Bridget S Banach

Loyola University Chicago, Chicago, Illinois, United States

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Publications (5)14.71 Total impact

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    ABSTRACT: Viral protease inhibitors are remarkably effective at blocking the replication of viruses such as human immunodeficiency virus and hepatitis C virus, but inevitably lead to the selection of inhibitor resistant mutants, which may contribute to ongoing disease. Protease inhibitors blocking the replication of coronavirus (CoV), including the causative agents of Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), provide a promising foundation for the development of anti-coronaviral therapeutics. However, the selection and consequences of inhibitor resistant CoVs are unknown. Here, we exploit the model coronavirus, mouse hepatitis virus (MHV) to investigate the genotype and phenotype of MHV quasispecies selected for resistance to a broad-spectrum CoV 3C-like protease (3CLpro) inhibitor. Clonal sequencing identified single or double mutations within the 3CLpro coding sequence of inhibitor resistant virus. Using reverse genetics to generate isogenic viruses with mutant 3CLpros, we found that viruses encoding double mutant 3CLpros are fully resistant to the inhibitor and exhibit a significant delay in proteolytic processing of the viral replicase polyprotein. The inhibitor resistant viruses also exhibited postponed and reduced production of infectious virus particles. Biochemical analysis verified double mutant 3CLpro enzyme as impaired for protease activity and exhibiting reduced sensitivity to the inhibitor, and revealed a delayed kinetics of inhibitor hydrolysis and activity restoration. Furthermore, the inhibitor resistant virus was shown to be highly attenuated in mice. Our study provides the first insight into the pathogenicity and mechanism of 3CLpro inhibitor resistant CoV mutants, revealing a low genetic barrier but high fitness cost of resistance.
    Journal of Virology 08/2014; 88(20). DOI:10.1128/JVI.01528-14 · 4.65 Impact Factor
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    ABSTRACT: Viruses have evolved elaborate mechanisms to evade or inactivate the complex system of sensors and signaling molecules that make up the host innate immune response. Here we show that human coronavirus (HCoV) NL63 and severe acute respiratory syndrome (SARS) CoV papain-like proteases (PLP) antagonize innate immune signaling mediated by STING (stimulator of interferon genes, also known as MITA/ERIS/MYPS). STING resides in the endoplasmic reticulum and upon activation, forms dimers which assemble with MAVS, TBK-1 and IKKε, leading to IRF-3 activation and subsequent induction of interferon (IFN). We found that expression of the membrane anchored PLP domain from human HCoV-NL63 (PLP2-TM) or SARS-CoV (PLpro-TM) inhibits STING-mediated activation of IRF-3 nuclear translocation and induction of IRF-3 dependent promoters. Both catalytically active and inactive forms of CoV PLPs co-immunoprecipitated with STING, and viral replicase proteins co-localize with STING in HCoV-NL63-infected cells. Ectopic expression of catalytically active PLP2-TM blocks STING dimer formation and negatively regulates assembly of STING-MAVS-TBK1/IKKε complexes required for activation of IRF-3. STING dimerization was also substantially reduced in cells infected with SARS-CoV. Furthermore, the level of ubiquitinated forms of STING, RIG-I, TBK1 and IRF-3 are reduced in cells expressing wild type or catalytic mutants of PLP2-TM, likely contributing to disruption of signaling required for IFN induction. These results describe a new mechanism used by CoVs in which CoV PLPs negatively regulate antiviral defenses by disrupting the STING-mediated IFN induction.
    PLoS ONE 02/2012; 7(2):e30802. DOI:10.1371/journal.pone.0030802 · 3.53 Impact Factor
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    ABSTRACT: Coronaviruses encode multifunctional proteins that are critical for viral replication and for blocking the innate immune response to viral infection. One such multifunctional domain is the coronavirus papain-like protease (PLP), which processes the viral replicase polyprotein, has deubiquitinating (DUB) activity, and antagonizes the induction of type I interferon (IFN). Here we characterized the DUB and IFN antagonism activities of the PLP domains of human coronavirus NL63 and severe acute respiratory syndrome (SARS) coronavirus to determine if DUB activity mediates interferon antagonism. We found that NL63 PLP2 deconjugated ubiquitin (Ub) and the Ub-line molecule ISG15 from cellular substrates and processed both lysine-48- and lysine-63- linked polyubiquitin chains. This PLP2 DUB activity was dependent on an intact catalytic cysteine residue. We demonstrated that in contrast to PLP2 DUB activity, PLP2-mediated interferon antagonism did not require enzymatic activity. Furthermore, addition of an inhibitor that blocks coronavirus protease/DUB activity did not abrogate interferon antagonism. These results indicated that a component of coronavirus PLP-mediated interferon antagonism was independent of protease and DUB activity. Overall, these results demonstrate the multifunctional nature of the coronavirus PLP domain as a viral protease, DUB, and IFN antagonist and suggest that these independent activities may provide multiple targets for antiviral therapies.
    Journal of Virology 02/2010; 84(9):4619-29. DOI:10.1128/JVI.02406-09 · 4.65 Impact Factor
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    ABSTRACT: Propagation of new human respiratory virus pathogens in established cell lines is hampered by a lack of predictability regarding cell line permissivity and by availability of suitable antibody reagents to detect infection in cell lines that do not exhibit significant cytopathic effect. Recently, molecular methods have been used to amplify and identify novel nucleic acid sequences directly from clinical samples, but these methods may be hampered by the quantity of virus present in respiratory secretions at different time points following the onset of infection. Human airway epithelial (HAE) cultures, which effectively mimic the human bronchial environment, allow for cultivation of a wide variety of human respiratory viral pathogens. The goal of the experiments described here was to determine if propagation and identification of a human respiratory virus may be achieved through inoculation of HAE cultures followed by whole transcriptome amplification (WTA) and sequence analysis. To establish proof-of-principle human coronavirus NL63 (HCoV-NL63) was evaluated, and the first visualization of HCoV-NL63 virus by transmission electron microscopy (TEM) is reported. Initial propagation of human respiratory secretions onto HAE cultures followed by TEM and WTA of culture supernatant may be a useful approach for visualization and detection of new human respiratory pathogens that have eluded identification by traditional approaches.
    Journal of Virological Methods 12/2008; 156(1-2):19-26. DOI:10.1016/j.jviromet.2008.10.022 · 1.88 Impact Factor
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    Jan M Orenstein, Bridget S. Banach, Susan C Baker
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    ABSTRACT: NL63 (HCoV-NL63) is a recently discovered human coronavirus that causes respiratory disease in infants and young children. NL63 productively infects LLCMK2 cells and ciliated epithelial cells of human airway cell cultures. Transmission electron microscopic (TEM) studies of NL63 infected LLCMK2 cells revealed that virions are spherical, spiked, and range from 75 to 115 nm in diameter. Virus replication predominantly occurs on the rough endoplasmic reticulum (RER), both perinuclear and cytoplasmic, and the Golgi. Plasma membrane budding was occasionally observed. As virus production increased, aberrant viral forms appeared with greater frequency. Unusual inclusions were present in infected cells including tubular and laminated structures. Pleomorphic double membrane-bound vesicles (DMV), measuring roughly 140 to 210 nm in diameter, were observed. The virus was released via exocytosis and cell lysis. In summary, we report the key morphologic characteristics of NL63 infection observed by TEM analysis.
    The Open Infectious Diseases Journal 01/2008; 2(1):52-58. DOI:10.2174/1874279300802010052