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    ABSTRACT: RNA folding pathways can be complex and even include kinetic traps or misfolded intermediates that can be slow to resolve. Characterizing these pathways is critical to understanding how RNA molecules acquire their biological function. We have previously developed a novel approach to help characterize such misfolded intermediates. Laser-assisted single-molecule refolding (LASR) is a powerful technique that combines temperature-jump (T-jump) kinetics with single-molecule detection. In a typical LASR experiment, the temperature is rapidly increased and conformational dynamics are characterized, in real-time, at the single-molecule level using single-molecule fluorescence resonance energy transfer (smFRET). Here, we provide detailed protocols for performing LASR experiments including sample preparation, temperature calibration, and data analysis.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1086:289-307.
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    ABSTRACT: Herpes simplex virus 1 (HSV1) is an enveloped virus that uses undefined transport carriers for trafficking of its glycoproteins to envelopment sites. Screening of an siRNA library against 60 Rab GTPases revealed Rab6 as the principal Rab involved in HSV1 infection, with its depletion preventing Golgi-to-plasma membrane transport of HSV1 glycoproteins in a pathway used by several integral membrane proteins but not the luminal secreted protein Gaussia luciferase. Knockdown of Rab6 reduced virus yield to 1% and inhibited capsid envelopment, revealing glycoprotein exocytosis as a prerequisite for morphogenesis. Rab6-dependent virus production did not require the effectors myosin-II, bicaudal-D, dynactin-1 or rabkinesin-6, but was facilitated by ERC1, a factor involved in linking microtubules to the cell cortex. Tubulation and exocytosis of Rab6-positive, glycoprotein-containing membranes from the Golgi was substantially augmented by infection, resulting in enhanced and targeted delivery to cell tips. This reveals HSV1 morphogenesis as one of the first biological processes shown to be dependent on the exocytic activity of Rab6.
    Traffic 10/2013;
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    ABSTRACT: The innate immune system is critical in the response to infection by pathogens and it is activated by pattern recognition receptors (PRRs) binding to pathogen associated molecular patterns (PAMPs). During viral infection, the direct recognition of the viral nucleic acids, such as the genomes of DNA viruses, is very important for activation of innate immunity. Recently, DNA-dependent protein kinase (DNA-PK), a heterotrimeric complex consisting of the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs was identified as a cytoplasmic PRR for DNA that is important for the innate immune response to intracellular DNA and DNA virus infection. Here we show that vaccinia virus (VACV) has evolved to inhibit this function of DNA-PK by expression of a highly conserved protein called C16, which was known to contribute to virulence but by an unknown mechanism. Data presented show that C16 binds directly to the Ku heterodimer and thereby inhibits the innate immune response to DNA in fibroblasts, characterised by the decreased production of cytokines and chemokines. Mechanistically, C16 acts by blocking DNA-PK binding to DNA, which correlates with reduced DNA-PK-dependent DNA sensing. The C-terminal region of C16 is sufficient for binding Ku and this activity is conserved in the variola virus (VARV) orthologue of C16. In contrast, deletion of 5 amino acids in this domain is enough to knockout this function from the attenuated vaccine strain modified vaccinia virus Ankara (MVA). In vivo a VACV mutant lacking C16 induced higher levels of cytokines and chemokines early after infection compared to control viruses, confirming the role of this virulence factor in attenuating the innate immune response. Overall this study describes the inhibition of DNA-PK-dependent DNA sensing by a poxvirus protein, adding to the evidence that DNA-PK is a critical component of innate immunity to DNA viruses.
    PLoS Pathogens 10/2013; 9(10):e1003649.
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    ABSTRACT: Herpes simplex virus 1 deleted for VP22 is proposed to require secondary mutation of VHS for viability. Here we show that a replication-competent Δ22 virus constructed by homologous recombination maintains a Wt VHS gene and has no other gross mutations. By contrast, Δ22 viruses recovered from a bacterial artificial chromosome contain multiple codon changes within a conserved region of VHS. Hence, the mode of virus rescue influences the acquisition of secondary mutations.
    Journal of Virology 07/2013;
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    ABSTRACT: H5N1 influenza viruses pose a pandemic threat but have not acquired the ability to support sustained transmission between mammals in nature. The restrictions to transmissibility of avian influenza viruses in mammals are multigenic and overcoming them requires adaptations in HA and PB2 genes. Here we propose that a further restriction to mammalian transmission of the majority of HPAI H5N1 viruses may be the short stalk length of the NA protein. This genetic feature is selected for when influenza viruses adapt to chickens. In our study a recombinant virus, with seven gene segments from a human isolate of the 2009 H1N1 pandemic combined with the NA gene from a typical chicken-adapted H5N1 virus with a short stalk, did not support transmission by respiratory droplet between ferrets. This virus was also compromised in multicycle replication in cultures of human airway epithelium at 32°C. These defects correlated with a reduction in the ability of virus with a short stalk NA to penetrate mucus and de-aggregate virions. The deficiency in transmission and in cleavage of tethered substrates was overcome by increasing the stalk length of the NA protein. These observations suggest that H5N1 viruses that acquire a long stalk NA through reassortment might be more likely to support transmission between humans. Phylogenetic analysis showed that reassortment with long stalk NA occurred sporadically and as recently as 2011. However, all identified H5N1 viruses with a long stalk NA lacked other mammalian adapting features and were thus several genetic steps away from becoming transmissible between humans.
    Journal of Virology 07/2013;
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    ABSTRACT: Clade 2.2 Eurasian lineage H5N1 highly pathogenic avian influenza viruses (HPAIV), were first detected in Qinghai Lake, China in 2005, and subsequently spread through Asia, Europe and Africa. Importantly, these viruses carried a lysine at position 627 of the PB2 protein (PB2 627K), a known mammalian adaptation motif. Previous avian influenza isolates have carried glutamic acid in this position (PB2 627E), commonly described to restrict virus polymerase function in the mammalian host. We sought to examine the effect of PB2 627K on viral maintenance in the avian reservoir. Reverse genetics viruses were engineered to contain converse PB2 627K/E mutations in a Eurasian H5N1 virus (A/turkey/Turkey/5/2005; Ty/05) and, for comparison, a historical pre-Asian HPAI H5N1 virus that naturally bears PB2 627E (A/turkey/England/50-92/1991; 50-92). 50-92 PB2 627K was genetically unstable during virus propagation resulting in reversion to PB2 627E or the accumulation of additional mutations PB2 628R and/or a synonymous mutation PB2 a1869g. Intriguingly PB2 628R and/or a1869g appeared to improve the genetic stability of 50-92 PB2 627K. However, the replication of 50-92 PB2 627K in conjunction with these stabilizing mutations was significantly restricted in experimentally infected chickens where reversion to PB2 627E occured. In contrast, no significant effects on viral fitness was observed for Ty/05 PB2 627E or K in in vitro or in vivo experiments. Our observations suggest PB2 627K is supported in Eurasian lineage viruses; in contrast PB2 627K carries a significant fitness cost in the historical pre-Asian 50-92 virus.
    Journal of Virology 07/2013;
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    ABSTRACT: In addition to transmission involving extracellular free particles, a generally accepted model of virus propagation is one wherein virus replicates in one cell producing infectious particles that transmit to the next cell via cell junctions or induced polarised contacts. This mechanism of spread is especially important in the presence of neutralising antibody and the concept underpins analysis of virus spread, plaque size, viral and host functions and general mechanisms of virus propagation. Here we demonstrate a novel process involved in cell-to-cell transmission of herpes simplex virus (HSV) in human skin cells that has not previously been appreciated. Using time lapse microscopy of fluorescent viruses we show that HSV infection induces the polarised migration of skin cells into the site of infection. In the presence of neutralising antibody, uninfected skin cells migrate to the initial site of infection and spread over infected cells, to become infected in a spatially confined cluster containing hundreds of cells. The cells in this cluster do not undergo cytocidal cell lysis but harbour abundant enveloped particles within cells and cell-free virus within interstitial regions below the cluster surface. Cells at the base and outside the cluster were generally negative for virus immediate-early expression. We further show using spatially separated monolayer assays, that at least one component of this induced migration is the paracrine stimulation of a cytotactic response from infected cells to uninfected cells. The existence of this process changes our concept of virus transmission and the potential functions, virus and host factors involved.
    Journal of Virology 05/2013;
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    ABSTRACT: Mammalian poxviruses, including vaccinia virus (VACV), have evolved multiple mechanisms to evade the host type I interferon (IFN) responses at different levels, with viral proteins targeting IFN induction, signaling and antiviral effector functions. Avian poxviruses (avipoxviruses), which have been developed as recombinant vaccine vectors for permissive (i.e. poultry) and non-permissive (i.e. mammals, including humans) species, encode no obvious equivalents of any of these proteins. We show that fowlpox virus (FWPV) fails to induce chicken IFN-β (ChIFN2) and is able to block its induction by transfected poly(I:C), an analog of cytoplasmic double-strand (ds) RNA. A broad-scale loss-of-function genetic screen was used to find FWPV-encoded modulators of poly(I:C)-mediated ChIFN2 induction. It identified fpv012, a member of a family of poxvirus genes, highly expanded in the avipoxviruses (31 in FWPV; 51 in canarypox virus (CNPV), representing 15% of the total gene complement), encoding proteins containing N-terminal ankyrin repeats (ANKs) and C-terminal F-box-like motifs. Under ectopic expression, the first ANK of fpv012 is dispensable for inhibitory activity and the CNPV ortholog is also able to inhibit induction of ChIFN2. FWPV defective in fpv012 replicate well in culture and barely induce ChIFN2 during infection, suggesting other factors are involved in blocking IFN induction and resisting the antiviral effectors. Nevertheless, unlike parental and revertant viruses, the mutants induce moderate levels of expression of interferon stimulated genes (ISG), suggesting either that there is sufficient ChIFN2 expression to partially induce the ISGs or the involvement of alternative, IFN-independent pathways, that are also normally blocked by fpv012.
    Journal of Virology 02/2013;
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    ABSTRACT: Viruses must be able to resist host innate responses, especially the type I interferon (IFN) response. They do so by preventing induction or activity of IFN and/or by resisting the antiviral effectors it induces. Poxviruses are no exception, with many mechanisms identified whereby mammalian poxviruses, notably vaccinia (VACV) but also cowpox and myxoma viruses, are able to evade host IFN responses. Similar mechanisms have not been described for avian poxviruses (avipoxviruses). Restricted for permissive replication to avian hosts, they have received less attention; moreover the avian host responses are less well characterised. We show that the prototypic avipoxvirus, fowlpox virus (FWPV) is highly resistant to the antiviral effects of avian IFN. A gain-of-function genetic screen identified fpv014 as contributing to increased resistance to exogenous recombinant chicken IFN-α (ChIFN1). Fpv014 is a member of the large family of poxvirus (especially avipoxvirus) genes that encode proteins containing N-terminal ankyrin repeats (ANKs) and C-terminal F-box-like motifs. By binding the Skp1/Cullin-1 complex, the F-box in such proteins appears to target ligands bound by the ANKs for ubiquitination. Mass spectrometry and immunoblotting demonstrated that tandem affinity-purified, tagged fpv014 was complexed with chicken cullin-1 and Skp-1. Prior infection with an fpv014 knockout mutant of FWPV still blocked transfected poly(I:C)-mediated induction of the IFNβ (ChIFN2) promoter as effectively as parental FWPV, but the mutant was more sensitive to exogenous ChIFN1. Therefore, unlike the related protein fpv012, fpv014 does not contribute to the FWPV block to induction of ChIFN2, but does confer resistance to an established antiviral state.
    Journal of Virology 02/2013;
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    ABSTRACT: To explore the role of p16(INK4a) as an intrinsic barrier to B cell transformation by EBV, we transformed primary B cells from an individual homozygous for a deletion in the CDKN2A locus encoding p16(INK4a) and p14(ARF). Using recombinant EBV-BAC viruses expressing conditional EBNA3C (3CHT), we developed a system that allows inactivation of EBNA3C in lymphoblastoid cell lines (LCLs) lacking active p16(INK4a) protein but expressing a functional 14(ARF)-fusion protein (p14/p16). The INK4a locus is epigenetically repressed by EBNA3C - in cooperation with EBNA3A - despite the absence of functional p16(INK4a). Although inactivation of EBNA3C in LCLs from normal B cells leads to an increase in p16(INK4a) and growth arrest, EBNA3C inactivation in the p16(INK4a)-null LCLs has no impact on the rate of proliferation, establishing that the repression of INK4a is a major function of EBNA3C in EBV-driven LCL proliferation. This conditional LCL system allowed us to use microarray analysis to identify and confirm genes regulated specifically by EBNA3C, independently of proliferation changes modulated by the p16(INK4a)-Rb-E2F axis. Infections of normal primary B cells with recombinant EBV-BAC virus from which EBNA3C is deleted or with 3CHT EBV in the absence of activating ligand 4-hydroxytamoxifen, revealed that EBNA3C is necessary to overcome an EBV-driven increase in p16(INK4a) expression and concomitant block to proliferation 2-4 weeks post-infection. If cells are p16(INK4a)-null, functional EBNA3C is dispensable for the outgrowth of LCLs.
    PLoS Pathogens 02/2013; 9(2):e1003187.
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