhinovirus C (RV-C) were discovered in 2006 and these agents are an important cause of respiratory morbidity. Little is known about their biology. RV-C15 (C15) can be produced by transfection of recombinant viral RNA into cells and subsequent purification over a 30% sucrose cushion, even though yields and infectivity of other RV-C genotypes with this protocol are low. The goal of this study was to determine whether poor RV-C yields were due to capsid instability, and moreover, to develop a robust protocol suitable for the purification of many RV-C types. Capsid stability assays indicated that virions of RV-C41 (refractory to purification) have similar tolerance for osmotic and temperature stress as RV-A16 (purified readily), although C41 is more sensitive to low pH. Modification to the purification protocol by removing detergent increased the yield of RV-C. Addition of nonfat dry milk to the sucrose cushion increased the virus yield but sacrificed purity of the viral suspension. Analysis of virus distribution following centrifugation indicated that the majority of detectable viral RNA (vRNA) was found in pellets refractory to resuspension. Reduction of the centrifugal force with commiserate increase in spin-time improved the recovery of RV-C for both C41 and C2. Transfection of primary lung fibroblasts (WisL cells) followed by the modified purification protocol further improved yields of infectious C41 and C2. Described herein is a higher-yield purification protocol suitable for RV-C types refractory to the standard purification procedure. The findings suggest that aggregation-adhesion problems rather than capsid instability influence RV-C yield during purification. Copyright © 2015. Published by Elsevier B.V.
    Journal of Virological Methods 02/2015; 217. DOI:10.1016/j.jviromet.2015.02.019 · 1.88 Impact Factor
  • Ann C Palmenberg, James E Gern
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    ABSTRACT: The historical classification of human rhinoviruses (RV) by serotyping has been replaced by a logical system of comparative sequencing. Given that strains must diverge within their capsid sequenced by a reasonable degree (>12-13 % pairwise base identities) before becoming immunologically distinct, the new nomenclature system makes allowances for the addition of new, future types, without compromising historical designations. Currently, three species, the RV-A, RV-B, and RV-C, are recognized. Of these, the RV-C, discovered in 2006, are the most unusual in terms of capsid structure, receptor use, and association with severe disease in children.
    Methods in Molecular Biology 01/2015; 1221:1-10. DOI:10.1007/978-1-4939-1571-2_1 · 1.29 Impact Factor
  • Bahar Inankur, Kelly Watters, Ann C Palmenberg, John Yin
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    ABSTRACT: Infections by human rhinovirus (HRV) contribute to a diversity of human illnesses that include the common cold, bronchitis, exacerbation of asthma and other chronic lung diseases. Previous studies have shown that HRVs grouped under three different species (HRV-A, B and C) span over 150 different genotypes and vary in their virulence and the severity of their infections. In parallel with clinical studies, genome sequencing has revealed a high level of diversity in protein sequences of the HRV 2Aprotease, an enzyme involved in viral polyprotein processing and shut off of host cell functions that can inhibit viral replication. Different 2Aproteases target and cleave different nuclear pore proteins (Nups), and thereby disrupt nucleocytoplasmic signaling and transport pathways. This suggests that the proteases might have different enzymatic activities and carry-out their anti-host functions with unique mechanistic signatures. Here we compared the ability of different HRV 2A proteases to disrupt nuclear transport pathways using a novel cellular time-lapse fluorescent transfection assay. We created stable HeLa cells expressing fluorescent proteins fused to four different nuclear localizations signals, and we transfected these host cells with RNAs encoding different HRV 2A proteases. We observed the nuclear reporter protein accumulation in the cytoplasm/nucleus in real-time, and we employed a custom-developed java-based graphical interface to extract and analyze the fluorescent intensities from individually identified cells. This enabled us to quantify differences between HRV 2A proteases in their ability to disrupt nuclear transport pathways. We found that all proteases disrupted active import of a fluorescent reporter protein by the importin-α/β, transportin-1, and transportin-3 pathways as well as active export by the Crm1 pathway; however, different proteases inhibited these nuclear transport pathways with different efficiencies. We extended these studies to probe the spatial and temporal dynamics of spreading HRV infections using a dual color reporter system. Reporter HeLa cell monolayers were infected by fluorescent HRV strains where individual cells were tracked through the course of the infection; analysis of the spatial and temporal expression behaviors of individual cells was obtained for HRV strains expressing different 2A proteases, and these single-cell profiles were correlated with different spatial patterns of HRV infection spread. Overall, this work provides a foundation for linking how HRV diversity at the level of encoded 2A proteases may contribute to differential severity of HRV infections as they spread, which can guide the development of improved treatment strategies for HR-related illnesses.
    14 AIChE Annual Meeting; 11/2014
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    ABSTRACT: Cardiovirus Leader (L) proteins induce potent antihost inhibition of active cellular nucleocytoplasmic trafficking by triggering aberrant hyperphosphorylation of nuclear pore proteins (Nup). To achieve this, L binds protein RanGTPase (Ran), a key trafficking regulator, and diverts it into tertiary or quaternary complexes with required kinases. The activity of L is regulated by two phosphorylation events not required for Ran binding. Matched NMR studies on the unphosphorylated, singly, and doubly phosphorylated variants of Mengovirus L (LM) show both modifications act together to partially stabilize a short internal α-helix comprising LM residues 43-46. This motif implies that ionic and Van der Waals forces contributed by phosphorylation help organize downstream residues 48-67 into a new interface. The full structure of LM as bound to Ran (unlabeled) and Ran (216 aa) as bound by LM (unlabeled) places LM into the BP1 binding site of Ran, wrapped by the conformational flexible COOH tail. The arrangement explains the tight KD for this complex and places the LM zinc finger and phosphorylation interface as surface exposed and available for subsequent reactions. The core structure of Ran, outside the COOH tail, is not altered by LM binding and remains accessible for canonical RanGTP partner interactions. Pull-down assays identify at least one putative Ran:LM partner as an exportin, Crm1, or CAS. A model of Ran:LM:Crm1, based on the new structures suggests LM phosphorylation status may mediate Ran's selection of exportin(s) and cargo(s), perverting these native trafficking elements into the lethal antihost Nup phosphorylation pathways.
    Proceedings of the National Academy of Sciences 10/2014; 111(44). DOI:10.1073/pnas.1411098111 · 9.81 Impact Factor
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    ABSTRACT: The Leader (L) and 2A proteins of cardioviruses are the primary anti-host agents produced during infection. For encephalomyocarditis virus (EMCV), the prototype of this genus, 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 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 RanGTPase in the nucleus, and Ran can displace 2A from L. The equivalent proteins from TMEV (BeAn) and Saffold virus interact similarly in any subtype combination, with varying 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 co-dependent phenotypes of L and 2A mutations.
    Journal of Virology 09/2014; 88(22). DOI:10.1128/JVI.02148-14 · 4.65 Impact Factor
  • Holly A Basta, Ann C Palmenberg
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    ABSTRACT: Cardioviruses of the Encephalomyocarditis virus (EMCV) and Theilovirus species encode small, amino-terminal proteins called Leaders (L). Phosphorylation of the EMCV L (LE) at two distinct sites by CK2 and Syk kinases is important for virus-induced Nup phosphorylation and nucleocytoplasmic trafficking inhibition. Despite similar biological activities, the LE phosphorylation sites are not conserved in the Theiloviruses, Saffold virus (LS, SafV) or Theiler׳s murine encephalitis virus (LT, TMEV) sequences even though these proteins also become phosphorylated in cells and cell-free extracts. Site prediction algorithms, combined with panels of site-specific protein mutations now identify analogous, but not homologous phosphorylation sites in the Ser/Thr and Theilo protein domains of LT and LS, respectively. In both cases, recombinant AMP-activated kinase (AMPK) was reactive with the proteins at these sites, and also with LE, modifying the same residue recognized by CK2.
    Virology 07/2014; 462-463C(1):236-240. DOI:10.1016/j.virol.2014.06.026 · 3.28 Impact Factor
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    ABSTRACT: Human rhinovirus strains differ greatly in their virulence, and this has been correlated with the differing substrate specificity of the respective 2A protease (2Apro). Rhinoviruses use their 2Apro to cleave a spectrum of cellular proteins important to virus replication and anti-host activities. These enzymes share a chymotrypsin-like fold stabilized by a tetra-coordinated zinc ion. The catalytic triad consists of conserved Cys (C105), His (H34), and Asp (D18) residues. We used a semi-automated NMR protocol developed at NMRFAM to determine the solution structure of 2Apro (C105A variant) from an isolate of the clinically important rhinovirus C species (RV-C). The backbone of C2 2Apro superimposed closely (1.41-1.81 Å rmsd) with those of orthologs from RV-A2, coxsackie B4 (CB4), and enterovirus 71 (EV71) having sequence identities between 40% and 60%. Comparison of the structures suggest that the differential functional properties of C2 2Apro stem from its unique surface charge, high proportion of surface aromatics, and sequence surrounding the di-tyrosine flap.
    PLoS ONE 06/2014; 9(6):e97198. DOI:10.1371/journal.pone.0097198 · 3.53 Impact Factor
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    ABSTRACT: Human rhinovirus (RV) isolates from the RV-C species are recently discovered infectious agents that are closely linked to asthma and wheezing etiologies in infants. Clinical study samples collected at the University of Wisconsin-Madison describe 41 nearly complete genome sequences representing 21 RV-C genotypes.
    Genome Announcements 03/2014; 2(2). DOI:10.1128/genomeA.00203-14
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    ABSTRACT: Full-length or nearly full-length RNA genome sequences for 98 rhinovirus (RV) A isolates (from the Enterovirus genus of the Picornaviridae family), representing 43 different genotypes, were resolved as part of ongoing studies to define RV genetic diversity and its potential link to respiratory disease.
    Genome Announcements 03/2014; 2(2). DOI:10.1128/genomeA.00200-14
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    ABSTRACT: Nearly full-length RNA genome sequences for 39 rhinovirus B isolates (RV-B), representing 13 different genotypes, were resolved as part of ongoing studies at the University of Wisconsin that attempt to link rhinovirus (RV) diversity and respiratory disease in infants.
    Genome Announcements 03/2014; 2(2). DOI:10.1128/genomeA.00202-14
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    Holly A. Basta, Jean-Yves Sgro, Ann C. Palmenberg
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    ABSTRACT: Features of human rhinovirus (RV)-C virions that allow them to use novel cell receptors and evade immune responses are unknown. Unlike the RV-A+B, these isolates cannot be propagated in typical culture systems or grown for structure studies. Comparative sequencing, I-TASSER, MODELLER, ROBETTA, and refined alignment techniques led to a structural approximation for C15 virions, based on the extensive, resolved RV-A+B datasets. The model predicts that all RV-C VP1 proteins are shorter by 21 residues relative to the RV-A, and 35 residues relative to the RV-B, effectively shaving the RV 5-fold plateau from the particle. There are major alterations in VP1 neutralizing epitopes and the structural determinants for ICAM-1 and LDLR receptors. The VP2 and VP3 elements are similar among all RV, but the loss of sequence “words” contributing Nim1ab has increased the apparent selective pressure among the RV-C to fix mutations elsewhere in the VP1, creating a possible compensatory epitope.
    Virology 01/2014; 448:176–184. DOI:10.1016/j.virol.2013.10.006 · 3.28 Impact Factor
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    ABSTRACT: Human rhinoviruses of the RV-C species are recently discovered pathogens with greater clinical significance than isolates in the RV-A+B species. The RV-C cannot be propagated in typical culture systems; so much of the virology is necessarily derivative, relying on comparative genomics, relative to the better studied RV-A+B. We developed a bioinformatics-based structural model for a C15 isolate. The model showed the VP1-3 capsid proteins retain their fundamental cores relative to the RV-A+B, but conserved, internal RV-C residues affect the shape and charge of the VP1 hydrophobic pocket that confers antiviral drug susceptibility. When predictions of the model were tested in organ cultures or ALI systems with recombinant C15 virus, there was a resistance to capsid-binding drugs, including pleconaril, BTA-188, WIN56291, WIN52035 and WIN52084. Unique to all RV-C, the model predicts conserved amino acids within the pocket and capsid surface pore leading to the pocket may correlate with this activity.
    Virology 01/2014; 448C:82-90. DOI:10.1016/j.virol.2013.10.004 · 3.28 Impact Factor
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    ABSTRACT: 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 NMR structure of LE from encephalomyocarditis virus (EMCV) has been determined. The protein has an NH2-proximal CHCC zinc-finger, 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, anti-host 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 anti-viral 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), 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 LS and LT are both activated by alternative kinases in different cell types, probably reactive within the Theilo-specific domains.
    Journal of Virology 12/2013; 88(4). DOI:10.1128/JVI.03150-13 · 4.65 Impact Factor
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    ABSTRACT: The CACNA1A gene, encoding the voltage-gated calcium channel subunit α1A, is involved in pre- and postsynaptic Ca(2+) signaling, gene expression, and several genetic neurological disorders. We found that CACNA1A coordinates gene expression using a bicistronic mRNA bearing a cryptic internal ribosomal entry site (IRES). The first cistron encodes the well-characterized α1A subunit. The second expresses a transcription factor, α1ACT, which coordinates expression of a program of genes involved in neural and Purkinje cell development. α1ACT also contains the polyglutamine (polyQ) tract that, when expanded, causes spinocerebellar ataxia type 6 (SCA6). When expressed as an independent polypeptide, α1ACT-bearing an expanded polyQ tract-lacks transcription factor function and neurite outgrowth properties, causes cell death in culture, and leads to ataxia and cerebellar atrophy in transgenic mice. Suppression of CACNA1A IRES function in SCA6 may be a potential therapeutic strategy.
    Cell 07/2013; 154(1):118-33. DOI:10.1016/j.cell.2013.05.059 · 33.12 Impact Factor
  • Valjean R Bacot-Davis, Ann C Palmenberg
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    ABSTRACT: 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.
    Virology 05/2013; 443(1). DOI:10.1016/j.virol.2013.05.002 · 3.28 Impact Factor
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    Ryan V Petty, Ann C Palmenberg
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    ABSTRACT: The Leader (L) protein of encephalomyocarditis virus (EMCV) shuts off host cell nucleocytoplasmic trafficking (NCT) by inducing hyperphosphorylation of nuclear pore proteins. This dramatic effect by a non-enzymatic protein of 6 kDa is not well understood, but clearly involves L binding to cellular Ran GTPase, a critical factor of active NCT. Exogenous GDP and GTP are inhibitory to L-Ran binding, but guanine-nucleotide exchange factor, RCC1, can relieve this inhibition. In the presence of RCC1, L binds Ran with a KD of ∼3 nM and reaches saturation within 20 minutes. Fluorescent nucleotide experiments suggest that L-Ran interactions impact the nucleotide-binding pocket of Ran.
    Journal of Virology 03/2013; 87(11). DOI:10.1128/JVI.02493-12 · 4.65 Impact Factor
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    ABSTRACT: Although picornavirus RNA genomes contain a 3'-terminal poly(A) tract that is critical for their replication, the impact of encephalomyocarditis virus (EMCV) infection on the host poly(A)-binding protein (PABP) remains unknown. Here, we establish that EMCV infection stimulates site-specific PABP proteolysis, resulting in accumulation of a 45-kDa N-terminal PABP fragment in virus-infected cells. Expression of a functional EMCV 3C proteinase was necessary and sufficient to stimulate PABP cleavage in uninfected cells, and bacterially expressed 3C cleaved recombinant PABP in vitro in the absence of any virus-encoded or eukaryotic cellular cofactors. N-terminal sequencing of the resulting C-terminal PABP fragment identified a 3C(pro) cleavage site on PABP between amino acids Q437 and G438, severing the C-terminal protein-interacting domain from the N-terminal RNA binding fragment. Single amino acid substitution mutants with changes at Q437 were resistant to 3C(pro) cleavage in vitro and in vivo, validating that this is the sole detectable PABP cleavage site. Finally, while ongoing protein synthesis was not detectably altered in EMCV-infected cells expressing a cleavage-resistant PABP variant, viral RNA synthesis and infectious virus production were both reduced. Together, these results establish that the EMCV 3C proteinase mediates site-specific PABP cleavage and demonstrate that PABP cleavage by 3C regulates EMCV replication.
    Journal of Virology 07/2012; 86(19):10686-94. DOI:10.1128/JVI.00896-12 · 4.65 Impact Factor

Publication Stats

5k Citations
639.57 Total Impact Points


  • 1980–2015
    • University of Wisconsin–Madison
      • • Institute for Molecular Virology
      • • Department of Biochemistry
      • • Graduate School
      Madison, Wisconsin, United States
  • 2010
    • The Pirbright Institute
      Woking, England, United Kingdom
  • 2000
    • Medical College of Wisconsin
      Milwaukee, Wisconsin, United States
  • 1997
    • Loyola University Chicago
      • Department of Microbiology and Immunology
      Chicago, Illinois, United States
  • 1987
    • Louisiana State University
      Baton Rouge, Louisiana, United States
  • 1986
    • The University of Chicago Medical Center
      • Department of Neurology
      Chicago, Illinois, United States