Publications by authors named "Shihyun You"

13 Publications

  • Page 1 of 1

Stimulator of interferon genes (STING) is an essential proviral host factor for human rhinovirus species A and C.

Proc Natl Acad Sci U S A 2020 11 15;117(44):27598-27607. Epub 2020 Oct 15.

Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599;

Human rhinoviruses (RVs) are positive-strand RNA viruses that cause respiratory tract disease in children and adults. Here we show that the innate immune signaling protein STING is required for efficient replication of members of two distinct RV species, RV-A and RV-C. The host factor activity of STING was identified in a genome-wide RNA interference (RNAi) screen and confirmed in primary human small airway epithelial cells. Replication of RV-A serotypes was strictly dependent on STING, whereas RV-B serotypes were notably less dependent. Subgenomic RV-A and RV-C RNA replicons failed to amplify in the absence of STING, revealing it to be required for a step in RNA replication. STING was expressed on phosphatidylinositol 4-phosphate (PI4P)-enriched membranes and was enriched in RV-A16 compared with RV-B14 replication organelles isolated in isopycnic gradients. The host factor activity of STING was species-specific, as murine STING (mSTING) did not rescue RV-A16 replication in STING-deficient cells. This species specificity mapped primarily to the cytoplasmic, ligand-binding domain of STING. Mouse-adaptive mutations in the RV-A16 2C protein allowed for robust replication in cells expressing mSTING, suggesting a role for 2C in recruiting STING to RV-A replication organelles. Palmitoylation of STING was not required for RV-A16 replication, nor was the C-terminal tail of STING that mediates IRF3 signaling. Despite co-opting STING to promote its replication, interferon signaling in response to STING agonists remained intact in RV-A16 infected cells. These data demonstrate a surprising requirement for a key host mediator of innate immunity to DNA viruses in the life cycle of a small pathogenic RNA virus.
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http://dx.doi.org/10.1073/pnas.2014940117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959528PMC
November 2020

ZAP's stress granule localization is correlated with its antiviral activity and induced by virus replication.

PLoS Pathog 2019 05 22;15(5):e1007798. Epub 2019 May 22.

The Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, United States of America.

Cellular antiviral programs encode molecules capable of targeting multiple steps in the virus lifecycle. Zinc-finger antiviral protein (ZAP) is a central and general regulator of antiviral activity that targets pathogen mRNA stability and translation. ZAP is diffusely cytoplasmic, but upon infection ZAP is targeted to particular cytoplasmic structures, termed stress granules (SGs). However, it remains unclear if ZAP's antiviral activity correlates with SG localization, and what molecular cues are required to induce this localization event. Here, we use Sindbis virus (SINV) as a model infection and find that ZAP's localization to SGs can be transient. Sometimes no apparent viral infection follows ZAP SG localization but ZAP SG localization always precedes accumulation of SINV non-structural protein, suggesting virus replication processes trigger SG formation and ZAP recruitment. Data from single-molecule RNA FISH corroborates this finding as the majority of cells with ZAP localization in SGs contain low levels of viral RNA. Furthermore, ZAP recruitment to SGs occurred in ZAP-expressing cells when co-cultured with cells replicating full-length SINV, but not when co-cultured with cells replicating a SINV replicon. ZAP recruitment to SGs is functionally important as a panel of alanine ZAP mutants indicate that the anti-SINV activity is correlated with ZAP's ability to localize to SGs. As ZAP is a central component of the cellular antiviral programs, these data provide further evidence that SGs are an important cytoplasmic antiviral hub. These findings provide insight into how antiviral components are regulated upon virus infection to inhibit virus spread.
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http://dx.doi.org/10.1371/journal.ppat.1007798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548403PMC
May 2019

Multi-Modal Imaging with a Toolbox of Influenza A Reporter Viruses.

Viruses 2015 Oct 13;7(10):5319-27. Epub 2015 Oct 13.

Medical Microbiology and Immunology, University of Wisconsin Madison, Madison, WI 53706, USA.

Reporter viruses are useful probes for studying multiple stages of the viral life cycle. Here we describe an expanded toolbox of fluorescent and bioluminescent influenza A reporter viruses. The enhanced utility of these tools enabled kinetic studies of viral attachment, infection, and co-infection. Multi-modal bioluminescence and positron emission tomography-computed tomography (PET/CT) imaging of infected animals revealed that antiviral treatment reduced viral load, dissemination, and inflammation. These new technologies and applications will dramatically accelerate in vitro and in vivo influenza virus studies.
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http://dx.doi.org/10.3390/v7102873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632381PMC
October 2015

The Role of Phosphodiesterase 12 (PDE12) as a Negative Regulator of the Innate Immune Response and the Discovery of Antiviral Inhibitors.

J Biol Chem 2015 Aug 8;290(32):19681-96. Epub 2015 Jun 8.

Antiviral Discovery Performance Unit, GlaxoSmithKline, Research Triangle Park, North Carolina 27709 and.

2',5'-Oligoadenylate synthetase (OAS) enzymes and RNase-L constitute a major effector arm of interferon (IFN)-mediated antiviral defense. OAS produces a unique oligonucleotide second messenger, 2',5'-oligoadenylate (2-5A), that binds and activates RNase-L. This pathway is down-regulated by virus- and host-encoded enzymes that degrade 2-5A. Phosphodiesterase 12 (PDE12) was the first cellular 2-5A- degrading enzyme to be purified and described at a molecular level. Inhibition of PDE12 may up-regulate the OAS/RNase-L pathway in response to viral infection resulting in increased resistance to a variety of viral pathogens. We generated a PDE12-null cell line, HeLaΔPDE12, using transcription activator-like effector nuclease-mediated gene inactivation. This cell line has increased 2-5A levels in response to IFN and poly(I-C), a double-stranded RNA mimic compared with the parental cell line. Moreover, HeLaΔPDE12 cells were resistant to viral pathogens, including encephalomyocarditis virus, human rhinovirus, and respiratory syncytial virus. Based on these results, we used DNA-encoded chemical library screening to identify starting points for inhibitor lead optimization. Compounds derived from this effort raise 2-5A levels and exhibit antiviral activity comparable with the effects observed with PDE12 gene inactivation. The crystal structure of PDE12 complexed with an inhibitor was solved providing insights into the structure-activity relationships of inhibitor potency and selectivity.
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http://dx.doi.org/10.1074/jbc.M115.653113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528132PMC
August 2015

Preclinical characterization of GSK2336805, a novel inhibitor of hepatitis C virus replication that selects for resistance in NS5A.

Antimicrob Agents Chemother 2014 14;58(1):38-47. Epub 2013 Oct 14.

GlaxoSmithKline, Research Triangle Park, North Carolina, USA.

GSK2336805 is an inhibitor of hepatitis C virus (HCV) with picomolar activity on the standard genotype 1a, 1b, and 2a subgenomic replicons and exhibits a modest serum shift. GSK2336805 was not active on 22 RNA and DNA viruses that were profiled. We have identified changes in the N-terminal region of NS5A that cause a decrease in the activity of GSK2336805. These mutations in the genotype 1b replicon showed modest shifts in compound activity (<13-fold), while mutations identified in the genotype 1a replicon had a more dramatic impact on potency. GSK2336805 retained activity on chimeric replicons containing NS5A patient sequences from genotype 1 and patient and consensus sequences for genotypes 4 and 5 and part of genotype 6. Combination and cross-resistance studies demonstrated that GSK2336805 could be used as a component of a multidrug HCV regimen either with the current standard of care or in combination with compounds with different mechanisms of action that are still progressing through clinical development.
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http://dx.doi.org/10.1128/AAC.01363-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910799PMC
September 2014

In vitro characterization of GSK2485852, a novel hepatitis C virus polymerase inhibitor.

Antimicrob Agents Chemother 2013 Nov 12;57(11):5216-24. Epub 2013 Aug 12.

GlaxoSmithKline, Research Triangle Park, North Carolina, USA.

GSK2485852 (referred to here as GSK5852) is a hepatitis C virus (HCV) NS5B polymerase inhibitor with 50% effective concentrations (EC50s) in the low nanomolar range in the genotype 1 and 2 subgenomic replicon system as well as the infectious HCV cell culture system. We have characterized the antiviral activity of GSK5852 using chimeric replicon systems with NS5B genes from additional genotypes as well as NS5B sequences from clinical isolates of patients infected with HCV of genotypes 1a and 1b. The inhibitory activity of GSK5852 remained unchanged in these intergenotypic and intragenotypic replicon systems. GSK5852 furthermore displays an excellent resistance profile and shows a <5-fold potency loss across the clinically important NS5B resistance mutations P495L, M423T, C316Y, and Y448H. Testing of a diverse mutant panel also revealed a lack of cross-resistance against known resistance mutations in other viral proteins. Data from both the newer 454 sequencing method and traditional population sequencing showed a pattern of mutations arising in the NS5B RNA-dependent RNA polymerase in replicon cells exposed to GSK5852. GSK5852 was more potent than HCV-796, an earlier inhibitor in this class, and showed greater reductions in HCV RNA during long-term treatment of replicons. GSK5852 is similar to HCV-796 in its activity against multiple genotypes, but its superior resistance profile suggests that it could be an attractive component of an all-oral regimen for treating HCV.
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http://dx.doi.org/10.1128/AAC.00874-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811320PMC
November 2013

End game: getting the most out of microRNAs.

Proc Natl Acad Sci U S A 2011 Feb 9;108(8):3101-2. Epub 2011 Feb 9.

Infectious Diseases Center for Excellence in Drug Discovery, GlaxoSmithKline, Research Triangle Park, NC 27709, USA.

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http://dx.doi.org/10.1073/pnas.1019613108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3044409PMC
February 2011

Gene expression profiling indicates the roles of host oxidative stress, apoptosis, lipid metabolism, and intracellular transport genes in the replication of hepatitis C virus.

J Virol 2010 May 3;84(10):5404-14. Epub 2010 Mar 3.

Department of Hepatology, Faculty of Medicine, Imperial College London, St. Mary's Campus, Norfolk Place, London W2 1PG, United Kingdom.

Hepatitis C virus (HCV) is a leading cause of chronic liver disease. The identification and characterization of key host cellular factors that play a role in the HCV replication cycle are important for the understanding of disease pathogenesis and the identification of novel antiviral therapeutic targets. Gene expression profiling of JFH-1-infected Huh7 cells by microarray analysis was performed to identify host cellular genes that are transcriptionally regulated by infection. The expression of host genes involved in cellular defense mechanisms (apoptosis, proliferation, and antioxidant responses), cellular metabolism (lipid and protein metabolism), and intracellular transport (vesicle trafficking and cytoskeleton regulation) was significantly altered by HCV infection. The gene expression patterns identified provide insight into the potential mechanisms that contribute to HCV-associated pathogenesis. These include an increase in proinflammatory and proapoptotic signaling and a decrease in the antioxidant response pathways of the infected cell. To investigate whether any of the host genes regulated by infection were required by HCV during replication, small interfering RNA (siRNA) silencing of host gene expression in HCV-infected cells was performed. Decreasing the expression of host genes involved in lipid metabolism (TXNIP and CYP1A1 genes) and intracellular transport (RAB33b and ABLIM3 genes) reduced the replication and secretion of HCV, indicating that they may be important factors for the virus replication cycle. These results show that major changes in the expression of many different genes in target cells may be crucial in determining the outcome of HCV infection.
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http://dx.doi.org/10.1128/JVI.02529-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2863852PMC
May 2010

3' RNA elements in hepatitis C virus replication: kissing partners and long poly(U).

J Virol 2008 Jan 17;82(1):184-95. Epub 2007 Oct 17.

Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, 1230 York Ave., New York, NY 10065, USA.

The hepatitis C virus (HCV) genomic RNA possesses conserved structural elements that are essential for its replication. The 3' nontranslated region (NTR) contains several of these elements: a variable region, the poly(U/UC) tract, and a highly conserved 3' X tail, consisting of stem-loop 1 (SL1), SL2, and SL3. Studies of drug-selected, cell culture-adapted subgenomic replicons have indicated that an RNA element within the NS5B coding region, 5BSL3.2, forms a functional kissing-loop tertiary structure with part of the 3' NTR, 3' SL2. Recent advances now allow the efficient propagation of unadapted HCV genomes in the context of a complete infectious life cycle (HCV cell culture [HCVcc]). Using this system, we determine that the kissing-loop interaction between 5BSL3.2 and 3' SL2 is required for replication in the genotype 2a HCVcc context. Remarkably, the overall integrity of the 5BSL3 cruciform is not an absolute requirement for the kissing-loop interaction, suggesting a model in which trans-acting factor(s) that stabilize this interaction may interact initially with the 3' X tail rather than 5BSL3. The length and composition of the poly(U/UC) tract were also critical determinants of HCVcc replication, with a length of 33 consecutive U residues required for maximal RNA amplification. Interrupting the U homopolymer with C residues was deleterious, implicating a trans-acting factor with a preference for U over mixed pyrimidine nucleotides. Finally, we show that both the poly(U) and kissing-loop RNA elements can function outside of their normal genome contexts. This suggests that the poly(U/UC) tract does not function simply as an unstructured spacer to position the kissing-loop elements.
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http://dx.doi.org/10.1128/JVI.01796-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2224383PMC
January 2008

Studying hepatitis C virus: making the best of a bad virus.

J Virol 2007 Sep 23;81(17):8853-67. Epub 2007 May 23.

The Scripps Research Institute, 5353 Parkside Drive, RF-2, Jupiter, FL 33458, USA.

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http://dx.doi.org/10.1128/JVI.00753-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1951464PMC
September 2007

Treating hepatitis C: can you teach old dogs new tricks?

Hepatology 2005 Dec;42(6):1455-8

Center for the Study of Hepatitis C Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA.

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.
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http://dx.doi.org/10.1002/hep.20975DOI Listing
December 2005

A cis-acting replication element in the sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C virus RNA replication.

J Virol 2004 Feb;78(3):1352-66

Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York 10021, USA.

RNA structures play key roles in the replication of RNA viruses. Sequence alignment software, thermodynamic RNA folding programs, and classical comparative phylogenetic analysis were used to build models of six RNA elements in the coding region of the hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B. The importance of five of these elements was evaluated by site-directed mutagenesis of a subgenomic HCV replicon. Mutations disrupting one of the predicted stem-loop structures, designated 5BSL3.2, blocked RNA replication, implicating it as an essential cis-acting replication element (CRE). 5BSL3.2 is about 50 bases in length and is part of a larger predicted cruciform structure (5BSL3). As confirmed by RNA structure probing, 5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop, and an 8-base internal loop. Mutational analysis and structure probing were used to explore the importance of these features. Primary sequences in the loops were shown to be important for HCV RNA replication, and the upper helix appears to serve as an essential scaffold that helps maintain the overall RNA structure. Unlike certain picornavirus CREs, whose function is position independent, 5BSL3.2 function appears to be context dependent. Understanding the role of 5BSL3.2 and determining how this new CRE functions in the context of previously identified elements at the 5' and 3' ends of the RNA genome should provide new insights into HCV RNA replication.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC321395PMC
http://dx.doi.org/10.1128/jvi.78.3.1352-1366.2004DOI Listing
February 2004

De novo synthesis of negative-strand RNA by Dengue virus RNA-dependent RNA polymerase in vitro: nucleotide, primer, and template parameters.

J Virol 2003 Aug;77(16):8831-42

Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.

By using a purified dengue virus RNA-dependent RNA polymerase and a subgenomic 770-nucleotide RNA template, it was shown previously that the ratio of the de novo synthesis product to hairpin product formed was inversely proportional to increments of assay temperatures (20 to 40 degrees C). In this study, the components of the de novo preinitiation complex are defined as ATP, a high concentration of GTP (500 micro M), the polymerase, and the template RNA. Even when the 3'-terminal sequence of template RNA was mutated from -GGUUCU-3' to -GGUUUU-3', a high GTP concentration was required for de novo initiation, suggesting that high GTP concentration plays a conformational role. Furthermore, utilization of synthetic primers by the polymerase indicated that AGAA is the optimal primer whereas AG, AGA, and AGAACC were inefficient primers. Moreover, mutational analysis of the highly conserved 3'-terminal dinucleotide CU of the template RNA indicated that change of the 3'-terminal nucleotide from U to C reduced the efficiency about fivefold. The order of preference for the 3'-terminal nucleotide, from highest to lowest, is U, A - G, and C. However, change of the penultimate nucleotide from C to U did not affect the template activity. A model consistent with these results is that the active site of the polymerase switches from a "closed" form, catalyzing de novo initiation through synthesis of short primers, to an "open" form for elongation of a double-stranded template-primer.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC167251PMC
http://dx.doi.org/10.1128/jvi.77.16.8831-8842.2003DOI Listing
August 2003