vaccinia-reporter-viruses-for-quantifying-viral-function-at-all

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

We have characterized transcripts synthesized in vivo by bacteriophage T7 RNA polymerase to investigate yeast mRNA processing. T7 transcripts are not capped, consistent with capping being tightly coupled to RNA polymerase II (pol II) transcription. In contrast to higher eukaryotic non-pol II transcripts, yeast T7 transcripts are spliced as well as cleaved and polyadenylated. However, T7 and pol II transcripts are affected differently in cleavage and polyadenylation mutant strains, indicating that pol II may have a role in yeast 3' end formation. T7 transcripts with 3' ends directed by a polyadenylation signal are exported from the nucleus, and this export is dependent on the canonical cleavage and polyadenylation machinery. Importantly, transcripts with T7 terminator-directed 3' ends are unadenylated and predominantly nuclear in wild-type cells. Our results suggest that transcription by pol II is required for neither the nuclear export of an in vivo-transcribed mRNA nor for the retention of transcripts with aberrant 3' ends. Moreover, proper 3' end formation may be necessary and sufficient to promote mRNA export in yeast.

T7 RNA polymerase-directed transcripts are processed in yeast and link 3' end formation to mRNA nuclear export.

Several aspects of eukaryotic mRNA processing are linked to transcription. In Saccharomyces cerevisiae, overexpression of the mRNA export factor Sub2p suppresses the growth defect of hpr1 null cells, yet the protein Hpr1p and the associated THO protein complex are implicated in transcriptional elongation. Indeed, we find that a pool of heat shock HSP104 transcripts are 3'-end truncated in THO complex mutant as well as sub2 mutant backgrounds. Surprisingly, however, this defect can be suppressed by deletion of the 3'-5' exonuclease Rrp6p. This indicates that incomplete RNAs result from nuclear degradation rather than from a failure to efficiently elongate transcription. RNAs that are not degraded are retained at the transcription site in a Rrp6p-dependent manner. Interestingly, the addition of a RRP6 deletion to sub2 or to THO complex mutants shows a strong synthetic growth phenotype, suggesting that the failure to retain and/or degrade defective mRNAs is deleterious. mRNAs produced in the 3'-end processing mutants rna14-3 and rna15-2, as well as an RNA harboring a 3' end generated by a self-cleaving hammerhead ribozyme, are also retained in Rrp6p-dependent transcription site foci. Taken together, our results show that several classes of defective RNPs are subject to a quality control step that impedes release from transcription site foci and suggest that suboptimal messenger ribonucleoprotein assembly leads to RNA degradation by Rrp6p.

Interactions between mRNA export commitment, 3'-end quality control, and nuclear degradation.

Eukaryotic mRNA is processed by enzymes and packaged with proteins within nuclei to generate functional messenger ribonucleoprotein (mRNP) particles. Processing and packaging factors can interact with mRNA cotranscriptionally to form an early mRNP. Erroneous mRNP formation leads to nuclear retention and degradation of the mRNA. It therefore appears that one function of cotranscriptional mRNP assembly is to discard aberrant mRNPs early in their biogenesis. Cotranscriptional mRNP assembly may also enable the transcription machinery to respond to improper mRNP formation.

Early formation of mRNP: license for export or quality control?

To investigate the role of 3' end formation in yeast mRNA export, we replaced the mRNA cleavage and polyadenylation signal with a self-cleaving hammerhead ribozyme element. The resulting RNA is unadenylated and accumulates near its site of synthesis. Nonetheless, a significant fraction of this RNA reaches the cytoplasm. Nuclear accumulation was relieved by insertion of a stretch of DNA-encoded adenosine residues immediately upstream of the ribozyme element (a synthetic A tail). This indicates that a 3' stretch of adenosines can promote export, independently of cleavage and polyadenylation. We further show that a synthetic A tail-containing RNA is unaffected in 3' end formation mutant strains, in which a normally cleaved and polyadenylated RNA accumulates within nuclei. Our results support a model in which a polyA tail contributes to efficient mRNA progression away from the gene, most likely through the action of the yeast polyA-tail binding protein Pab1p.

A synthetic A tail rescues yeast nuclear accumulation of a ribozyme-terminated transcript.

Nonsense-mediated decay (NMD) is a eukaryotic regulatory process that degrades mRNAs with premature termination codons (PTCs). Although NMD is a translation-dependent process, there is evidence from mammalian systems that PTC recognition and mRNA degradation takes place in association with nuclei. Consistent with this notion, degradation of mammalian PTC-containing mRNAs occurs when they are bound by the cap binding complex (CBC) during a "pioneer" round of translation. Moreover, there are reports indicating that a PTC can trigger other nuclear events such as alternative splicing, abnormal 3' end processing, and accumulation of pre-mRNA at transcription sites. To examine whether a PTC can elicit similar nuclear events in yeast, we used RNA export-defective mutants to sequester mRNAs within nuclei. The results indicate that nuclear PTC-containing yeast RNAs are NMD insensitive. We also observed by fluorescent in situ hybridization that there was no PTC effect on mRNA accumulated at the site of transcription. Finally, we show that yeast NMD occurs minimally if at all on CBC-bound transcripts, arguing against a CBC-mediated pioneer round of translation in yeast. The data taken together indicate that there are no direct consequences of a PTC within the yeast nucleus.

Nonsense-mediated decay does not occur within the yeast nucleus.

Multiple studies indicate that mRNA processing defects cause mRNAs to accumulate in discrete nuclear foci or dots, in mammalian cells as well as yeast. To investigate this phenomenon, we have studied a series of GAL reporter constructs integrated into the yeast genome adjacent to an array of TetR-GFP-bound TetO sites. mRNA within dots is predominantly post-transcriptional, and dots are adjacent to but distinct from their transcription site. These reporter genes also localize to the nuclear periphery upon gene induction, like their endogenous GAL counterparts. Surprisingly, this peripheral localization persists long after transcriptional shutoff, and there is a comparable persistence of the RNA in the dots. Moreover, dot disappearance and gene delocalization from the nuclear periphery occur with similar kinetics after transcriptional shutoff. Both kinetics depend in turn on reporter gene 3'-end formation signals. Our experiments indicate that gene association with the nuclear periphery does not require ongoing transcription and suggest that the mRNPs within dots may make a major contribution to the gene-nuclear periphery tether.

3'-end formation signals modulate the association of genes with the nuclear periphery as well as mRNP dot formation.

TREM-1 (triggering receptor expressed on myeloid cells-1) is an orphan immunoreceptor expressed on monocytes, macrophages, and neutrophils. TREM-1 associates with and signals via the adapter protein DAP12/TYROBP, which contains an ITAM. TREM-1 activation by receptor cross-linking has been shown to be proinflammatory and to amplify some cellular responses to TLR ligands such as bacterial LPS. To investigate the cellular consequences of TREM-1 activation, we have characterized global gene expression changes in human monocytes in response to TREM-1 cross-linking in comparison to and combined with LPS. Both TREM-1 activation and LPS up-regulate chemokines, cytokines, matrix metalloproteases, and PTGS/COX2, consistent with a core inflammatory response. However, other immunomodulatory factors are selectively induced, including SPP1 and CSF1 (i.e., M-CSF) by TREM-1 activation and IL-23 and CSF3 (i.e., G-CSF) by LPS. Additionally, cross-talk between TREM-1 activation and LPS occurs on multiple levels. Although synergy in GM-CSF protein production is reflected in commensurate mRNA abundance, comparable synergy in IL-1beta protein production is not. TREM-1 activation also attenuates the induction of some LPS target genes, including those that encode IL-12 cytokine family subunits. Where tested, positive TREM-1 outputs are greatly reduced by the PI3K inhibitor wortmannin, whereas this attenuation is largely PI3K independent. These experiments provide a detailed analysis of the cellular consequences of TREM-1 activation and highlight the complexity in signal integration between ITAM- and TLR-mediated signaling.

Innate immune responses to TREM-1 activation: overlap, divergence, and positive and negative cross-talk with bacterial lipopolysaccharide.

To investigate the expression and function of triggering receptor expressed on myeloid cells-1 (TREM-1) in the synovium of human RA patients as well as the level of soluble TREM-1 in the plasma of RA patients.

TREM-1 expression is increased in the synovium of rheumatoid arthritis patients and induces the expression of pro-inflammatory cytokines.

Vaccinia virus is the prototypical orthopoxvirus of Poxviridae, a family of viruses that includes the human pathogens Variola (smallpox) and Monkeypox. Core viral functions are conserved among orthopoxviruses, and consequently Vaccinia is routinely used to study poxvirus biology and screen for novel antiviral compounds. Here we describe the development of a series of fluorescent protein-based reporter Vaccinia viruses that provide unprecedented resolution for tracking viral function. The reporter viruses are divided into two sets: (1) single reporter viruses that utilize temporally regulated early, intermediate, or late viral promoters; and (2) multi-reporter viruses that utilize multiple temporally regulated promoters. Promoter and reporter combinations were chosen that yielded high signal-to-background for stage-specific viral outputs. We provide examples for how these viruses can be used in the rapid and accurate monitoring of Vaccinia function and drug action.

Development of Vaccinia reporter viruses for rapid, high content analysis of viral function at all stages of gene expression.

Vaccinia-Virus ist die prototypische Orthopoxvirus Poxviridae, eine Familie von Viren, die die menschliche Krankheitserreger Variola (Pocken) und Monkeypox enth&auml;lt. Virale Kernfunktionen sind unter Orthopoxviruses konserviert, und folglich Vaccinia wird routinem&auml;&szlig;ig verwendet, um die Pockenviren Biologie und Bildschirm f&uuml;r neuartige antivirale Substanzen zu studieren. Hier beschreiben wir die Entwicklung einer Reihe von fluoreszierenden Protein-basierte Reporter Vaccinia-Viren, die bis dahin unerreichter Aufl&ouml;sung bieten f&uuml;r die Verfolgung von viralen Funktion. Die Reporter-Viren sind in zwei Gruppen unterteilt: (1) einzelne Reporter-Viren, die zeitlich nutzen geregelt fr&uuml;he, mittlere oder sp&auml;te virale Promotoren; und (2) multi-reporter Viren, die mehrere zeitlich nutzen reguliert F&ouml;rderer. Projekttr&auml;ger und Reporter-Kombinationen wurden ausgew&auml;hlt, dass das hohen Signal-zu-Hintergrund f&uuml;r B&uuml;hne-spezifische virale Ausg&auml;nge ergab. Wir bieten Beispiele wie diese Viren in die schnelle und genaue &Uuml;berwachung der Vaccinia-Funktion und Drogen-Aktion verwendet werden k&ouml;nnen.

Entwicklung von Vaccinia Reporter Viren f&uuml;r schnelle, hohe Inhaltsanalyse der viral-Funktion auf allen Stufen der Genexpression.

Orthopoxviruses include the prototypical vaccinia virus, the emerging infectious agent monkeypox virus, and the potential biothreat variola virus (the causative agent of smallpox). There is currently no FDA-approved drug for humans infected with orthopoxviruses. We screened a diversity-oriented synthesis library for new scaffolds with activity against vaccinia virus. This screen identified a nonnucleoside analog that blocked postreplicative intermediate and late gene expression. Viral genome replication was unaffected, and inhibition could be elicited late in infection and persisted upon drug removal. Sequencing of drug-resistant viruses revealed mutations predicted to be on the periphery of the highly conserved viral RNA polymerase large subunit. Consistent with this, the compound had broad-spectrum activity against orthopoxviruses in vitro. These findings indicate that novel chemical synthesis approaches are a potential source for new infectious disease therapeutics and identify a potentially promising candidate for development to treat orthopoxvirus-infected individuals.

Identification of a pyridopyrimidinone inhibitor of orthopoxviruses from a diversity-oriented synthesis library.

Orthopoxviruses geh&ouml;ren das prototypische Vaccinia-Virus, der aufstrebenden infekti&ouml;ses Agens Monkeypox Virus und die potenziellen Biothreat Variola-Virus (der Erreger der Pocken). Es gibt derzeit kein FDA zugelassene Medikament f&uuml;r Menschen, die mit Orthopoxviruses infiziert. Wir sehen eine Vielfalt-orientierte Synthese-Bibliothek f&uuml;r neue Ger&uuml;ste mit Aktivit&auml;t gegen des Vaccinia Virus. Dieser Bildschirm identifiziert eine analoge Nonnucleoside, die postreplicative zwischen- und sp&auml;ten Genexpression blockiert. Virale Genom-Replikation nicht betroffen war, und Hemmung l&ouml;ste in der Infektion und nach der Droge Entfernung beibehalten werden konnte. Sequenzierung von Medikamenten-resistenten Viren offenbart Mutationen, die voraussichtlich am Ortsrand der gro&szlig;en stark konservierten virale RNA-Polymerase-Untereinheit. Folgerichtig hatte das Compound Breitspektrum Aktivit&auml;t gegen Orthopoxviruses in-vitro. Diese Befunde zeigen, dass neuartige chemische Synthese Ans&auml;tze eine potenzielle Quelle f&uuml;r neue Infektionskrankheiten-Therapeutika sind und identifizieren einen potenziell vielversprechenden Kandidaten f&uuml;r die Entwicklung Orthopoxvirus-infizierten Personen zu behandeln.

Kennung f&uuml;r ein Pyridopyrimidinone-Inhibitor der Orthopoxviruses aus einer Vielfalt-orientierte Synthese-Bibliothek.

Toll-like receptor 4 (TLR4) is activated by bacterial endotoxin, a prototypical pathogen-associated molecular pattern (PAMP). It has been suggested that TLR4 can also be activated by damage-associated molecular pattern (DAMP) proteins such as HSP70. It remains a challenge to provide unequivocal evidence that DAMP proteins themselves play a role in TLR4 activation, as the DAMP proteins used are often contaminated with endotoxin and other TLR ligands introduced during protein expression and/or purification.

Stimulation of TLR4 by recombinant HSP70 requires structural integrity of the HSP70 protein itself.

Myxoma virus (MYXV) and vaccinia virus (VACV), two distinct members of the family Poxviridae, are both currently being developed as oncolytic virotherapeutic agents. Recent studies have demonstrated that ex vivo treatment with MYXV can selectively recognize and kill contaminating cancerous cells from autologous bone marrow transplants without perturbing the engraftment of normal CD34(+) hematopoietic stem and progenitor cells. However, the mechanism(s) by which MYXV specifically recognizes and eliminates the cancer cells in the autografts is not understood. While little is known about the cellular attachment factor(s) exploited by MYXV for entry into any target cells, VACV has been shown to utilize cell surface glycosaminoglycans such as heparan sulfate (HS), the extracellular matrix protein laminin, and/or integrin β1. We have constructed MYXV and VACV virions tagged with the Venus fluorescent protein and compared their characteristics of binding to various human cancer cell lines as well as to primary human leukocytes. We report that the binding of MYXV or VACV to some adherent cell lines could be partially inhibited by heparin, but laminin blocked only VACV binding. In contrast to cultured fibroblasts, the binding of MYXV and VACV to a wide spectrum of primary human leukocytes could not be competed by either HS or laminin. Additionally, MYXV and VACV exhibited very different binding characteristics against certain select human leukocytes, suggesting that the two poxviruses utilize different cell surface determinants for the attachment to these cells. These results indicate that VACV and MYXV can exhibit very different oncolytic tropisms against some cancerous human leukocytes.

Myxoma and vaccinia viruses bind differentially to human leukocytes.

The genus Orthopoxviridae contains a diverse group of human pathogens including monkeypox, smallpox and vaccinia. These viruses are presumed to be less dependent on host functions than other DNA viruses because they have large genomes and replicate in the cytoplasm, but a detailed understanding of the host factors required by orthopoxviruses is lacking. To address this topic, we performed an unbiased, genome-wide pooled RNAi screen targeting over 17,000 human genes to identify the host factors that support orthopoxvirus infection. We used secondary and tertiary assays to validate our screen results. One of the strongest hits was heat shock factor 1 (HSF1), the ancient master regulator of the cytoprotective heat-shock response. In investigating the behavior of HSF1 during vaccinia infection, we found that HSF1 was phosphorylated, translocated to the nucleus, and increased transcription of HSF1 target genes. Activation of HSF1 was supportive for virus replication, as RNAi knockdown and HSF1 small molecule inhibition prevented orthopoxvirus infection. Consistent with its role as a transcriptional activator, inhibition of several HSF1 targets also blocked vaccinia virus replication. These data show that orthopoxviruses co-opt host transcriptional responses for their own benefit, thereby effectively extending their functional genome to include genes residing within the host DNA. The dependence on HSF1 and its chaperone network offers multiple opportunities for antiviral drug development.

The master regulator of the cellular stress response (HSF1) is critical for orthopoxvirus infection.

IRAK4 is a central kinase in innate immunity, but the role of its kinase activity is controversial. The mechanism of activation for IRAK4 is currently unknown, and little is known about the role of IRAK4 kinase in cytokine production, particularly in different human cell types. We show IRAK4 autophosphorylation occurs by an intermolecular reaction and that autophosphorylation is required for full catalytic activity of the kinase. Phosphorylation of any two of the residues Thr-342, Thr-345, and Ser-346 is required for full activity, and the death domain regulates the activation of IRAK4. Using antibodies against activated IRAK4, we demonstrate that IRAK4 becomes phosphorylated in human cells following stimulation by IL-1R and Toll-like receptor agonists, which can be blocked pharmacologically by a dual inhibitor of IRAK4 and IRAK1. Interestingly, in dermal fibroblasts, although complete inhibition of IRAK4 kinase activity does not inhibit IL-1-induced IL-6 production, NF-κB, or MAPK activation, there is complete ablation of these processes in IRAK4-deficient cells. In contrast, the inhibition of IRAK kinase activity in primary human monocytes reduces R848-induced IL-6 production with minimal effect on NF-κB or MAPK activation. Taken together, these studies define the mechanism of IRAK4 activation and highlight the differential role of IRAK4 kinase activity in different human cell types as well as the distinct roles IRAK4 scaffolding and kinase functions play.

Interleukin 1/Toll-like Receptor-induced Autophosphorylation Activates Interleukin 1 Receptor-associated Kinase 4 and Controls Cytokine Induction in a Cell Type-specific Manner.

Article	Year
T7 RNA polymerase-directed transcripts are processed in yeast and link 3' end formation to mRNA nuclear export. RNA (New York, N.Y.)\| PubMed ID: 12022234	2002
Interactions between mRNA export commitment, 3'-end quality control, and nuclear degradation. Molecular and cellular biology\| PubMed ID: 12417728	2002
Early formation of mRNP: license for export or quality control? Molecular cell\| PubMed ID: 12769839	2003
A synthetic A tail rescues yeast nuclear accumulation of a ribozyme-terminated transcript. RNA (New York, N.Y.)\| PubMed ID: 15547135	2004
Nonsense-mediated decay does not occur within the yeast nucleus. RNA (New York, N.Y.)\| PubMed ID: 15547136	2004
3'-end formation signals modulate the association of genes with the nuclear periphery as well as mRNP dot formation. The EMBO journal\| PubMed ID: 16946703	2006
Innate immune responses to TREM-1 activation: overlap, divergence, and positive and negative cross-talk with bacterial lipopolysaccharide. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 18292579	2008
TREM-1 expression is increased in the synovium of rheumatoid arthritis patients and induces the expression of pro-inflammatory cytokines. Rheumatology (Oxford, England)\| PubMed ID: 19713442	2009
Development of Vaccinia reporter viruses for rapid, high content analysis of viral function at all stages of gene expression. Antiviral research\| PubMed ID: 21569797	2011
Identification of a pyridopyrimidinone inhibitor of orthopoxviruses from a diversity-oriented synthesis library. Journal of virology\| PubMed ID: 22205744	2012
Stimulation of TLR4 by recombinant HSP70 requires structural integrity of the HSP70 protein itself. Journal of inflammation (London, England)\| PubMed ID: 22448747	2012
Myxoma and vaccinia viruses bind differentially to human leukocytes. Journal of virology\| PubMed ID: 23388707	2013
The master regulator of the cellular stress response (HSF1) is critical for orthopoxvirus infection. PLoS pathogens\| PubMed ID: 24516381	2014
Interleukin 1/Toll-like Receptor-induced Autophosphorylation Activates Interleukin 1 Receptor-associated Kinase 4 and Controls Cytokine Induction in a Cell Type-specific Manner. The Journal of biological chemistry\| PubMed ID: 24567333	2014

Ken Dower

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