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Papers by John Connor

Mechanisms regulating protein phosphatase-1

The growth arrest and DNA-damage inducible gene product, GADD-34, recruits protein phosphatase-1 and its regulator, Inhibitor-1, to control protein synthesis

Paradoxical alpha IIb beta 3 priming and clustering by orally active integrin antagonists

Treatment of ige-mediated disease

Integrating microfluidic sample concentrator with Interferometric Reflectance Imaging Sensor for point-of-care viral identification

ABSTRACT We integrated a portable, disposable, simple to fabricate, and highly effective polymeri... more ABSTRACT We integrated a portable, disposable, simple to fabricate, and highly effective polymeric microfluidic viral sample concentration device to the Interferometric Reflectance Imaging Sensor (IRIS) to increase infectious disease diagnostic speed and sensitivity at the point-of-care.

Ultrasensitive Plasmonic Biosensors for Direct Detection of Biomarker Proteins with The Naked Eye

We introduce an ultrasensitive label free biodetection technique based on asymmetric plasmonic Fa... more We introduce an ultrasensitive label free biodetection technique based on asymmetric plasmonic Fano resonances. Our sensors bring a number of advantages: (i) ultrasensitive detection limits surpassing gold standard Kretschmann configuration plasmon sensors, (ii) detection of biomarker molecules with ``the naked eye&#x27;&#x27;, (iii) massive multiplexing capabilities. By exploiting extraordinary light transmission phenomena through high quality factor sub-radiant dark modes, we experimentally demonstrate record high figures of merits for intrinsic detection limits surpassing the gold ...

Pyridopyrimidinone inhibitors of viruses

Approaches for antiviral probe development: new libraries, new mechanisms

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 a... more Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

Lassa and Marburg viruses elicit distinct host transcriptional responses early after infection

Lassa virus and Marburg virus are two causative agents of viral hemorrhagic fever. Their diagnosi... more Lassa virus and Marburg virus are two causative agents of viral hemorrhagic fever. Their diagnosis is difficult because patients infected with either pathogen present similar nonspecific symptoms early after infection. Current diagnostic tests are based on detecting viral proteins or nucleic acids in the blood, but these cannot be found during the early stages of disease, before the virus starts replicating in the blood. Using the transcriptional response of the host during infection can lead to earlier diagnoses compared to those of traditional methods. In this study, we use RNA sequencing to obtain a high-resolution view of the in vivo transcriptional dynamics of peripheral blood mononuclear cells (PBMCs) throughout both types of infection. We report a subset of host mRNAs, including heat-shock proteins like HSPA1B, immunoglobulins like IGJ, and cell adhesion molecules like SIGLEC1, whose differences in expression are strong enough to distinguish Lassa infection from Marburg infection in non-human primates. We have validated these infection-specific expression differences by using microarrays on a larger set of samples, and by quantifying the expression of individual genes using RT-PCR. These results suggest that host transcriptional signatures are correlated with specific viral infections, and that they can be used to identify highly pathogenic viruses during the early stages of disease, before standard detection methods become effective.

The formation of protein-RNA granules is a part of both natural cellular function (P-bodies and n... more The formation of protein-RNA granules is a part of both natural cellular function (P-bodies and nuclear HNRNPs) and the response to cellular stress (stress granules and ND10 bodies). To better understand the role of stress-induced granules in viral infection, we have studied the ability of cells to restrict poxvirus replication through the formation of antiviral granules (AVGs). Of cells infected with a wild-type poxvirus, a small number spontaneously formed AVGs. In these AVG-positive cells, viral gene expression was inhibited. The addition of compounds that altered RNA helicase activity, induced oxidative stress, or stimulated translation initiation factor phosphorylation significantly increased the number of AVG-positive cells. When AVGs formed, both viral translation and titers were decreased even when host translation persisted. Treatment with the antiviral compound isatin ␤-thiosemicarbazone (IBT), a compound that was used to treat smallpox infections, induced AVGs, suggesting a role for these structures in the pharmacological inhibition of poxvirus replication. These findings provide evidence that AVGs are an innate host response that can be exogenously stimulated to combat virus infection. Since small molecules are able to stimulate AVG formation, it is a potential target for new antiviral development. FIG 2 Antiviral granule formation is enhanced during alterations of translation initiation. HeLa and U2OS cells were infected with WT-VACV at an MOI of 10. At 6 hpi, cells were treated with PBS, 500 M arsenite (Ars), 1 M thapsigargin (Thap), 1 M hippuristanol (Hipp), or 1 M rohinitib (RHT) and fixed an hour later at 7 hpi. Alternatively, cells were pretreated with 200 U/ml beta 1 interferon for 24 h before infection. Cells were stained with antibodies to G3BP1, TIA1, or CAPRIN1 and with DAPI. (A) Schematic describing parallel methods to alter translation initiation with small molecules. The activation of multiple stress kinases results in the phosphorylation of the alpha subunit of eIF2, preventing the formation of the ternary complex necessary for translation initiation. Alternatively, eIF4A is inhibited by hippuristanol and RHT in an eIF2␣-independent manner. (B) Whole-cell extracts from mock-infected and infected cells treated with 500 M Ars were separated by SDS-PAGE and blotted with an antibody against phosphorylated eIF2␣ (S51) (i) and total eIF2␣ (ii). A band densitometry comparison Rozelle et al. 2006 mcb.asm.org Molecular and Cellular Biology on June 17, 2016 by guest

Background: The Orthopoxvirus genus contains numerous virus species that are capable of causing d... more Background: The Orthopoxvirus genus contains numerous virus species that are capable of causing disease in humans, including variola virus (the etiological agent of smallpox), monkeypox virus, cowpox virus, and vaccinia virus (the prototypical member of the genus). Monkeypox is a zoonotic disease that is endemic in the Democratic Republic of the Congo and is characterized by systemic lesion development and prominent lymphadenopathy. Like variola virus, monkeypox virus is a high priority pathogen for therapeutic development due to its potential to cause serious disease with significant health impacts after zoonotic, accidental, or deliberate introduction into a naïve population. Results: The purpose of this study was to investigate the prophylactic and therapeutic potential of interferon-β (IFN-β) for use against monkeypox virus. We found that treatment with human IFN-β results in a significant decrease in monkeypox virus production and spread in vitro. IFN-β substantially inhibited monkeypox virus when introduced 6-8 h post infection, revealing its potential for use as a therapeutic. IFN-β induced the expression of the antiviral protein MxA in infected cells, and constitutive expression of MxA was shown to inhibit monkeypox virus infection. Conclusions: Our results demonstrate the successful inhibition of monkeypox virus using human IFN-β and suggest that IFN-β could potentially serve as a novel safe therapeutic for human monkeypox disease.

demonstrating that the enhanced translation of viral mRNA is genetically separable from inhibitio... more demonstrating that the enhanced translation of viral mRNA is genetically separable from inhibition of translation of host mRNA.

The recent solution of the crystal structure of a fragment of the vesicular stomatitis virus matr... more The recent solution of the crystal structure of a fragment of the vesicular stomatitis virus matrix (M) protein suggested that amino acids 121 to 124, located on a solvent-exposed loop of the protein, are important for M protein self-association and association with membranes. These residues were mutated from the hydrophobic AVLA sequence to the polar sequence DKQQ. Expression and purification of this mutant from bacteria showed that it was structurally stable and that the mutant M protein had self-association kinetics similar to those of the wild-type M protein. Analysis of the membrane association of M protein in the context of infection with isogenic recombinant viruses showed that both wild-type and mutant M proteins associated with membranes to the same extent. Virus expressing the mutant M protein did show an approximately threefold-lower binding affinity of M protein for nucleocapsid-M complexes. In contrast to the relatively minor effects of the M protein mutation on virus assembly, the mutant virus exhibited growth restriction in MDBK but not BHK cells, a slower induction of apoptosis, and lower viral-protein synthesis. Despite translating less viral protein, the mutant virus produced more viral mRNA, showing that the mutant virus could not effectively promote viral translation. These results demonstrate that the 121-to-124 region of the VSV M protein plays a minor role in virus assembly but is involved in virus-host interactions and VSV replication by augmenting viral-mRNA translation.

proposed that viral mRNAs are subjected to the same inhibition but are predominantly translated b... more proposed that viral mRNAs are subjected to the same inhibition but are predominantly translated because of their abundance. To compare translation efficiencies of viral and host mRNAs during infection, we used an enhanced green fluorescent protein (EGFP) reporter expressed from a recombinant virus or from the host nucleus in stably transfected cells. Translation efficiency of host-derived EGFP mRNA was reduced more than threefold at eight hours postinfection, while viral-derived mRNA was translated around sevenfold more efficiently than host-derived EGFP mRNA in VSV-infected cells. To test whether mRNAs transcribed in the cytoplasm are resistant to shutoff of translation during VSV infection, HeLa cells were infected with a recombinant simian virus 5 (rSV5) that expressed GFP. Cells were then superinfected with VSV or mock superinfected.

Cellular Mechanisms Regulating Protein Phosphatase-1 A KEY FUNCTIONAL INTERACTION BETWEEN INHIBITOR-2 AND THE TYPE 1 PROTEIN PHOSPHATASE CATALYTIC SUBUNIT

Inhibitor-1 (I-1) and inhibitor-2 (I-2) selectively inhibit type 1 protein serine/threonine phosp... more Inhibitor-1 (I-1) and inhibitor-2 (I-2) selectively inhibit type 1 protein serine/threonine phosphatases (PP1). To define the molecular basis for PP1 inhibition by I-1 and I-2 charged-to-alanine substitutions in the Saccharomyces cerevisiae, PP1 catalytic subunit (GLC7), were analyzed. Two PP1 mutants, E53A/E55A and K165A/E166A/K167A, showed reduced sensitivity to I-2 when compared with wild-type PP1. Both mutants were effectively inhibited by I-1. Two-hybrid analysis and coprecipitation or pull-down assays established that wild-type and mutant PP1 catalytic subunits bound I-2 in an identical manner and suggested a role for the mutated amino acids in enzyme inhibition. Inhibition of wild-type and mutant PP1 enzymes by full-length I-2(1-204), I-2(1-114), and I-2(36-204) indicated that the mutant enzymes were impaired in their interaction with the N-terminal 35 amino acids of I-2. Site-directed mutagenesis of amino acids near the N terminus of I-2 and competition for PP1 binding by a synthetic peptide encompassing an I-2 N-terminal sequence suggested that a PP1 domain composed of amino acids Glu-53, Glu-55, Asp-165, Glu-166, and Lys-167 interacts with the N terminus of I-2. This defined a novel regulatory interaction between I-2 and PP1 that determines I-2 potency and perhaps selectivity as a PP1 inhibitor.

Inhibition of Host and Viral Translation during Vesicular Stomatitis Virus Infection eIF2 IS RESPONSIBLE FOR THE INHIBITION OF VIRAL BUT NOT HOST TRANSLATION

In cells that allow replication of vesicular stomatitis virus (VSV), there are two phases of tran... more In cells that allow replication of vesicular stomatitis virus (VSV), there are two phases of translation inhibition: an early block of host translation and a later inhibition of viral translation. We investigated the phosphorylation of the alpha subunit of the eIF2 complex during these two phases of viral infection. In VSV-infected cells, the accumulation of phosphorylated (inactivated) eIF2alpha did not begin until well after host protein synthesis was inhibited, suggesting that it only plays a role in blocking viral translation later after infection. Consistent with this, cells expressing an unphosphorylatable eIF2alpha showed prolonged viral protein synthesis without an effect on host protein synthesis inhibition. Induction of eIF2alpha phosphorylation at early times of viral infection by treatment with thapsigargin showed that virus and host translation are similarly inhibited, demonstrating that viral and host messages are similarly sensitive to eIF2alpha phosphorylation. A recombinant virus that expresses a mutant matrix protein and is defective in the inhibition of host and virus protein synthesis showed an altered phosphorylation of eIF2alpha, demonstrating an involvement of viral protein function in inducing this antiviral response. This analysis of eIF2alpha phosphorylation, coupled with earlier findings that the eIF4F complex is modified earlier during VSV infection, supports a temporal/kinetic model of translation control, where at times soon after infection, changes in the eIF4F complex result in the inhibition of host protein synthesis; at later times, inactivation of the eIF2 complex blocks VSV protein synthesis.

Synaptic plasticity is thought to be a key process for learning, memory and other cognitive funct... more Synaptic plasticity is thought to be a key process for learning, memory and other cognitive functions of the nervous system. The initial events of plasticity require the conversion of brief electrical signals into alterations of the biochemical properties of synapses that last for much longer than the initial stimuli. Here we show that a regulator of synaptic plasticity, calcium/calmodulin-dependent protein kinase IIα (CaMKII), sequentially translocates to postsynaptic sites, undergoes autophosphorylation and gets trapped for several minutes until its dissociation is induced by secondary autophosphorylation and phosphatase 1 action. Once dissociated, CaMKII shows facilitated translocation for several minutes. This suggests that trapping of CaMKII by its targets and priming of CaMKII translocation may function as biochemical memory mechanisms that change the signaling capacity of synapses.