Bio

Academic Appointments


Administrative Appointments


  • Chair, Stanford University School of Medicine - Microbiology & Immunology (2006 - 2010)

Research & Scholarship

Current Research and Scholarly Interests


For many subcellular viruses and parasites, RNA, not DNA, is the carrier of genetic information. This has several interesting consequences for the genetics and biology of the virus. Poliovirus serves as a model to increase our understanding of positive-strand RNA viruses for which no vaccine is available and which remain a significant health hazard: examples include other picornaviruses, such as rhinoviruses, coxsackieviruses and the deadly enterovirus 71 as well as more distantly related positive-strand RNA viruses such as hepatitis C and Dengue fever.

Questions currently under scrutiny are posed below, and discussed in greater detail in our web site.

1. How does the biochemistry of RNA-dependent RNA polymerases affect the biology of RNA viruses?

2. How are the membranous structures on which viral RNA replication complexes assemble form, and
from what intracellular organelles do they derive?

3. Why are the genetic properties of many RNA genomes different from DNA genomes? How does the error-prone nature of RNA-dependent RNA replication and the membrane association of the RNA replication complexes affect these genetic properties?

4. How does the inhibition of the protein secretory apparatus by the 3A and 2B proteins of picornaviruses such as poliovirus affect their pathogenesis? What would happen to the secretion of interferons, and to the presentation of antigens in the context of MHC class I molecules, if the host secretory pathway were not inhibited during infection by polioviruses, rhinoviruses and coxsackieviruses?

Teaching

2013-14 Courses


Postdoctoral Advisees


Graduate and Fellowship Programs


Publications

Journal Articles


  • Potential subversion of autophagosomal pathway by picornaviruses AUTOPHAGY Taylor, M. P., Kirkegaard, K. 2008; 4 (3): 286-289

    Abstract

    The RNA replication complexes of small positive-strand RNA viruses such as poliovirus are known to form on the surfaces of membranous vesicles in the cytoplasm of infected mammalian cells. These membranes resemble cellular autophagosomes in their double-membraned morphology, cytoplasmic lumen, lipid-rich composition and the presence of cellular proteins LAMP 1 and LC3. Furthermore, LC3 protein is covalently modified during poliovirus infection in a manner indistinguishable from that observed during bona fide autophagy. This covalent modification can also be induced by the expression of viral protein 2BC in isolation. However, differences between poliovirus-induced vesicles and autophagosomes also exist: the viral-induced membranes are smaller, at 200-400 nm in diameter, and can be induced by the combination of two viral proteins, termed 2BC and 3A. Experimental suppression of expression of proteins in the autophagy pathway was found to reduce viral yield, arguing that this pathway facilitates viral infection, rather than clearing it. We have hypothesized that, in addition to providing membranous surfaces for assembly of viral RNA replication complexes, double-membraned vesicles provide a topological mechanism to deliver cytoplasmic contents, including mature virus, to the extracellular milieu without lysing the cell.

    View details for Web of Science ID 000254477400006

    View details for PubMedID 18094610

  • Modification of cellular autophagy protein LC3 by poliovirus JOURNAL OF VIROLOGY Taylor, M. P., Kirkegaard, K. 2007; 81 (22): 12543-12553

    Abstract

    Poliovirus infection remodels intracellular membranes, creating a large number of membranous vesicles on which viral RNA replication occurs. Poliovirus-induced vesicles display hallmarks of cellular autophagosomes, including delimiting double membranes surrounding the cytosolic lumen, acquisition of the endosomal marker LAMP-1, and recruitment of the 18-kDa host protein LC3. Autophagy results in the covalent lipidation of LC3, conferring the property of membrane association to this previously microtubule-associated protein and providing a biochemical marker for the induction of autophagy. Here, we report that a similar modification of LC3 occurs both during poliovirus infection and following expression of a single viral protein, a stable precursor termed 2BC. Therefore, one of the early steps in cellular autophagy, LC3 modification, can be genetically separated from the induction of double-membraned vesicles that contain the modified LC3, which requires both viral proteins 2BC and 3A. The existence of viral inducers that promote a distinct aspect of the formation of autophagosome-like membranes both facilitates the dissection of this cellular process and supports the hypothesis that this branch of the innate immune response is directly subverted by poliovirus.

    View details for DOI 10.1128/JVI.00755-07

    View details for Web of Science ID 000254065400045

    View details for PubMedID 17804493

  • Intramolecular and intermolecular uridylylation by poliovirus RNA-dependent RNA polymerase JOURNAL OF VIROLOGY Richards, O. C., Spagnolo, J. F., Lyle, J. M., Vleck, S. E., Kuchta, R. D., Kirkegaard, K. 2006; 80 (15): 7405-7415

    Abstract

    The 22-amino-acid protein VPg can be uridylylated in solution by purified poliovirus 3D polymerase in a template-dependent reaction thought to mimic primer formation during RNA amplification in infected cells. In the cell, the template used for the reaction is a hairpin RNA termed 2C-cre and, possibly, the poly(A) at the 3' end of the viral genome. Here, we identify several additional substrates for uridylylation by poliovirus 3D polymerase. In the presence of a 15-nucleotide (nt) RNA template, the poliovirus polymerase uridylylates other polymerase molecules in an intermolecular reaction that occurs in a single step, as judged by the chirality of the resulting phosphodiester linkage. Phosphate chirality experiments also showed that VPg uridylylation can occur by a single step; therefore, there is no obligatory uridylylated intermediate in the formation of uridylylated VPg. Other poliovirus proteins that could be uridylylated by 3D polymerase in solution were viral 3CD and 3AB proteins. Strong effects of both RNA and protein ligands on the efficiency and the specificity of the uridylylation reaction were observed: uridylylation of 3D polymerase and 3CD protein was stimulated by the addition of viral protein 3AB, and, when the template was poly(A) instead of the 15-nt RNA, the uridylylation of 3D polymerase itself became intramolecular instead of intermolecular. Finally, an antiuridine antibody identified uridylylated viral 3D polymerase and 3CD protein, as well as a 65- to 70-kDa host protein, in lysates of virus-infected human cells.

    View details for DOI 10.1128/JVI.02533-05

    View details for Web of Science ID 000239189100013

    View details for PubMedID 16840321

  • Bottleneck-mediated quasispecies restriction during spread of an RNA virus from inoculation site to brain PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pfeiffer, J. K., Kirkegaard, K. 2006; 103 (14): 5520-5525

    Abstract

    The amplification of RNA viruses such as poliovirus is associated with high error rates, and the resulting diversity likely facilitates viral survival within an infected host. However, within individual tissues of infected hosts, there may be barriers to viral spread that limit genome sampling. We tested whether poliovirus population diversity was maintained during viral spread to the brain of poliovirus receptor-expressing mice. Each of four restriction enzyme site-tagged viruses was shown to be able to replicate in the mouse brain. However, when infection was initiated by i.m., i.v., or i.p. routes, only a subset of the members of the injected pool was detectable in the brain. This jackpot effect was the result of a bottleneck in viral transit from the inoculation site to the brain. The bottleneck was difficult to overcome, requiring a 10(7) increase in viral inoculum to allow representation of all or most members of the infecting pool. Therefore, the bottleneck is not likely to be a physical barrier but an antiviral state induced by a founder virus. We suggest that the innate immune response can limit viral pathogenicity by limiting the number and therefore the diversity of viruses during spread to vulnerable tissues.

    View details for DOI 10.1073/pnas.0600834103

    View details for Web of Science ID 000236636400051

    View details for PubMedID 16567621

  • Nucleotide channel of RNA-dependent RNA polymerase used for intermolecular uridylylation of protein primer JOURNAL OF MOLECULAR BIOLOGY Tellez, A. B., Crowder, S., Spagnolo, J. F., Thompson, A. A., Peersen, O. B., Brutlag, D. L., Kirkegaard, K. 2006; 357 (2): 665-675

    Abstract

    Poliovirus VPg is a 22 amino acid residue peptide that serves as the protein primer for replication of the viral RNA genome. VPg is known to bind directly to the viral RNA-dependent RNA polymerase, 3D, for covalent uridylylation, yielding mono and di-uridylylated products, VPg-pU and VPg-pUpU, which are subsequently elongated. To model the docking of the VPg substrate to a putative VPg-binding site on the 3D polymerase molecule, we performed a variety of structure-based computations followed by experimental verification. First, potential VPg folded structures were identified, yielding a suite of predicted beta-hairpin structures. These putative VPg structures were then docked to the region of the polymerase implicated by genetic experiments to bind VPg, using grid-based and fragment-based methods. Residues in VPg predicted to affect binding were identified through molecular dynamics simulations, and their effects on the 3D-VPg interaction were tested computationally and biochemically. Experiments with mutant VPg and mutant polymerase molecules confirmed the predicted binding site for VPg on the back side of the polymerase molecule during the uridylylation reaction, opposite to that predicted to bind elongating RNA primers.

    View details for DOI 10.1016/j.jmb.2005.12.044

    View details for Web of Science ID 000236120200027

    View details for PubMedID 16427083

  • Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice PLOS PATHOGENS Pfeiffer, J. K., Kirkegaard, K. 2005; 1 (2): 102-110

    Abstract

    RNA viruses have high error rates, and the resulting quasispecies may aid survival of the virus population in the presence of selective pressure. Therefore, it has been theorized that RNA viruses require high error rates for survival, and that a virus with high fidelity would be less able to cope in complex environments. We previously isolated and characterized poliovirus with a mutation in the viral polymerase, 3D-G64S, which confers resistance to mutagenic nucleotide analogs via increased fidelity. The 3D-G64S virus was less pathogenic than wild-type virus in poliovirus-receptor transgenic mice, even though only slight growth defects were observed in tissue culture. To determine whether the high-fidelity phenotype of the 3D-G64S virus could decrease its fitness under a defined selective pressure, we compared growth of the 3D-G64S virus and 3D wild-type virus in the context of a revertible attenuating point mutation, 2C-F28S. Even with a 10-fold input advantage, the 3D-G64S virus was unable to compete with 3D wild-type virus in the context of the revertible attenuating mutation; however, in the context of a non-revertible version of the 2C-F28S attenuating mutation, 3D-G64S virus matched the replication of 3D wild-type virus. Therefore, the 3D-G64S high-fidelity phenotype reduced viral fitness under a defined selective pressure, making it likely that the reduced spread in murine tissue could be caused by the increased fidelity of the viral polymerase.

    View details for DOI 10.1371/journal.ppat.0010011

    View details for Web of Science ID 000202893800002

    View details for PubMedID 16220146

  • Topology of double-membraned vesicles and the opportunity for non-lytic release of cytoplasm AUTOPHAGY Kirkegaard, K., Jackson, W. T. 2005; 1 (3): 182-184

    Abstract

    Infection of mammalian cells with several positive-strand RNA viruses induces double-membraned vesicles whose cytosolic surfaces serve as platforms for viral RNA replication. Our recent publication (Jackson et al. PLoS Biol 2005; 3:861-71) chronicled several similarities between poliovirus-induced membranes and autophagosomes, including induced co-localization of GFP-LC3 and LAMP1. Occasionally, the cytosolic lumen of these structures also contains viral particles; this likely results from wrapping of cytosol, which can contain high viral concentrations late in infection, by newly formed double membranes. Interestingly, RNAi treatment to reduce LC3 or Atg12p concentrations reduced yields of extracellular virus even more than intracellular virus. It is often assumed that exit of non-enveloped viruses such as poliovirus requires cell lysis. However, we hypothesize that autophagosome-like double-membranes, which can become single-membraned upon maturation, provide a long-sought mechanism for the observed non-lytic release of cytoplasmic viruses and possibly other cytoplasmic material resistant to the environment of maturing autophagosomes.

    View details for Web of Science ID 000238564100011

    View details for PubMedID 16874042

  • Trans-dominant inhibition of RNA viral replication can slow growth of drug-resistant viruses NATURE GENETICS Crowder, S., Kirkegaard, K. 2005; 37 (7): 701-709

    Abstract

    The high error rates of viral RNA-dependent RNA polymerases create heterogeneous viral populations whose disparate RNA genomes affect each other's survival. We systematically screened the poliovirus genome and identified four sets of dominant mutations. Mutated alleles in capsid- and polymerase-coding regions resulted in dominant negative phenotypes, probably due to the proteins' oligomeric properties. We also identified dominant mutations in an RNA element required for priming RNA synthesis (CRE) and in the protein primer (VPg), suggesting that nonproductive priming intermediates are inhibitory. Mutations that inhibit the activity of viral proteinase 2A were dominant, arguing that inhibition of its known intramolecular activity creates a toxic product. Viral products that, when defective, dominantly interfere with growth of nondefective viruses will probably be excellent drug targets because drug-sensitive viruses should be dominant over drug-resistant variants. Accordingly, a virus sensitive to anticapsid compound WIN51711 dominantly inhibited the intracellular growth of a drug-resistant virus. Therefore, dominant inhibitor screening should validate or predict targets for antiviral therapy with reduced risk for drug resistance.

    View details for DOI 10.1038/ng1583

    View details for Web of Science ID 000230196400016

    View details for PubMedID 15965477

  • Inhibition of cellular protein secretion by picornaviral 3A proteins VIROLOGY Choe, S. S., Dodd, D. A., Kirkegaard, K. 2005; 337 (1): 18-29

    Abstract

    During poliovirus infection, anterograde traffic between the endoplasmic reticulum and the Golgi is inhibited due to the action of 3A, an 87 amino acid viral protein. The ability of poliovirus protein 3A to inhibit ER-to-Golgi traffic is not required for virus growth. Instead, we have suggested that the inhibition of host protein secretion, shown to reduce the secretion of interferon-beta, IL-6, and IL-8 and the expression of both newly synthesized MHC class I and TNF receptor in the plasma membrane of infected cells, affects growth in host organisms. To determine whether the ability of poliovirus 3A to inhibit ER-to-Golgi traffic is conserved, the ability of 3A proteins from several picornaviruses, including human rhinovirus 14, foot-and-mouth disease virus, enterovirus 71, hepatitis A, and Theiler's virus, was tested. Only the 3A proteins from another poliovirus, Sabin 3, and closely related coxsackievirus B3 inhibited ER-to-Golgi traffic as effectively as the 3A protein from poliovirus Mahoney type 1. Site-directed mutagenesis based on these findings and the three-dimensional structure of the amino-terminal domain of poliovirus 3A protein revealed that residues in the unstructured amino terminus of 3A are critical for the inhibition of host protein secretion.

    View details for DOI 10.1016/j.virol.2005.03.036

    View details for Web of Science ID 000229670700003

    View details for PubMedID 15914217

  • Allosteric effects of ligands and mutations on poliovirus RNA-dependent RNA polymerase JOURNAL OF VIROLOGY Boerner, J. E., Lyle, J. M., Daijogo, S., Semler, B. L., SCHULTZ, S. C., Kirkegaard, K., Richards, O. C. 2005; 79 (12): 7803-7811

    Abstract

    Protein priming of viral RNA synthesis plays an essential role in the replication of picornavirus RNA. Both poliovirus and coxsackievirus encode a small polypeptide, VPg, which serves as a primer for addition of the first nucleotide during synthesis of both positive and negative strands. This study examined the effects on the VPg uridylylation reaction of the RNA template sequence, the origin of VPg (coxsackievirus or poliovirus), the origin of 3D polymerase (coxsackievirus or poliovirus), the presence and origin of interacting protein 3CD, and the introduction of mutations at specific regions in the poliovirus 3D polymerase. Substantial effects associated with VPg origin were traced to differences in VPg-polymerase interactions. The effects of 3CD proteins and mutations at polymerase-polymerase intermolecular Interface I were most consistent with allosteric effects on the catalytic 3D polymerase molecule. In conclusion, the efficiency and specificity of VPg uridylylation by picornavirus polymerases is greatly influenced by allosteric effects of ligand binding that are likely to be relevant during the viral replicative cycle.

    View details for DOI 10.1128/JVI.79.12.7803-7811.2005

    View details for Web of Science ID 000229416100052

    View details for PubMedID 15919933

  • Subversion of cellular autophagosomal machinery by RNA viruses PLOS BIOLOGY Jackson, W. T., Giddings, T. H., Taylor, M. P., Mulinyawe, S., RABINOVITCH, M., Kopito, R. R., Kirkegaard, K. 2005; 3 (5): 861-871

    Abstract

    Infection of human cells with poliovirus induces the proliferation of double-membraned cytoplasmic vesicles whose surfaces are used as the sites of viral RNA replication and whose origin is unknown. Here, we show that several hallmarks of cellular autophagosomes can be identified in poliovirus-induced vesicles, including colocalization of LAMP1 and LC3, the human homolog of Saccharomyces cerevisiae Atg8p, and staining with the fluorophore monodansylcadaverine followed by fixation. Colocalization of LC3 and LAMP1 was observed early in the poliovirus replicative cycle, in cells infected with rhinoviruses 2 and 14, and in cells that express poliovirus proteins 2BC and 3A, known to be sufficient to induce double-membraned vesicles. Stimulation of autophagy increased poliovirus yield, and inhibition of the autophagosomal pathway by 3-methyladenine or by RNA interference against mRNAs that encode two different proteins known to be required for autophagy decreased poliovirus yield. We propose that, for poliovirus and rhinovirus, components of the cellular machinery of autophagosome formation are subverted to promote viral replication. Although autophagy can serve in the innate immune response to microorganisms, our findings are inconsistent with a role for the induced autophagosome-like structures in clearance of poliovirus. Instead, we argue that these double-membraned structures provide membranous supports for viral RNA replication complexes, possibly enabling the nonlytic release of cytoplasmic contents, including progeny virions, from infected cells.

    View details for DOI 10.1371/journal.pbio.0030156

    View details for Web of Science ID 000229125400014

    View details for PubMedID 15884975

  • Ribavirin resistance in hepatitis C virus replicon-containing cell lines conferred by changes in the cell line or mutations in the replicon RNA JOURNAL OF VIROLOGY Pfeiffer, J. K., Kirkegaard, K. 2005; 79 (4): 2346-2355

    Abstract

    Ribavirin (RBV), used in combination with alpha interferon to treat hepatitis C virus (HCV) infections, is a guanosine nucleotide analog that can increase the error rate of viral RNA-dependent RNA polymerases, imbalance intracellular nucleotide pools, and cause toxicity in many cell types. To determine potential mechanisms of RBV resistance during HCV RNA replication, we passaged HCV replicon-containing cell lines in the presence of increasing concentrations of RBV. RBV-resistant, HCV replicon-containing cell lines were generated, and the majority of RBV resistance was found to be conferred by changes in the cell lines. The resistant cell lines were defective in RBV import, as measured by [(3)H]RBV uptake experiments. These cell lines displayed reduced RBV toxicity and reduced error accumulation during infection with poliovirus, whose replication is known to be sensitive to RBV-induced error. For one RBV-resistant isolate, two mutations in the replicon RNA contributed to the observed phenotype. Two responsible mutations resided in the C-terminal region of NS5A, G404S, and E442G and were each sufficient for low-level RBV resistance. Therefore, RBV resistance in HCV replicon cell lines can be conferred by changes in the cell line or by mutations in the HCV replicon.

    View details for DOI 10.1128/JVI.79.4.2346-2355.2005

    View details for Web of Science ID 000226772100036

    View details for PubMedID 15681435

  • Intracellular topology and epitope shielding of poliovirus 3A protein JOURNAL OF VIROLOGY Choe, S. S., Kirkegaard, K. 2004; 78 (11): 5973-5982

    Abstract

    The poliovirus RNA replication complex comprises multiple viral and possibly cellular proteins assembled on the cytoplasmic surface of rearranged intracellular membranes. Viral proteins 3A and 3AB perform several functions during the poliovirus replicative cycle, including significant roles in rearranging membranes, anchoring the viral polymerase to these membranes, inhibiting host protein secretion, and possibly providing the 3B protein primer for RNA synthesis. During poliovirus infection, the immunofluorescence signal of an amino-terminal epitope of 3A-containing proteins is markedly shielded compared to 3A protein expressed in the absence of other poliovirus proteins. This is not due to luminal orientation of all or a subset of the 3A-containing polypeptides, as shown by immunofluorescence following differential permeabilization and proteolysis experiments. Shielding of the 3A epitope is more pronounced in cells infected with wild-type poliovirus than in cells with temperature-sensitive mutant virus that contains a mutation in the 3D polymerase coding region adjacent to the 3AB binding site. Therefore, it is likely that direct binding of the poliovirus RNA-dependent RNA polymerase occludes the amino terminus of 3A-containing polypeptides in the RNA replication complex.

    View details for DOI 10.1128/JVI.78.11.5973-5982.2004

    View details for Web of Science ID 000221513400046

    View details for PubMedID 15140995

  • Cellular autophagy: Surrender, avoidance and subversion by microorganisms NATURE REVIEWS MICROBIOLOGY Kirkegaard, K., Taylor, M. P., Jackson, W. T. 2004; 2 (4): 301-314

    View details for DOI 10.1038/nrmicro865

    View details for Web of Science ID 000220714800013

    View details for PubMedID 15031729

  • Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus JOURNAL OF VIROLOGY Konan, K. V., Giddings, T. H., Ikeda, M., Li, K., Lemon, S. M., Kirkegaard, K. 2003; 77 (14): 7843-7855

    Abstract

    The nonstructural proteins of hepatitis C virus (HCV) have been shown previously to localize to the endoplasmic reticulum (ER) when expressed singly or in the context of other HCV proteins. To determine whether the expression of HCV nonstructural proteins alters ER function, we tested the effect of expression of NS2/3/4A, NS4A, NS4B, NS4A/B, NS4B/5A, NS5A, and NS5B from genotype 1b HCV on anterograde traffic from the ER to the Golgi apparatus. Only the nominal precursor protein NS4A/B affected the rate of ER-to-Golgi traffic, slowing the rate of Golgi-specific modification of the vesicular stomatitis virus G protein expressed by transfection by approximately threefold. This inhibition of ER-to-Golgi traffic was not observed upon expression of the processed proteins NS4A and NS4B, singly or in combination. To determine whether secretion of other cargo proteins was inhibited by NS4A/B expression, we monitored the appearance of newly synthesized proteins on the cell surface in the presence and absence of NS4A/B expression; levels of all were reduced in the presence of NS4A/B. This reduction is also seen in cells that contain genome length HCV replicons: the rate of appearance of major histocompatibility complex class I (MHC-I) on the cell surface was reduced by three- to fivefold compared to that for a cured cell line. The inhibition of protein secretion caused by NS4A/B does not correlate with the ultrastructural changes leading to the formation a "membranous web" (D. Egger et al., J. Virol. 76:5974-5984, 2002), which can be caused by expression of NS4B alone. Inhibition of global ER-to-Golgi traffic could, by reducing cytokine secretion, MHC-I presentation, and transport of labile membrane proteins to the cell surface, have significant effects on the host immune response to HCV infection.

    View details for DOI 10.1128/JVI.77.14.7843-7855.2003

    View details for Web of Science ID 000183899200016

    View details for PubMedID 12829824

  • A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pfeiffer, J. K., Kirkegaard, K. 2003; 100 (12): 7289-7294

    Abstract

    Ribavirin is a nucleotide analog that can be incorporated by viral polymerases, causing mutations by allowing base mismatches. It is currently used therapeutically as an antiviral drug during hepatitis C virus infections. During the amplification of poliovirus genomic RNA or hepatitis C replicons, error frequency is known to increase upon ribavirin treatment. This observation has led to the hypothesis that ribavirin's antiviral activity results from error catastrophe caused by increased mutagenesis of viral genomes. Here, we describe the generation of ribavirin-resistant poliovirus by serial viral passage in the presence of increasing concentrations of the drug. Ribavirin resistance can be caused by a single amino acid change, G64S, in the viral polymerase in an unresolved portion of the fingers domain. Compared with wild-type virus, ribavirin-resistant poliovirus displays increased fidelity of RNA synthesis in the absence of ribavirin and increased survival both in the presence of ribavirin and another mutagen, 5-azacytidine. Ribavirin-resistant poliovirus represents an unusual class of viral drug resistance: resistance to a mutagen through increased fidelity.

    View details for DOI 10.1073/pnas.1232294100

    View details for Web of Science ID 000183493500071

    View details for PubMedID 12754380

  • Visualization and functional analysis of RNA-dependent RNA polymerase lattices SCIENCE Lyle, J. M., Bullitt, E., Bienz, K., Kirkegaard, K. 2002; 296 (5576): 2218-2222

    Abstract

    Positive-strand RNA viruses such as poliovirus replicate their genomes on intracellular membranes of their eukaryotic hosts. Electron microscopy has revealed that purified poliovirus RNA-dependent RNA polymerase forms planar and tubular oligomeric arrays. The structural integrity of these arrays correlates with cooperative RNA binding and RNA elongation and is sensitive to mutations that disrupt intermolecular contacts predicted by the polymerase structure. Membranous vesicles isolated from poliovirus-infected cells contain structures consistent with the presence of two-dimensional polymerase arrays on their surfaces during infection. Therefore, host cytoplasmic membranes may function as physical foundations for two-dimensional polymerase arrays, conferring the advantages of surface catalysis to viral RNA replication.

    View details for Web of Science ID 000176379000058

    View details for PubMedID 12077417

  • Similar structural basis for membrane localization and protein priming by an RNA-dependent RNA polymerase JOURNAL OF BIOLOGICAL CHEMISTRY Lyle, J. M., Clewell, A., Richmond, K., Richards, O. C., Hope, D. A., SCHULTZ, S. C., Kirkegaard, K. 2002; 277 (18): 16324-16331

    Abstract

    Protein primers are used to initiate genomic synthesis of several RNA and DNA viruses, although the structural details of the primer-polymerase interactions are not yet known. Poliovirus polymerase binds with high affinity to the membrane-bound viral protein 3AB but uridylylates only the smaller peptide 3B in vitro. Mutational analysis of the polymerase identified four surface residues on the three-dimensional structure of poliovirus polymerase whose wild-type identity is required for 3AB binding. These mutants also decreased 3B uridylylation, arguing that the binding sites for the membrane tether and the protein primer overlap. Mutation of flanking residues between the 3AB binding site and the polymerase active site specifically decreased 3B uridylylation, likely affecting steps subsequent to binding. The physical overlap of sites for protein priming and membrane association should facilitate replication initiation in the membrane-associated complex.

    View details for DOI 10.1074/jbc.M112429200

    View details for Web of Science ID 000175510400143

    View details for PubMedID 11877407

  • Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase JOURNAL OF VIROLOGY Wang, Q. M., Hockman, M. A., Staschke, K., Johnson, R. B., Case, K. A., Lu, J. R., Parsons, S., Zhang, F. M., Rathnachalam, R., Kirkegaard, K., Colacino, J. M. 2002; 76 (8): 3865-3872

    Abstract

    The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, K(d), of approximately 22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 A. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.

    View details for DOI 10.1128/JVI.76.8.3865-3872.2002

    View details for Web of Science ID 000174520600029

    View details for PubMedID 11907226

  • Poliovirus 3A protein limits interleukin-6 (IL-6), IL-8, and beta interferon secretion during viral infection JOURNAL OF VIROLOGY Dodd, D. A., Giddings, T. H., Kirkegaard, K. 2001; 75 (17): 8158-8165

    Abstract

    During viral infections, the host secretory pathway is crucial for both innate and acquired immune responses. For example, the export of most proinflammatory and antiviral cytokines, which recruit lymphocytes and initiate antiviral defenses, requires traffic through the host secretory pathway. To investigate potential effects of the known inhibition of cellular protein secretion during poliovirus infection on pathogenesis, cytokine secretion from cells infected with wild-type virus and with 3A-2, a mutant virus carrying an insertion in viral protein 3A which renders the virus defective in the inhibition of protein secretion, was tested. We show here that cells infected with 3A-2 mutant virus secrete greater amounts of cytokines interleukin-6 (IL-6), IL-8, and beta interferon than cells infected with wild-type poliovirus. Increased cytokine secretion from the mutant-infected cells can be attributed to the reduced inhibition of host protein secretion, because no significant differences between 3A-2- and wild-type-infected cells were observed in the inhibition of viral growth, host cell translation, or the ability of wild-type- or 3A-2-infected cells to support the transcriptional induction of beta interferon mRNA. We surmise that the wild-type function of 3A in inhibiting ER-to-Golgi traffic is not required for viral replication in tissue culture but, by altering the amount of secreted cytokines, could have substantial effects on pathogenesis within an infected host. The global inhibition of protein secretion by poliovirus may reflect a general mechanism by which pathogens that do not require a functional protein secretory apparatus can reduce the native immune response and inflammation associated with infection.

    View details for Web of Science ID 000170343900040

    View details for PubMedID 11483761

  • MHC I-dependent antigen presentation is inhibited by poliovirus protein 3A PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Deitz, S. B., Dodd, D. A., Cooper, S., Parham, P., Kirkegaard, K. 2000; 97 (25): 13790-13795

    Abstract

    The effects of poliovirus 3A protein expression and poliovirus infection on the presentation of hepatitis C virus antigens in cultured chimpanzee cells were examined. Expression of poliovirus 3A protein inhibits protein secretion when expressed in isolation and was sufficient to protect chimpanzee cells from lysis by hepatitis C virus-specific cytotoxic T cells in standard (51)Cr-release assays. Poliovirus infection also inhibited antigen presentation, as determined by decreased cytotoxic T cell activation. A mutation in 3A that abrogates the inhibition of protein secretion also abolished the effects of poliovirus on antigen presentation. These results demonstrate that the inhibition of secretion observed in poliovirus-infected cells substantially reduces the presentation of new antigens on the cell surface. These observations may reflect a general mechanism by which nonenveloped viruses such as poliovirus and other viruses that do not require a functional protein secretory apparatus can evade detection by the cellular immune response.

    View details for Web of Science ID 000165728800061

    View details for PubMedID 11095746

  • Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles JOURNAL OF VIROLOGY Suhy, D. A., Giddings, T. H., Kirkegaard, K. 2000; 74 (19): 8953-8965

    Abstract

    All positive-strand RNA viruses of eukaryotes studied assemble RNA replication complexes on the surfaces of cytoplasmic membranes. Infection of mammalian cells with poliovirus and other picornaviruses results in the accumulation of dramatically rearranged and vesiculated membranes. Poliovirus-induced membranes did not cofractionate with endoplasmic reticulum (ER), lysosomes, mitochondria, or the majority of Golgi-derived or endosomal membranes in buoyant density gradients, although changes in ionic strength affected ER and virus-induced vesicles, but not other cellular organelles, similarly. When expressed in isolation, two viral proteins of the poliovirus RNA replication complex, 3A and 2C, cofractionated with ER membranes. However, in cells that expressed 2BC, a proteolytic precursor of the 2B and 2C proteins, membranes identical in buoyant density to those observed during poliovirus infection were formed. When coexpressed with 2BC, viral protein 3A was quantitatively incorporated into these fractions, and the membranes formed were ultrastructurally similar to those in poliovirus-infected cells. These data argue that poliovirus-induced vesicles derive from the ER by the action of viral proteins 2BC and 3A by a mechanism that excludes resident host proteins. The double-membraned morphology, cytosolic content, and apparent ER origin of poliovirus-induced membranes are all consistent with an autophagic origin for these membranes.

    View details for Web of Science ID 000089244300020

    View details for PubMedID 10982339

  • Functional coupling between replication and packaging of poliovirus replicon RNA JOURNAL OF VIROLOGY Nugent, C. I., Johnson, K. L., Sarnow, P., Kirkegaard, K. 1999; 73 (1): 427-435

    Abstract

    Poliovirus RNA genomes that contained deletions in the capsid-coding regions were synthesized in monkey kidney cells that constitutively expressed T7 RNA polymerase. These replication-competent subgenomic RNAs, or replicons (G. Kaplan and V. R. Racaniello, J. Virol. 62:1687-1696, 1988), were encapsidated in trans by superinfecting polioviruses. When superinfecting poliovirus resistant to the antiviral compound guanidine was used, the RNA replication of the replicon RNAs could be inhibited independently of the RNA replication of the guanidine-resistant helper virus. Inhibiting the replication of the replicon RNA also profoundly inhibited its trans-encapsidation, even though the capsid proteins present in the cells could efficiently encapsidate the helper virus. The observed coupling between RNA replication and RNA packaging could account for the specificity of poliovirus RNA packaging in infected cells and the observed effects of mutations in the coding regions of nonstructural proteins on virion morphogenesis. It is suggested that this coupling results from direct interactions between the RNA replication machinery and the capsid proteins. The coupling of RNA packaging to RNA replication and of RNA replication to translation (J. E. Novak and K. Kirkegaard, Genes Dev. 8:1726-1737, 1994) could serve as mechanisms for late proofreading of poliovirus RNA, allowing only those RNA genomes capable of translating a full complement of functional RNA replication proteins to be propagated.

    View details for Web of Science ID 000077461700049

    View details for PubMedID 9847348

  • KH domain integrity is required for wild-type localization of Sam68 EXPERIMENTAL CELL RESEARCH McBride, A. E., Taylor, S. J., Shalloway, D., Kirkegaard, K. 1998; 241 (1): 84-95

    Abstract

    The protein Sam68 (Src-associated in mitosis, 68 kDa) has been found to bind to SH2 and to SH3 domain-containing proteins and to RNA. Although its protein-protein interactions implicate Sam68 in cell signaling, the significance of its RNA binding remains obscure. In most cells, Sam68 shows diffuse nucleoplasmic staining. Upon treatment with transcription inhibitors, however, Sam68 localize into punctate nuclear structures. Mutant forms of mouse Sam68 were overexpressed in human cells to test the importance of the KH domain, which is required for RNA binding, in the intracellular localization of Sam68. A small deletion within the KH domain (delta 206-218) or point mutation I184N had no effect upon the localization of overexpressed Sam68. Sam68 that contained a deletion of the entire KH domain (delta KH, delta 157-256) or point mutation G178E, however, localized to distinct nuclear spots. Furthermore, delta KH Sam68, unlike wild-type Sam68 and several other mutant Sam68 proteins, did not relocalize upon poliovirus infection and caused the normally cytoplasmic viral polymerase to localize to the nuclear spots. Thus both ongoing transcription and an intact KH domain are crucial determinants of the dynamic intracellular localization of Sam68.

    View details for Web of Science ID 000074191200009

    View details for PubMedID 9633516

  • Site size of cooperative single-stranded RNA binding by poliovirus RNA-dependent RNA polymerase JOURNAL OF BIOLOGICAL CHEMISTRY Beckman, M. T., Kirkegaard, K. 1998; 273 (12): 6724-6730

    Abstract

    The poliovirus RNA-dependent RNA polymerase binds cooperatively to single-stranded RNA. We have determined the minimal RNA-binding site size of the poliovirus polymerase using binding titration with oligonucleotides of increasing length. A dramatic increase in affinity was observed when the length of the oligo(U) increased from 8 to 10 nucleotides (nt), arguing that the minimal size of RNA for polymerase binding is 10 nt. Another increase in affinity seen as the oligo(U) reached 24 nt suggests that a 24-nucleotide RNA can be occupied by two polymerase molecules. Direct binding of wild-type polymerase to oligo(U)12 and oligo(U)24 RNAs showed differences in affinity and cooperativity consistent with this model. The increase in binding affinity seen for oligo(U)10 suggests either that the RNA-binding determinants are widely spaced on the polymerase structure or that a substantial conformational change in the polymerase occurs upon the filling of its RNA-binding site.

    View details for Web of Science ID 000072775900021

    View details for PubMedID 9506971

  • Inhibition of endoplasmic reticulum-to-Golgi traffic by poliovirus protein 3A: Genetic and ultrastructural analysis JOURNAL OF VIROLOGY Doedens, J. R., Giddings, T. H., Kirkegaard, K. 1997; 71 (12): 9054-9064

    Abstract

    Poliovirus protein 3A, only 87 amino acids in length, is a potent inhibitor of protein secretion in mammalian cells, blocking anterograde protein traffic from the endoplasmic reticulum (ER) to the Golgi complex. The function of viral protein 3A in blocking protein secretion is extremely sensitive to mutations near the N terminus of the protein. Deletion of the first 10 amino acids or insertion of a single amino acid between amino acids 15 and 16, a mutation that causes a cold-sensitive defect in poliovirus RNA replication, abrogates the inhibition of protein secretion although wild-type amounts of the mutant proteins are expressed. Immunofluorescence light microscopy and immunoelectron microscopy demonstrate that 3A protein, expressed in the absence of other viral proteins, colocalizes with membranes derived from the ER. The precise topology of 3A with respect to ER membranes is not known, but it is likely to be associated with the cytosolic surface of the ER. Although the glycosylation of 3A in translation extracts has been reported, we show that tunicamycin, under conditions in which glycosylation of cellular proteins is inhibited, has no effect on poliovirus growth. Therefore, glycosylation of 3A plays no functional role in the viral replicative cycle. Electron microscopy reveals that the ER dilates dramatically in the presence of 3A protein. The absence of accumulated vesicles and the swelling of the ER-derived membranes argues that ER-to-Golgi traffic is inhibited at the step of vesicle formation or budding from the ER.

    View details for Web of Science ID A1997YF89600012

    View details for PubMedID 9371562

  • Poliovirus RNA recombination in cell-free extracts RNA-A PUBLICATION OF THE RNA SOCIETY Tang, R. S., Barton, D. J., Flanegan, J. B., Kirkegaard, K. 1997; 3 (6): 624-633

    Abstract

    Poliovirus RNA has been shown to undergo homologous genetic recombination at a high frequency in infected human cells. Recently it has become possible to mimic the entire intracellular replicative cycle of poliovirus replication in cytoplasmic extracts prepared from HeLa cells, resulting in the generation of infectious poliovirions. The mechanism of poliovirus RNA recombination has been shown previously to be coupled to RNA replication, presumably by template switching during the replication of parental RNAs. Experiments were designed to test whether recombinant poliovirus RNA molecules are produced in a cell-free environment. Recombinant molecules generated bear marker sequences that can be detected physically by reverse transcription and PCR. We report here successful detection of poliovirus RNA recombination in a cell-free replication system. The frequency measured for cell-free RNA recombination between two polymorphic marker loci 656 nt apart was between 10(-2) and 10(-3) recombinants/genome, a frequency comparable to or slightly higher than that measured for RNA recombination in infected cells.

    View details for Web of Science ID A1997XB20800008

    View details for PubMedID 9174097

  • Cellular origin and ultrastructure of membranes induced during poliovirus infection JOURNAL OF VIROLOGY Schlegel, A., Giddings, T. H., Ladinsky, M. S., Kirkegaard, K. 1996; 70 (10): 6576-6588

    Abstract

    Poliovirus RNA replicative complexes are associated with cytoplasmic membranous structures that accumulate during viral infection. These membranes were immunoisolated by using a monoclonal antibody against the viral nonstructural protein 2C. Biochemical analysis of the isolated membranes revealed that several organelles of the host cell (lysosomes, trans-Golgi stack and trans-Golgi network, and endoplasmic reticulum) contributed to the virus-induced membranous structures. Electron microscopy of infected cells preserved by high-pressure freezing revealed that the virus-induced membranes contain double lipid bilayers that surround apparently cytosolic material. Immunolabeling experiments showed that poliovirus proteins 2C and 3D were localized to the same membranes as the cellular markers tested. The morphological and biochemical data are consistent with the hypothesis that autophagy or a similar host process is involved in the formation of the poliovirus-induced membranes.

    View details for Web of Science ID A1996VG12700006

    View details for PubMedID 8794292

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