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?
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?
Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication.
How do viruses spread from cell to cell? Enveloped viruses acquire their surrounding membranes by budding. If a newly enveloped virus has budded through the plasma membrane, it finds itself outside the cell immediately. If it has budded through the bounding membrane of an internal compartment such as the ER, the virus finds itself in the lumen, from which it can exit the cell via the conventional secretion pathway. Thus, although some enveloped viruses destroy the cells they infect, there is no topological need to do so. On the other hand, naked viruses such as poliovirus lack an external membrane. They are protein-nucleic acid complexes within the cytoplasm or nucleus of the infected cell, like a ribosome, a spliceosome or an aggregate of Huntingtin protein. The simplest way for such a particle to pass through the single lipid bilayer that separates it from the outside of the cell would be to violate the integrity of that bilayer. Thus, it is not surprising that the primary mode of exit for non-enveloped viruses is cell lysis. However, more complex exit strategies are possible, such as the creation of new compartments whose complex topologies allow the exit of cytoplasm and its contents without violating the integrity of the cell. Here we will discuss the non-lytic spread of poliovirus and recent observations of such compartments during viral infection with several different picornaviruses.
How do viruses spread from cell to cell? Enveloped viruses acquire their surrounding membranes by budding: either through the plasma membrane or an internal membrane of infected cells. Thus, a newly budded enveloped virus finds itself either in the extracellular milieu or in a lumenal compartment from which it can exit the cell by conventional secretion. On the other hand, naked viruses such as poliovirus, nodavirus, adenovirus, and SV40 lack an external membrane. They are simply protein-nucleic acid complexes within the cytoplasm or nucleus of the infected cell, and thus would seem to have no other exit route than cell lysis. We have presented the first documentation of nonlytic spread of a naked virus, and showed the interconnections between this event and the process or components of the autophagy pathway.
The emergence of drug resistance can defeat the successful treatment of pathogens that display high mutation rates, as exemplified by RNA viruses. Here we detail a new paradigm in which a single compound directed against a 'dominant drug target' suppresses the emergence of naturally occurring drug-resistant variants in mice and cultured cells. All new drug-resistant viruses arise during intracellular replication and initially express their phenotypes in the presence of drug-susceptible genomes. For the targets of most anti-viral compounds, the presence of these drug-susceptible viral genomes does not prevent the selection of drug resistance. Here we show that, for an inhibitor of the function of oligomeric capsid proteins of poliovirus, the expression of drug-susceptible genomes causes chimeric oligomers to form, thus rendering the drug-susceptible genomes dominant. The use of dominant drug targets should suppress drug resistance whenever multiple genomes arise in the same cell and express products in a common milieu.
The cell-to-cell spread of cytoplasmic constituents such as nonenveloped viruses and aggregated proteins is usually thought to require cell lysis. However, mechanisms of unconventional secretion have been described that bypass the secretory pathway for the extracellular delivery of cytoplasmic molecules. Components of the autophagy pathway, an intracellular recycling process, have been shown to play a role in the unconventional secretion of cytoplasmic signaling proteins. Poliovirus is a lytic virus, although a few examples of apparently nonlytic spread have been documented. Real demonstration of nonlytic spread for poliovirus or any other cytoplasmic constituent thought to exit cells via unconventional secretion requires demonstration that a small amount of cell lysis in the cellular population is not responsible for the release of cytosolic material. Here, we use quantitative time-lapse microscopy to show the spread of infectious cytoplasmic material between cells in the absence of lysis. siRNA-mediated depletion of autophagy protein LC3 reduced nonlytic intercellular viral transfer. Conversely, pharmacological stimulation of the autophagy pathway caused more rapid viral spread in tissue culture and greater pathogenicity in mice. Thus, the unconventional secretion of infectious material in the absence of cell lysis is enabled by components of the autophagy pathway. It is likely that other nonenveloped viruses also use this pathway for nonlytic intercellular spread to affect pathogenesis in infected hosts.
Newly observed mechanisms for viral entry, assembly, and exit are challenging our current understanding of the replication cycle of different viruses. To address and better understand these mechanisms, a Keystone Symposium was organized in the snowy mountains of Colorado ("The Ins and Outs of Viral Infection: Entry, Assembly, Exit, and Spread"; 30 March-4 April 2014, Beaver Run Resort, Breckenridge, Colorado, organized by Karla Kirkegaard, Mavis Agbandje-McKenna, and Eric O. Freed). The meeting served to bring together cell biologists, structural biologists, geneticists, and scientists expert in viral pathogenesis to discuss emerging mechanisms of viral ins and outs. The conference was organized around different phases of the viral replication cycle, including cell entry, viral assembly and post-assembly maturation, virus structure, cell exit, and virus spread. This review aims to highlight important topics and themes that emerged during the conference.
Infection with many positive-strand RNA viruses dramatically remodels cellular membranes, resulting in the accumulation of double-membraned vesicles that resemble cellular autophagosomes. In this study, a single protein encoded by poliovirus, 3AB, is shown to be sufficient to induce the formation of double-membraned liposomes via the invagination of single-membraned liposomes. Poliovirus 3AB is a 109-amino acid protein with a natively unstructured N-terminal domain. HeLa cells transduced with 3AB protein displayed intracellular membrane disruption; specifically, the formation of cytoplasmic invaginations. The ability of a single viral protein to produce structures of similar topology to cellular autophagosomes should facilitate the understanding of both cellular and viral mechanisms for membrane remodeling.
Long noncoding RNAs (lncRNAs) are increasingly appreciated as regulators of cell-specific gene expression. Here, an enhancer-like lncRNA termed NeST (nettoie Salmonella pas Theiler's [cleanup Salmonella not Theiler's]) is shown to be causal for all phenotypes conferred by murine viral susceptibility locus Tmevp3. This locus was defined by crosses between SJL/J and B10.S mice and contains several candidate genes, including NeST. The SJL/J-derived locus confers higher lncRNA expression, increased interferon-γ (IFN-γ) abundance in activated CD8(+) T cells, increased Theiler's virus persistence, and decreased Salmonella enterica pathogenesis. Transgenic expression of NeST lncRNA alone was sufficient to confer all phenotypes of the SJL/J locus. NeST RNA was found to bind WDR5, a component of the histone H3 lysine 4 methyltransferase complex, and to alter histone 3 methylation at the IFN-γ locus. Thus, this lncRNA regulates epigenetic marking of IFN-γ-encoding chromatin, expression of IFN-γ, and susceptibility to a viral and a bacterial pathogen.
Autophagy is an important component of the innate immune response, directly destroying many intracellular pathogens. However, some pathogens, including several RNA viruses, subvert the autophagy pathway, or components of the pathway, to facilitate their replication. In the present study, the effect of inhibiting autophagy on the growth of dengue virus was tested using a novel inhibitor, spautin-1 (specific and potent autophagy inhibitor 1). Inhibition of autophagy by spautin-1 generated heat-sensitive, noninfectious dengue virus particles, revealing a large effect of components of the autophagy pathway on viral maturation. A smaller effect on viral RNA accumulation was also observed. Conversely, stimulation of autophagy resulted in increased viral titers and pathogenicity in the mouse. We conclude that the presence of functional autophagy components facilitates viral RNA replication and, more importantly, is required for infectious dengue virus production. Pharmacological inhibition of host processes is an attractive antiviral strategy to avoid selection of treatment-resistant variants, and inhibitors of autophagy may prove to be valuable therapeutics against dengue virus infection and pathogenesis.
The lesions of Parkinson disease spread through the brain in a characteristic pattern that corresponds to axonal projections. Previous observations suggest that misfolded α-synuclein could behave as a prion, moving from neuron to neuron and causing endogenous α-synuclein to misfold. Here, we characterized and quantified the axonal transport of α-synuclein fibrils and showed that fibrils could be transferred from axons to second-order neurons following anterograde transport.We grew primary cortical mouse neurons in microfluidic devices to separate somata from axonal projections in fluidically isolated microenvironments. We used live-cell imaging and immunofluorescence to characterize the transport of fluorescent α-synuclein fibrils and their transfer to second-order neurons.Fibrillar α-synuclein was internalized by primary neurons and transported in axons with kinetics consistent with slow component-b of axonal transport (fast axonal transport with saltatory movement). Fibrillar α-synuclein was readily observed in the cell bodies of second-order neurons following anterograde axonal transport. Axon-to-soma transfer appeared not to require synaptic contacts.These results support the hypothesis that the progression of Parkinson disease can be caused by neuron-to-neuron spread of α-synuclein aggregates and that the anatomical pattern of progression of lesions between axonally connected areas results from the axonal transport of such aggregates. That the transfer did not appear to be trans-synaptic gives hope that α-synuclein fibrils could be intercepted by drugs during the extracellular phase of their journey.
Viral infection depends on a complex interplay between host and viral factors. Here, we link host susceptibility to viral infection to a network encompassing sulfur metabolism, tRNA modification, competitive binding, and programmed ribosomal frameshifting (PRF). We first demonstrate that the iron-sulfur cluster biosynthesis pathway in Escherichia coli exerts a protective effect during lambda phage infection, while a tRNA thiolation pathway enhances viral infection. We show that tRNA(Lys) uridine 34 modification inhibits PRF to influence the ratio of lambda phage proteins gpG and gpGT. Computational modeling and experiments suggest that the role of the iron-sulfur cluster biosynthesis pathway in infection is indirect, via competitive binding of the shared sulfur donor IscS. Based on the universality of many key components of this network, in both the host and the virus, we anticipate that these findings may have broad relevance to understanding other infections, including viral infection of humans.
Catalytic activities can be facilitated by ordered enzymatic arrays that co-localize and orient enzymes and their substrates. The purified RNA-dependent RNA polymerase from poliovirus self-assembles to form two-dimensional lattices, possibly facilitating the assembly of viral RNA replication complexes on the cytoplasmic face of intracellular membranes. Creation of a two-dimensional lattice requires at least two different molecular contacts between polymerase molecules. One set of polymerase contacts, between the "thumb" domain of one polymerase and the back of the "palm" domain of another, has been previously defined. To identify the second interface needed for lattice formation and to test its function in viral RNA synthesis, we used a hybrid approach of electron microscopic and biochemical evaluation of both wild-type and mutant viral polymerases to evaluate computationally generated models of this second interface. A unique solution satisfied all constraints and predicted a two-dimensional structure formed from antiparallel arrays of polymerase fibers that use contacts from the flexible amino-terminal region of the protein. Enzymes that contained mutations in this newly defined interface did not form lattices and altered the structure of wild-type lattices. When reconstructed into virus, mutations that disrupt lattice assembly exhibited growth defects, synthetic lethality or both, supporting the function of the oligomeric lattice in infected cells. Understanding the structure of polymerase lattices within the multimeric RNA-dependent RNA polymerase complex should facilitate antiviral drug design and provide a precedent for other positive-strand RNA viruses.
Few antivirals are effective against positive-strand RNA viruses, primarily because the high error rate during replication of these viruses leads to the rapid development of drug resistance. One of the favored current targets for the development of antiviral compounds is the active site of viral RNA-dependent RNA polymerases. However, like many subcellular processes, replication of the genomes of all positive-strand RNA viruses occurs in highly oligomeric complexes on the cytosolic surfaces of the intracellular membranes of infected host cells. In this study, catalytically inactive polymerases were shown to participate productively in functional oligomer formation and catalysis, as assayed by RNA template elongation. Direct protein transduction to introduce either active or inactive polymerases into cells infected with mutant virus confirmed the structural role for polymerase molecules during infection. Therefore, we suggest that targeting the active sites of polymerase molecules is not likely to be the best antiviral strategy, as inactivated polymerases do not inhibit replication of other viruses in the same cell and can, in fact, be useful in RNA replication complexes. On the other hand, polymerases that could not participate in functional RNA replication complexes were those that contained mutations in the amino terminus, leading to altered contacts in the folded polymerase and mutations in a known polymerase-polymerase interaction in the two-dimensional protein lattice. Thus, the functional nature of multimeric arrays of RNA-dependent RNA polymerase supplies a novel target for antiviral compounds and provides a new appreciation for enzymatic catalysis on membranous surfaces within cells.
The rate of protein secretion in host cells is inhibited during infection with several different picornaviruses, with consequences likely to have significant effects on viral growth, spread, and pathogenesis. This Sin(+) (secretion inhibition) phenotype has been documented for poliovirus, foot-and-mouth disease virus, and coxsackievirus B3 and can lead to reduced cell surface expression of major histocompatibility complex class I and tumor necrosis factor receptor as well as reduced extracellular secretion of induced cytokines such as interleukin-6 (IL-6), IL-8, and beta interferon. The inhibition of protein secretion is global, affecting the movement of all tested cargo proteins through the cellular secretion apparatus. To test the physiological significance of the Sin(-) and Sin(+) phenotypes in animal models, Sin(-) mutant viruses are needed that fail to inhibit host protein secretion and also exhibit robust growth properties. To identify such Sin(-) mutant polioviruses, we devised a fluorescence-activated cell sorter-based screen to select virus-infected cells that nevertheless expressed newly synthesized surface proteins. After multiple rounds of selection, candidate Sin(-) mutant viruses were screened for genetic stability, increased secretion of cargo molecules and wild-type translation and growth properties. A newly identified Sin(-) mutant poliovirus that contained coding changes in nonstructural proteins 2A (N32D) and 2C (E253G) was characterized. In this virus, the 2C mutation is responsible for the Sin(-) phenotype and the 2A mutation suppresses a resulting growth defect by increasing the rate of cell death and therefore the rate of viral spread. The 2A-N32D suppressor mutation was not allele specific and, by increasing the rate of cellular apoptosis, affected a completely different pathway than the 2C-E253G Sin(-) mutation. Therefore, the 2A mutation suppresses the 2C-E253G mutant phenotype by a bypass suppression mechanism.
Cellular autophagy, a process that directs cytosolic contents to the endosomal and lysosomal pathways via the formation of double-membraned vesicles, is a crucial aspect of innate immunity to many intracellular pathogens. However, evidence is accumulating that certain RNA viruses, such as poliovirus, subvert this pathway to facilitate viral growth. The autophagosome-like membranes induced during infection with wild-type poliovirus were found to be, unlike cellular autophagosomes, relatively immobile. Their mobility increased upon nocodazole treatment, arguing that vesicular tethering is microtubule dependent. In cells infected with a mutant virus that is defective in its interaction with the host cytoskeleton and secretory pathway, vesicle movement increased, indicating reduced tethering. In all cases, the release of tethering correlated with increased amounts of extracellular virus, which is consistent with the hypothesis that small amounts of cytosol and virus entrapped by double-membraned structures could be released via fusion with the plasma membrane. We propose that this extracellular delivery of cytoplasmic contents be termed autophagosome-mediated exit without lysis (AWOL). This pathway could explain the observed exit, in the apparent absence of cellular lysis, of other cytoplasmic macromolecular complexes, including infectious agents and complexes of aggregated proteins.
Autophagy is a cellular process that creates double-membraned vesicles, engulfs and degrades cytoplasmic material, and generates and recycles nutrients. A recognized participant in the innate immune response to microbial infection, a functional autophagic response can help to control the replication of many viruses. However, for several viruses, there is functional and mechanistic evidence that components of the autophagy pathway act as host factors in viral replicative cycles, viral dissemination, or both. Investigating the mechanisms by which viruses subvert or imitate autophagy, as well as the mechanisms by which they inhibit autophagy, will reveal cell biological tools and processes that will be useful for understanding the many functional ramifications of the double-membraned vesicle formation and cytosolic entrapment unique to the autophagy pathway.
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.
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.
Cells infected with poliovirus exhibit a rapid inhibition of protein secretion and disruption of the Golgi complex. Neither the precise step at which the virus inhibits protein secretion nor the fate of the Golgi complex during infection has been determined. We find that transport-vesicle exit from the endoplasmic reticulum (ER) and trafficking to the ER-Golgi intermediate compartment (ERGIC) are unaffected in the poliovirus-infected cell. By contrast, poliovirus infection blocks transport from the ERGIC to the Golgi complex. Poliovirus infection also induces fragmentation of the Golgi complex resulting in diffuse distribution of both large and small vesicles throughout the cell. Pre-treatment with nocodazole prevents complete fragmentation, indicating that microtubules are required for poliovirus-induced Golgi dispersion. However, virally induced inhibition of the secretory pathway is not affected by nocodazole, and Golgi dispersion was found to occur during infection with mutant viruses with reduce ability to inhibit protein secretion. We conclude that the dispersion of the Golgi complex is not in itself the cause of inhibition of traffic between the ERGIC and the Golgi. Instead, these phenomena are independent effects of poliovirus infection on the host secretory complex.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Central to the replication of poliovirus and other positive-strand RNA viruses is the virally encoded RNA-dependent RNA polymerase. Previous biochemical studies have suggested that direct polymerase- polymerase interactions might be important for polymerase function, and the structure of poliovirus polymerase has revealed two regions of extensive polymerase-polymerase interaction. To explore potential functional roles for the structurally observed polymerase-polymerase interactions, we have performed RNA binding and extension studies of mutant polymerase proteins in solution, disulfide cross-linking studies, mutational analyses in cells, in vitro activity analyses and RNA substrate modeling studies. The results of these studies indicate that both regions of polymerase-polymerase interaction observed in the crystals are indeed functionally important and, furthermore, reveal specific functional roles for each. One of the two regions of interaction provides for efficient substrate RNA binding and the second is crucial for forming catalytic sites. These studies strongly support the hypothesis that the polymerase- polymerase interactions discovered in the crystal structure provide an exquisitely detailed structural context for poliovirus polymerase function and for poliovirus RNA replication in cells.
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.
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.
Poliovirus has evolved to maximize its genomic information by producing multifunctional viral proteins. The P3 nonstructural proteins harbor various activities when paired with different binding partners. These viral polypeptides regulate host cell macromolecular synthesis and function as proteinases, as RNA binding proteins, or as RNA-dependent RNA polymerase. A cleavage product of the P3 region is the genome-linked protein VPg that is essential in the initiation of RNA synthesis. We have used an inducible yeast two-hybrid system to analyze directly protein-protein interactions among P3 proteins. Sixteen signals of homo- or heterodimer interactions have been observed and have been divided into three groups. Of interest is the newly discovered affinity of VPg to 3Dpol that suggests direct interaction between these molecules in genome replication. A battery of 3AB variants (eight clustered-charge-to-alanine changes and five single-amino-acid mutations) has been used to map the binding determinants of 3AB-3AB interaction which were found to differ from the amino acids critical for the 3AB-3Dpol interaction. The viral proteinase 3Cpro was not found to interact with other 3Cpro molecules or with any other P3 polypeptide in yeast cells, a result confirmed by glutaraldehyde cross-linking. The weak apparent interaction between 3AB and 3CDpro scored in the yeast two-hybrid system was in contrast to a strong signal by far-Western blotting. The results elucidate, in part, previous results of biochemical and genetic analyses. The role of the interactions in RNA replication is addressed.
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.
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.
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.
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.
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.
Poliovirus RNA replication occurs on the surface of membranous vesicles that proliferate throughout the cytoplasm of the infected cell. Since at least some of these vesicles are thought to originate within the secretory pathway of the host cell, we examined the effect of poliovirus infection on protein transport through the secretory pathway. We found that transport of both plasma membrane and secretory proteins was inhibited by poliovirus infection early in the infectious cycle. Transport inhibition did not require viral RNA replication or the inhibition of host cell translation by poliovirus. The viral proteins 2B and 3A were each sufficient to inhibit transport in the absence of viral infection. The intracellular localization of a secreted protein in the presence of 3A with the endoplasmic reticulum suggested that 3A directly blocks transport from the endoplasmic reticulum to the Golgi apparatus.
We examined the importance of two interactions between poliovirus and its host cell: the putative association between viral proteins and a rearranged intermediate filament (IF) network and the apparent requirement for functional vesicle budding machinery within the host-cell secretory pathway. Poliovirus capsid proteins appeared to associate with reorganized IF proteins during infection. Treatment of cells with cytochalasin D and nocodazole in combination disrupted normal cytoskeletal organization and prevented the poliovirus-induced redistribution of IF proteins to a juxtanuclear location. However, this treatment had no effect on viral yields from single-cycle infections, indicating that neither cytoskeletal integrity nor a specific poliovirus-induced rearrangement of IF proteins is required in the poliovirus life cycle. In contrast, we report that the inhibition of poliovirus replication by brefeldin A (BFA), an inhibitor of secretory membrane traffic, is specific to the host cell. Polioviral yields were not affected by BFA in two BFA-resistant cell lines, demonstrating that BFA targets a host protein or process required by poliovirus. No BFA-resistant virus was detected in these experiments, further supporting the hypothesis that poliovirus replication requires secretory pathway function, perhaps for the generation of vesicles on which viral RNA replication complexes are assembled.


Should you take a much-needed vacation or save money for your children's education? Should you protect the endangered owl or maintain jobs for loggers?
How do you handle questions such as these? We frequently face ethical dilemmas in our daily lives, and few have trouble with the "right vs. wrong" choices. However, the "right vs. right" dilemmas, in which neither choice is clearly or widely accepted as wrong, many times present obstacles that call for value-based decisions, and that's where we often need help.
Kidder -- the founder of the Institute for Global Ethics -- teaches us how to think for ourselves in order to resolve any ethical dilemma, from the personal to the philosophical. Unique in its approach and full of illustrative anecdotes, How Good People Make Tough Choices is an indispensable resource for arriving at sound conclusions when facing tough choices.
How Good People Make Tough Choices: Resolving the Dilemmas of Ethical Living.


Sean Hannity and Jeanine Pirro appeared at a pre-election Trump rally, Monday and delivered speeches in favor of the President. But that didn't sit too well with FOX executives.
Introducing the president as he stumped for Republican candidates was Rush Limbaugh, the radio host who was born and raised in Cape Girardeau. Then after Mr. Trump took the microphone, he invited two Fox News personalities, Sean Hannity and Jeanine Pirro, to join him onstage, where each also delivered a short speech backing the president.
How it was addressed the network did not say. Mr. Hannity followed up with his own statement saying that Mr. Trump's invitation to come onstage was spontaneous, but the popular conservative host expressed no regret about accepting. The only thing he lamented was that some took his onstage attack on the "fake news" at the back of the room to include his colleagues from Fox who were covering the event.
Is FOX right to distance themselves from Trump? Let us know in the comments.


The right tablet case will provide protection for your tablet computer and make any brand and model of the device easier to carry and use. We rank 10 of the best tablet cases to help you select a case that will provide the protection and style you want.
1. What is a tablet case?
A tablet case is a protective accessory made out of cushioning materials that protects tablet computers from bumps or falls and shields the screen from scratches.
2. What does a tablet case do?
In addition to providing protection, tablet cases may also make it easier to safely transport these devices or prop a tablet up at an angle suitable for hands-free viewing or typing.
3. How does a tablet case work?
Most cases wrap around or snap onto these devices. Cases designed for specific tablet models feature cut-outs that provide easy access to cameras and ports.
4. Where can you buy a tablet case?
Purchase a tablet case from an electronics retailer that sells accessories or from an online marketplace.
5. How much do tablet cases cost?
Cases for tablets can be priced as low as $5.99 or as high as $50, depending on the brand, materials, and which device the case is for.
We base our rankings on pricing, features, and customer reviews and testimonials. Most of the cases we review are made by manufacturers that sell designs for a variety of tablet brands. Even if the model in our review is not your tablet, we make a note of the other brands for which each type of case is available.
Tablet cases priced under $10 may not be made out of materials that are as high quality as more expensive cases. The price of a case typically corresponds to the specificity of the design and the materials used. Most of the cases in this ranking are priced between $10 and $20, though some models with special features or heavy-duty designs range up to $49.90.
MoKo is one of the top-rated tablet case manufacturers. The brand makes cases for many major tablet brands and models. This folding cover protector case has a synthetic leather exterior, soft interior lining, and comes in a variety of colors and designs. These cases feature cut-outs suitable for particular models and a stand for hands-free viewing or typing. When used on a compatible tablet, these cases automatically wake or put the screen to sleep and provide impact and shock resistance. Some models also feature holders for a pen stylus or Apple Pencil.
The MoKo folio-style tablet case ranges from $9.99 to $13.99.
Purchase MoKo cases on Amazon's marketplace from a seller called BSCstore. Most models are eligible for Prime shipping.
ProCase makes slim and lightweight folio cases with external synthetic PU leather, microfiber interiors, and the ability to support a tablet at a natural angle. Universal tablet cases fit a range of devices based on measurements. Keep in mind that a universal case will not have specialized cut-outs for cameras or controls. You may want to consider a brand- and model-specific case such as ProCase's designs for the ASUS ZenPad, Samsung Galaxy Tab, or Lenovo Tab.
A universal 9- to 10- inch ProCase tablet case retails for $16.99. Specialized products range in price from $10.99 to $17.99.
Order ProCase tablet cases from Amazon or other online electronics accessory vendors.
Tablet owners seeking a heavy-duty case capable of providing 360 degrees of protection should consider Unicorn Beetle series cases by SUPCASE. These cases combine a hard polycarbonate shell with flexible TPU and a screen protector designed to prevent scratches while preserving sensitivity. SUPCASE makes cases with precise bezels and slip-resistant stands for Amazon Fire, Kindle, iPad and Samsung tablets.
Heavy-duty SUPCASES are priced in the $24.99 - 29.99 range depending on the device the case protects.
Purchase SUPCASES on Amazon, where most models qualify for Prime shipping and some qualify for coupon discounts.
[Scratch Protection]: Powerful magnets keep the front cover sealed and your iPad safe from scratches.
[Reliable Auto Sleep/Wake]: Stronger magnets ensure the auto sleep/wake function engages quickly every time.
ESR makes lightweight trifold cases for iPads. These affordable cases come in a wide variety of colors and feature a magnetic closure to provide automatic sleep and wake functions and to provide stability when standing the tablet.
ESR iPad cases range in price from $12.99 to $20.99.
Purchase ESR cases from Amazon. Most models are eligible for Prime shipping.
Slim and lightweight hard back design adds minimal bulk while protecting your precious device.
MoKo's line of smart shell tablet cases feature a translucent frosted back protector and many options for solid color or patterned folio-style screen covers. These cases do not have bezels around the screen, which allows for gesture swipes without restrictions. MoKo makes cases for most major tablet brands and models.
MoKo Smart Shell cases are priced from $11.99 to $12.99, depending on the type of tablet the case is for.
Purchase MoKo cases on Amazon from the seller BSCstore. Most of these case designs qualify for free two-day Prime shipping.
Fintie's durable iPad Keyboard Case comes with a detachable wireless Bluetooth keyboard. This high-quality hard ABS laptop-style keyboard with spring mechanisms provides easy and reliable input for the 9.7-inch iPad and iPad Air 1 and 2. This case offers automatic sleep and wake functionality when the keyboard is not attached and features a kickstand for hands-free viewing and typing.
Purchase this combined case and wireless keyboard set for $37.99.
Find a full selection of Fintie cases for iPads and other tablets on Amazon.
Belt-clip holster included that doubles as a kickstand for hands-free media viewing.
OtterBox has a great reputation for making some of the most protective cases on the market. The Defender series features a three-layer design with an inner shell, outer cover, and touchscreen protector. These cases feature built-in screen protectors, port and jack covers, and a durable shield stand.
Cases may range from $19.99 up to $45.99 depending on the tablet model.
Purchase OtterBox cases from any electronics retailer that sells tablet accessories or online marketplaces.
Built from premium composition leather exterior and soft microfiber interior - snug fit, durable and protective.
This Fintie case designed for the 10.5″ iPad Pro provides reliable multi-angle viewing. This case has a durable synthetic leather exterior and microfiber interior with a magnetic strip to support automatic sleep and wake features.
The price of this tablet cover varies depending on whether you choose a solid color for $15.99 to $16.99 or a patterned case for $17.99.
Purchase this Fintie iPad Pro case from Amazon.com. All colors and designs are eligible for Prime shipping.
If you want the durability of an OtterBox with a lighter weight folio-style case, consider the Profile series. Profile cases are available in several color combinations for the iPad Air and mini models. This model is designed to allow easy access to the cameras on compatible iPad models and features auto-wake and sleep magnets.
OtterBox Profile series cases for iPad Air or mini models cost between $16.95 and $29.95.
Purchase Otterbox Profile series cases from the manufacturer, tablet accessory retailers or online marketplaces.
Microsoft Surface Pro users may be interested in a tablet case that is compatible with or without the Type Cover keyboard on the 2017 and 4 models. JETech's Microsoft Surface case features precisely aligned cut-outs, adjustable viewing angles, a pen loop, and hook and loop fasteners that secure the tablet in place. JETech makes highly-rated and affordable cases for major tablet brands including Apple, Samsung, and Microsoft.
The JETech is a great value at $9.99 for the Microsoft Surface case. Models for other tablet brands range from $7.99 up to $14.99.
Order JETech tablet cases from Amazon. Most of the cases made by this brand qualify for Prime shipping.
The MoKo Folio is a basic and affordable tablet case available for most major brands and models. MoKo cases provide a reasonable amount of protection for the price and are available in many colors and patterns. The runner-up from ProCase is also a great option, particularly if you order a case designed for your tablet model. A universal case will not support automatic waking or sleeping functions or provide easy access to cameras or ports.
If you want more protection, consider the SUPCASE Heavy Duty Unicorn Beetle Series or OtterBox Defender Series of cases. The Fintie iPad Keyboard Case includes a detachable wireless keyboard, while the JETech Case fits the Microsoft Surface Type Cover keyboard. No matter what your priorities are, one of these tablet cases should provide the right fit and make it easy to keep track of the accessories you use on a regular basis.


text:
Star Trek 4 might lose Chris Pine and Chris Hemsworth
Chrises Pine and Hemsworth exit Star Trek 4 after budget changes
The fourth instalment in the Star Trek film series looks to be in trouble before it has even started: Chris Pine and Chris Hemsworth probably won't be appearing, according to The Hollywood Reporter.
Both Chrises were originally in negotiations to appear in Star Trek 4, but sources say that they were unable to come to a good agreement for Paramount Pictures and Skydance Media, the studios set to produce the film.
The reason for the oncoming parting of the ways is reportedly down to the studios going back on existing deals following budget changes caused by the previous film, Star Trek Beyond, failing to wow at the box office. The new deals would have seen Pine and Hemsworth receiving pay cuts.
However, the film is currently still going ahead, and Star Trek, Star Trek Into Darkness and Star Trek Beyond cast members Zoe Saldana, Zachary Quinto, Karl Urban, John Cho and Simon Pegg are all expected to return. Director SJ Clarkson is helming the film from a script by Patrick McKay and John D Payne.
It's a pity the actors and the studios were unable to come to an agreement, especially with Pine playing iconic Star Trek mainstay Captain James T Kirk.
But maybe this will give the film series a chance to rejig and try something new, and actually boldly go somewhere.
Star Trek 4 doesn't yet have a release date. Get all the latest sci-fi news with every issue of SciFiNow.
Noah Hawley to direct next Star Trek film Super Bowl 2021 Genre Trailers
Tags: Star Trek 4

redpajama_set_name: RedPajamaCommonCrawl


text:
Solo, Small Firm and General Practice Division
Young Lawyers Fellowship Program
The ABA Solo, Small Firm and General Practice Division (GPSolo) Young Lawyers Fellowship Program provides leadership opportunities for young lawyers. GPSolo is committed to increasing the participation of young lawyers in Division activities. This program will provide young lawyers the opportunity to become integrally involved in the Division.
A candidate must be an active member of the ABA Young Lawyers Division (YLD), or a previously active member who has aged out within the past three years. Successful candidates will have demonstrated involvement in bar association and non-bar related activities. Candidates do not need to be current members of GPSolo, but must join upon selection.
GPSolo's Young Lawyers Committee will select two applicants to serve as fellows for the following bar year. The selection will be based on the individual's achievement and commitment to the organized bar and GPSolo's mission and goals.
Fellows will be required to attend the Division's Fall meeting (October 17 - 19, 2019 at the Park Hyatt Aviara in Carlsbad, CA) and Spring meeting (April 30 - May 2, 2020 at the La Concha Resort in San Juan, Puerto Rico) with reimbursement of up to $500 for airfare and a $100 per diem for two days for each meeting. Fellows may also show their commitment to the Division by attending the ABA Annual Meeting; however, reimbursement for this meeting is not available.
The selected fellows will be appointed to the GPSolo Young Lawyers Committee and will be expected to complete a substantive project for the Division. Each fellow will also be paired with a mentor. Finally, fellows must remain an active member of the Division for two years after their Fellowship concludes.
For further information, please contact the Division at gpsolo@americanbar.org.
2018-2019 Young Lawyers Fellows
Julie T. Houth, La Jolla, CA (left) and Amber M. Rush, Vancouver, WA (right)
American Bar Association TM American Bar Association|/content/aba-cms-dotorg/en/groups/gpsolo/initiatives/young_lawyers_fellowship


text:
CORONAVIRUSHealthmedicaidpandemic
Workers Signing Up The Uninsured For Health Coverage Worry About Getting Sick
House Democrats blast the Centers for Medicare and Medicaid Services for keeping workers at call centers during the pandemic.
Dave Jamieson
Labor Reporter, HuffPost
Apr 9, 2020, 01:05 PM EDT | Updated Apr 10, 2020
A worker at a call center.
ImagesBazaar via Getty Images
The workers who help Americans sign up for health care coverage through Medicaid and the Affordable Care Act's health insurance exchanges are going to get awfully busy as more jobless claims pile up. Now, members of Congress are worried those workers will end up sick themselves as they guide the newly uninsured through the coverage process.
More than 40 Democrats signed a letter Thursday to the head of the Centers for Medicare and Medicaid Services expressing concern that the agency's call center workers were being put in "grave danger" during the coronavirus pandemic. The lawmakers said CMS' private contractor, the call center operator Maximus, still hadn't developed a plan for employees to telework during the public health crisis.
″The cruel irony of forcing call center workers to come to work in unsafe conditions, while they help people nationwide on Medicare, Medicaid and the ACA during a pandemic, is astounding," Rep. Mark Pocan (D-Wis.) told HuffPost in a statement.
Pocan and his colleagues noted in their letter that Maximus had to temporarily close some call centers since the coronavirus outbreak began. They said that thousands of workers "are now at risk of contracting the virus and continuing to spread it in their communities" due to having to work onsite rather than at home.
Call centers have emerged as a major concern during the pandemic because they can be crowded workplaces with less-than-generous sick leave policies. Call center workers have told HuffPost and In These Times they were reluctant to continue clocking in and working in such close proximity to their colleagues, especially when some people already exhibited cold and flu symptoms.
According to the Democrats' letter, CMS had ordered state and local governments to allow 11 of its call centers to continue operating because they were not set up for telework at any of those locations. The lawmakers asked CMS Administrator Seema Verma to alter that guidance to allow for telework for "every worker possible," and to guarantee sick leave without penalty for anyone who needs it.
Rep. Jan Schakowsky (D-Ill.) said in a statement to HuffPost that Verma "has had her foot on the throat of these frontline call center workers" from "the second she was sworn in" in 2017. Schakowsky called it "politically motivated cruelty."
CMS spokesperson Johnathan Monroe said the agency "has been vigilant in ensuring that our call centers are following all CDC guidelines on workplace protections. The health and safety of our employees, including representatives working in our call centers, is a high priority for the agency as we also equally ensure eligible Medicare, Medicaid, Children's Health Insurance and other high-risk beneficiaries are able to receive continuous access to quality care."
Maximus did not respond to questions emailed Thursday. In a note to employees posted on its website April 2, Maximus said it was instituting a "no exception stance" on social distancing, requiring workers onsite to stay at least six feet apart from one another. The note said anyone who violated the policy would face disciplinary action.
"The cruel irony is astounding."
- Rep. Mark Pocan (D-Wis.)
The call center workers will be filling a critical role in the coming weeks as more Americans lose their employer-sponsored health care coverage, forcing them to look for insurance through the exchanges. The Labor Department said 6.6 million new jobless claims were filed last week, on top of 6.9 million the previous week. Roughly one in 10 U.S. workers have lost their jobs in less than a month.
The Communications Workers of America union, which represents thousands of call center employees, has been trying to organize workers at Maximus facilities under government contracts, saying the wages are too low and the benefits too skimpy. "Here we're trying to help low-income people get coverage, and we can hardly afford our own coverage," one Maximus employee told New York Magazine in February.
Chris Shelton, the union's president, has accused Maximus of refusing to engage in a dialogue with employees on safety during the pandemic. He has demanded the company meet with a workers' organizing committee.
Monica Harris, who works at a Maximus call center in Chester, Virginia, said in a statement through the union that the company waited too long in issuing its guidelines in early April, when the pandemic was already well underway.
"We shouldn't have to put our families, ourselves, and our communities at risk by coming to work when other critical government services like the Social Security Administration have moved to telework," Harris said.
This story has been updated with a statement from CMS.
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Alfred Tennyson, 1st Baron Tennyson
(Redirected from Alfred Lord Tennyson)
Tennyson.jpg
Lord Tennyson, Poet Laureate
Alfred Tennyson, 1st Baron Tennyson (August 6, 1809 – October 6, 1892) is generally regarded as one of the greatest English poets.
Much of his verse was based on classical or mythological themes. Idylls of the King (1859) takes its subject from Arthurian romance. Later in his career, he attempted drama, but his plays enjoyed little success even in his lifetime.
He was born in Lincolnshire, a rector's son. His father had fallen out with his family and been disinherited; he drank heavily and became mentally unstable. Tennyson and two of his elder brothers were writing poetry in their teens, and a collection of poems by all three was published locally when Alfred was only seventeen. One of those brothers, Charles Tennyson Turner, later married Louisa Sellwood, younger sister of Alfred's future wife; the other poet brother was Frederick Tennyson. Educated at Louth grammar school and at Trinity College, Cambridge, where he joined the secret society called the Cambridge Apostles, Alfred Tennyson published his first solo collection of poems in 1830. Although decried by critics as over-sentimental, his verse soon proved popular. One of his best-known poems, The Lady of Shalott, appeared in his second collection in 1833.
After Tennyson's father died, he shared the responsibility for his widowed mother and her large brood of children. They were allowed to stay in the rectory for some time, but later moved to Essex. An unwise investment in an ecclesiastical wood-carving enterprise resulted in the loss of much of their money, and this may have been one of the reasons why Tennyson was so late in marrying.
Alfred_Tennyson,_1st_Baron_Tennyson_-_Project_Gutenberg_eText_13619.jpg
Alfred Tennyson, 1st Baron Tennyson, English poet
It was in 1850 that Tennyson reached the pinnacle of his career, being appointed Poet Laureate in succession to William Wordsworth and in the same year producing his masterpiece, In Memoriam A.H.H., dedicated to a friend from his student days, Arthur Hallam, who was to have been married to Tennyson's sister, Emilia. In the same year, Tennyson himself married Emily Sellwood, whom he had known since childhood. They had two sons, Hallam -- named after his late friend -- and Lionel.
He held the position of Poet Laureate from 1850 until his death, turning out appropriate but mediocre verse, such as a poem of greeting to Alexandra of Denmark when she arrived in Britain to marry the future King Edward VII. Other works written as Laureate include Ode on the Death of the Duke of Wellington and Ode Sung at the Opening of the International Exhibition.
Queen Victoria was an ardent admirer of Tennyson's work, and in 1884 created him Baron Tennyson, of Aldworth in the County of Sussex and of Freshwater in the Isle of Wight. He was the first English writer raised to the Peerage.
Recordings exist of Lord Tennyson declaiming his own poetry, but they are of poor quality.
Lord Tennyson's death was widely mourned, and he was buried at Westminster Abbey. He was succeeded as 2nd Baron Tennyson by his son, Hallam, who produced an authorised biography of his father in 1897, and was later the second Governor-General of Australia.
StatueOfTennyson.jpg
Statue of Lord Tennyson in the chapel of Trinity College, Cambridge
Preceded by:
William Wordsworth British Poet Laureate
1850–1892 Succeeded by:
Alfred Austin
Template:End box
New creation Baron Tennyson Succeeded by:
Hallam Tennyson
The Kraken (1830)
Harold (1876) - began a revival of interest in King Harold
The Charge of the Light Brigade
The Lady of Shalott
In Memoriam A.H.H.
Locksley Hall
Crossing the Bar
Tithonus
The Lotus-Eaters (Wikisource (http://wikisource.org/wiki/The_Lotus-Eaters))
Idylls of the King
Template:Wikiquote Template:Wikisource author
Biography & Works (http://www.online-literature.com/tennyson/) (public domain)
Online copy of 'Locksley Hall' (http://eir.library.utoronto.ca/rpo/display/poem2161.html)
Selected Poems of A.Tennyson (http://www.poetseers.org/the_great_poets/br/alfred_tennyson/library/)de:Alfred Tennyson
eo:Alfred, Lord TENNYSON fr:Alfred Tennyson he:אלפרד טניסון nl:Alfred Tennyson pl:Alfred Tennyson sv:Alfred Tennyson
Retrieved from "https://tantalum.academickids.com:443/encyclopedia/index.php/Alfred_Tennyson%2C_1st_Baron_Tennyson"
Categories: 1809 births | 1892 deaths | British Poets Laureate | English poets | Fellows of the Royal Society | Barons in the Peerage of the United Kingdom | Isle of Wight


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Is Ndamukong Suh trolling the Lions?
Former Detroit Lion Ndamukong Suh was essentially trying to get out of the Motor City from the moment he was drafted here, and bolted...
Detroit Lions' fans let the refs have it nearly 1 hour before game vs. Vikings
Detroit Lions NewsMichael Whitaker - January 8, 2020 0
Former Detroit Lion Ndamukong Suh was essentially trying to get out of the Motor City from the moment he...
It has not been a fun week for the Detroit Lions and their fans.
On Monday, the Lions were screwed multiple times by the officials, costing them what would have been a huge win over the Green Bay Packers. The win would have put the Lions in first place in the NFC North, but instead, they are in last.
This week, the Lions will look to get back over the .500 mark when they square off against the Minnesota Vikings at Ford Field.
About 1-hour before kickoff, the officials came out onto the field and the Lions fans who were already in the stadium let them have it!
From MLive:
Ford Field was maybe 10% full when the officials emerged from the tunnel 53 minutes before the Lions' game on Sunday against Minnesota. Yet that was enough for the crowd to greet the men in stripes with a hearty and unmistakable torrent of boos.
Yeah, these guys and their every call — or non-call — are going to be under a microscope today.
Detroit has been victimized by some questionable officiating this season, including on a pair of highly dubious hands to the face calls against Trey Flowers on Monday Night Football. Both came on third-down stops late in the fourth quarter, and both led to scores, including the Packers' game-winning field goal as time expired.
You can bet Ford Field will be LOUD today!
GO LIONS!!
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"All is fair in love and war." Though the rivalry has been extremely one-sided for some time now, there is no question about it that...
Detroit Lions News Michael Whitaker - January 8, 2020 0
Detroit Lions News Don Drysdale - January 8, 2020 0
Detroit Lions News Arnold Powell - January 8, 2020 0


WILKES-BARRE, Pa. -- It was a homecoming for a Navy drag queen from Columbia County with impressive credentials.
Joshua Kelley from Berwick is in the United States Navy.
Kelley performs as a drag queen, under the name Harpy Daniels.
The administrative supervisor aboard the aircraft carrier The Ronald Reagan performed Saturday night at Heat, a club along North Main Street in downtown Wilkes-Barre.
"This is my passion, my thrill. As a sailor, it always comes first. I'm on leave here. I'm here to celebrate. I'm here to let my hair down," said Kelley.
Kelley said he's proud of his work on and off the stage.
He has helped to create an LGBTQ friendly environment aboard The Ronald Reagan.
This is an embarrassment and an abomination. Could you imagine what the Nazis or the Imperial forces would think of our military back in WW-2 if they saw this? Can you imagine what the enemys of our country are thinking now? Weak and mentally ill sailors, and weak leadership for allowing this thing to serve in our armed forces. What a joke.
To quote Archie Bunker: "The world's gone nuts".


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Gaming: Would you like to have an e-sports tournament at school!
October 23, 2022 September 10, 2021 by Payal Sharma
Have you at any point found the opportunity to play video games at school previously?
Many grade younger students in Wales have been finding the opportunity to contend in the very first very first public junior e-sports competition.
They've been playing Nintendo Switch games including Mario Kart 8 Deluxe, Race with Ryan, and Crayola Scoot.
The thought run by Digital Schoolhouse, in association with Nintendo UK and Outright Games, is to assist with giving youngsters matured 8-11 a greater amount of thought regarding the sort of occupations that exist in the computer games industry, to give them a shot for themselves. The youngsters will likewise do a scope of examples and exercises to show how computer games are associated with school subjects like Computing, English, Maths, Design, and Technology (DT), and Personal, Social, Health, and Economic Education (PSHE).
The dispatch follows an effective pilot conspire in 2020 which showed that the e-sports competition assisted kids with being more drawn in with exercises in school. An understudy who partook in the pilot plot said: "I love when I had the chance to play the game I additionally loved doing every one of the positions it was truly fun!
"My #1 occupation was creation team that was fun however I don't think I had the chance to do have which I was truly anticipating. I truly appreciated it I wish I did it consistently at school!"
Coordinators said they tracked down that 84% of students needed more examples in school like the e-sports competition, and 55% of educators said that youngsters were improving outcomes than expected. It additionally appeared to assist kids with developing their premium in the computer games industry, with 55% of educators saying youngsters were essentially more persuaded to examine processing subsequently.
The gaming fun doesn't end with the lesser competition by the same token!
The senior e-sports competition will likewise before long be making a return with the DSH Super Smash Bros. Extreme Team Battle going to fire up for 12-18 years across the UK. Computerized Schoolhouse (DSH), the instruction drive from UK games industry exchange body Ukie, has reported another lesser school esports competition.
The DSH junior esports competition rivalry has been assembled in association with Nintendo UK and British game designer Outright Games following a pilot last year.
It's for understudies matured 8 to 11 and provisions Switch games Mario Kart 8 Deluxe, Race with Ryan, and Crayola Scoot, as declared in this article on the Digital Schoolhouse site.
Instructors are additionally ready to utilize plugin assets to utilize various games as they show understudies jobs in the games business, with exercises planned to subjects on the educational program including Computing, English, Maths, Design and Technology (DT) and Personal, Social, Health and Economic Education (PSHE).
The lesser school esports pilot conspires last year tracked down that 84% of understudies needed more examples in school like the esports competition, while 55% of instructors detailed that their students' degree of fulfillment was higher than expected. Also, 91% of educators announced that students were either entirely or amazingly keen on professions in games. Computerized Schoolhouse will likewise run its different Super Smash Bros Ultimate Team Battle esports competition for 12 to long term holds
Kalpesh Tailor, head of interchanges of Nintendo UK, remarked: "We are excited in our job as a lead accomplice for Digital Schoolhouse to bring the one more groundbreaking and motivating system to youthful students across the UK with Nintendo Switch.
"In the previous year, our organization has reached more than 10,000 understudies and 1,000 groups in schools and universities across the UK with the Super Smash Bros. Extreme Team Battle competition. We have seen direct the positive effect this has had on students who have profited from collaboration, methodology, and social enhancements.
"It is extraordinary that we can convey a customized instructive program utilizing Mario Kart 8 Deluxe so grade school understudies can likewise connect with and get hands on encounters in a huge number of significant jobs creating and running a cross country imaginative competition."
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Summer Grades Are In--and Udacity's Scores Are Up
Udacity Scores Higher Pass Rates in Summer Term
After a poor grade this spring in its San Jose State University partnership, online education start-up Udacity reports new data with better results.
By Issie Lapowsky@issielapowsky
The ever-ballooning field of online education took a big hit this summer when news broke that more than half of the students who took Udacity's online courses at San Jose State University this spring failed the class. The poor results inspired the college to temporarily suspend its partnership with Udacity for the fall semester.
Now, the Palo Alto-based company, which is one of many providers of massively open online courses, or MOOCs, is making a comeback.
Udacity has just released the pass rates from its summer program at San Jose State, and the results are substantially more promising. Pass rates were up across the board, and in some classes, including Elementary Statistics and College Algebra, they even surpassed pass rates for on-campus students. Here's how they stacked up:
Elementary Statistics: Spring pass rate: 50.5 percent; summer pass rate: 83 percent; on-campus pass rate: 76.3 percent
College Algebra: Spring pass rate: 25.4 percent; summer pass rate: 72.6 percent; on-campus pass rate: 64.7 percent
Entry Level Math: Spring pass rate: 23.8 percent; summer pass rate: 29.8 percent; on-campus pass rate: 45.5 percent
General Psychology (not offered in the Spring): Summer pass rate: 67.3 percent; on-campus pass rate: 83 percent
Intro to Programming (not offered in the Spring): Summer pass rate: 70.4 percent; on-campus pass rate: 67.6 percent
According to Udacity co-founder and CEO Sebastian Thrun, the company changed a number of pieces of its strategy to improve pass rates this time around. For starters, Udacity re-recorded some of its least successful course videos. The company also changed the pacing of the courses, so students knew ahead of time when they were falling behind, and added more staff to support students online.
Though he's pleased with the results, Thrun recently told me by phone, "We're not perfect yet. There are a lot of improvements we can make, but invention is a process. You have to work really hard, look at data, and improve to get better and better and better."
Another big change this time around is the fact that for the pilot program this spring, Udacity made a concerted effort to recruit students from underserved high schools in California. After all, Thrun says, the purpose of MOOCs is not to replace a college education for people who have access to one, but to expand access to education to people don't. "A lot of people say, 'How does this compare to on campus?' but I think that's the wrong question," Thrun says. "The question is, 'How does it compare to nothing?'"
The summer session, however, which was open to the public, primarily consisted of students who already had some sort of post-secondary degree. This switch does call into question just how well MOOC providers like Udacity are able to serve communities that have little to no access to a college education. In a recent speech on affordability in higher education, President Obama referenced Udacity's upcoming partnership with Georgia Tech University, which, beginning this January, will offer a Master's in Computer Science for $7,000. President Obama listed this as one example of how universities are embracing "innovative new ways to prepare our students for a 21st century economy and maintain a high level of quality without breaking the bank."
While Thrun commends President Obama for encouraging technological innovation in education, he says the San Jose experiment has taught him that MOOC providers like Udacity, Coursera, and edX still have a long way to go before they're a viable substitute for a traditional college degree.
"I believe Obama is really after creative new solutions, and I'm extremely hopeful that we can bring a hardcore education to people who have no access whatsoever, but that doesn't mean we won't fail a lot," Thrun says. "We have to be humble. It's a serious topic. It's not going to be solved overnight. We have to be honest about the fact that we're experimenting, and we haven't solved the problem, but we're making progress."