Publications by authors named "Priya Luthra"

32 Publications

Cardiac inflammation in COVID-19: Lessons from heart failure.

Life Sci 2020 Nov 21;260:118482. Epub 2020 Sep 21.

Department of Biomedical Research and Translational Medicine, Masonic Medical Research Institute, Utica, NY, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA; Department of Medicine, Division of Cardiology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

Cardiovascular disease (CVD) is the most common co-morbidity associated with COVID-19 and the fatality rate in COVID-19 patients with CVD is higher compared to other comorbidities, such as hypertension and diabetes. Preliminary data suggest that COVID-19 may also cause or worsen cardiac injury in infected patients through multiple mechanisms such as 'cytokine storm', endotheliosis, thrombosis, lymphocytopenia etc. Autopsies of COVID-19 patients reveal an infiltration of inflammatory mononuclear cells in the myocardium, confirming the role of the immune system in mediating cardiovascular damage in response to COVID-19 infection and also suggesting potential causal mechanisms for the development of new cardiac pathologies and/or exacerbation of underlying CVDs in infected patients. In this review, we discuss the potential underlying molecular mechanisms that drive COVID-19-mediated cardiac damage, as well as the short term and expected long-term cardiovascular ramifications of COVID-19 infection in patients.
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http://dx.doi.org/10.1016/j.lfs.2020.118482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505073PMC
November 2020

High-Throughput Screening Assay to Identify Small Molecule Inhibitors of Marburg Virus VP40 Protein.

ACS Infect Dis 2020 10 16;6(10):2783-2799. Epub 2020 Sep 16.

Center for Microbial Pathogenesis, Institute for Biomedical Sciences, Georgia State University, Atlanta, Georgia 30302-3965, United States.

Marburg virus (MARV) causes sporadic outbreaks of severe disease with high case fatality rates in humans. To date, neither therapeutics nor prophylactic approaches have been approved for MARV disease. The MARV matrix protein VP40 (mVP40) plays central roles in virus assembly and budding. mVP40 also inhibits interferon signaling by inhibiting the function of Janus kinase 1. This suppression of host antiviral defenses likely contributes to MARV virulence and therefore is a potential therapeutic target. We developed and optimized a cell-based high-throughput screening (HTS) assay in 384-well format to measure mVP40 interferon (IFN) antagonist function such that inhibitors could be identified. We performed a pilot screen of 1280 bioactive compounds and identified 3 hits, azaguanine-8, tosufloxacin hydrochloride, and linezolid, with scores > 3 and no significant cytotoxicity. Of these, azaguanine-8 inhibited MARV growth at noncytotoxic concentrations. These data demonstrate the suitability of the HTS mVP40 assay for drug discovery and suggest potential directions for anti-MARV therapeutic development.
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http://dx.doi.org/10.1021/acsinfecdis.0c00512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793242PMC
October 2020

Impact of Měnglà Virus Proteins on Human and Bat Innate Immune Pathways.

J Virol 2020 06 16;94(13). Epub 2020 Jun 16.

Center for Microbial Pathogenesis, Institute for Biomedical Sciences, Georgia State University, Atlanta, Georgia, USA

Měnglà virus (MLAV), identified in bats, is a phylogenetically distinct member of the family Because the filoviruses Ebola virus (EBOV) and Marburg virus (MARV) modulate host innate immunity, MLAV VP35, VP40, and VP24 proteins were compared with their EBOV and MARV homologs for innate immune pathway modulation. In human and cells, MLAV VP35 behaved like EBOV and MARV VP35s, inhibiting virus-induced activation of the interferon beta (IFN-β) promoter and interferon regulatory factor 3 (IRF3) phosphorylation. MLAV VP35 also interacted with PACT, a host protein engaged by EBOV VP35 to inhibit RIG-I signaling. MLAV VP35 also inhibits PKR activation. MLAV VP40 was demonstrated to inhibit type I IFN-induced gene expression in human and bat cells. It blocked STAT1 tyrosine phosphorylation induced either by type I IFN or overexpressed Jak1, paralleling MARV VP40. MLAV VP40 also inhibited virus-induced IFN-β promoter activation, a property shared by MARV VP40 and EBOV VP24. A Jak kinase inhibitor did not recapitulate this inhibition in the absence of viral proteins. Therefore, inhibition of Jak-STAT signaling is insufficient to explain inhibition of IFN-β promoter activation. MLAV VP24 did not inhibit IFN-induced gene expression or bind karyopherin α proteins, properties of EBOV VP24. MLAV VP24 differed from MARV VP24 in that it failed to interact with Keap1 or activate an antioxidant response element reporter gene due to the absence of a Keap1-binding motif. These functional observations support a closer relationship of MLAV to MARV than to EBOV but also are consistent with MLAV belonging to a distinct genus. EBOV and MARV, members of the family , are highly pathogenic zoonotic viruses that cause severe disease in humans. Both viruses use several mechanisms to modulate the host innate immune response, and these likely contribute to the severity of disease. Here, we demonstrate that MLAV, a filovirus newly discovered in a bat, suppresses antiviral type I interferon responses in both human and bat cells. Inhibitory activities are possessed by MLAV VP35 and VP40, which parallels how MARV blocks IFN responses. However, whereas MARV activates cellular antioxidant responses through an interaction between its VP24 protein and host protein Keap1, MLAV VP24 lacks a Keap1-binding motif and fails to activate this cytoprotective response. These data indicate that MLAV possesses immune-suppressing functions that could facilitate human infection. They also support the placement of MLAV in a different genus than either EBOV or MARV.
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http://dx.doi.org/10.1128/JVI.00191-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307173PMC
June 2020

A VP35 Mutant Ebola Virus Lacks Virulence but Can Elicit Protective Immunity to Wild-Type Virus Challenge.

Cell Rep 2019 09;28(12):3032-3046.e6

Center for Microbial Pathogenesis, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA 30303, USA. Electronic address:

Zaire ebolavirus (EBOV) VP35 protein is a suppressor of type I interferon (IFN) production, an inhibitor of dendritic cell maturation, and a putative virulence determinant. Here, a recombinant EBOV encoding a mutant VP35 virus (VP35m) is demonstrated to activate RIG-I-like receptor signaling and innate antiviral pathways. When inoculated into cynomolgus macaques, VP35m exhibits dramatic attenuation as compared to wild-type EBOV (wtEBOV), with 20 or 300 times the standard 100% lethal challenge dose not causing EBOV disease (EVD). Further, VP35m infection, despite limited replication in vivo, activates antigen presentation and innate immunity pathways and elicits increased frequencies of proliferating memory T cells and B cells and production of anti-EBOV antibodies. Upon wtEBOV challenge, VP35m-immunized animals survive, exhibiting host responses consistent with an orderly immune response and the absence of excessive inflammation. These data demonstrate that VP35 is a critical EBOV immune evasion factor and provide insights into immune mechanisms of EBOV control.
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http://dx.doi.org/10.1016/j.celrep.2019.08.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886687PMC
September 2019

Severe influenza pneumonitis in children with inherited TLR3 deficiency.

J Exp Med 2019 09 19;216(9):2038-2056. Epub 2019 Jun 19.

Columbia Center for Translational Immunology, Columbia University Medical Center, New York, NY.

Autosomal recessive IRF7 and IRF9 deficiencies impair type I and III IFN immunity and underlie severe influenza pneumonitis. We report three unrelated children with influenza A virus (IAV) infection manifesting as acute respiratory distress syndrome (IAV-ARDS), heterozygous for rare variants (P554S in two patients and P680L in the third) causing autosomal dominant (AD) TLR3 deficiency. AD TLR3 deficiency can underlie herpes simplex virus-1 (HSV-1) encephalitis (HSE) by impairing cortical neuron-intrinsic type I IFN immunity to HSV-1. TLR3-mutated leukocytes produce normal levels of IFNs in response to IAV. In contrast, TLR3-mutated fibroblasts produce lower levels of IFN-β and -λ, and display enhanced viral susceptibility, upon IAV infection. Moreover, the patients' iPSC-derived pulmonary epithelial cells (PECs) are susceptible to IAV. Treatment with IFN-α2b or IFN-λ1 rescues this phenotype. AD TLR3 deficiency may thus underlie IAV-ARDS by impairing TLR3-dependent, type I and/or III IFN-mediated, PEC-intrinsic immunity. Its clinical penetrance is incomplete for both IAV-ARDS and HSE, consistent with their typically sporadic nature.
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http://dx.doi.org/10.1084/jem.20181621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719423PMC
September 2019

Inhibition of Marburg Virus RNA Synthesis by a Synthetic Anti-VP35 Antibody.

ACS Infect Dis 2019 08 4;5(8):1385-1396. Epub 2019 Jun 4.

Department of Medicine , Washington University School of Medicine , 660 South Euclid Avenue , St. Louis , Missouri 63110 , United States.

Marburg virus causes sporadic outbreaks of severe hemorrhagic fever with high case fatality rates. Approved, effective, and safe therapeutic or prophylactic countermeasures are lacking. To address this, we used phage display to engineer a synthetic antibody, sFab H3, which binds the Marburg virus VP35 protein (mVP35). mVP35 is a critical cofactor of the viral replication complex and a viral immune antagonist. sFab H3 displayed high specificity for mVP35 and not for the closely related Ebola virus VP35. sFab H3 inhibited viral-RNA synthesis in a minigenome assay, suggesting its potential use as an antiviral. We characterized sFab H3 by a combination of biophysical and biochemical methods, and a crystal structure of the complex solved to 1.7 Å resolution defined the molecular interface between the sFab H3 and mVP35 interferon inhibitory domain. Our study identifies mVP35 as a therapeutic target using an approach that provides a framework for generating engineered Fabs targeting other viral proteins.
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http://dx.doi.org/10.1021/acsinfecdis.9b00091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827488PMC
August 2019

Protein Interaction Mapping Identifies RBBP6 as a Negative Regulator of Ebola Virus Replication.

Cell 2018 12;175(7):1917-1930.e13

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA. Electronic address:

Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologics that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.
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http://dx.doi.org/10.1016/j.cell.2018.08.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366944PMC
December 2018

Inhibiting pyrimidine biosynthesis impairs Ebola virus replication through depletion of nucleoside pools and activation of innate immune responses.

Antiviral Res 2018 10 23;158:288-302. Epub 2018 Aug 23.

Center for Microbial Pathogenesis, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA. Electronic address:

Specific host pathways that may be targeted therapeutically to inhibit the replication of Ebola virus (EBOV) and other emerging viruses remain incompletely defined. A screen of 200,000 compounds for inhibition of an EBOV minigenome (MG) assay that measures the function of the viral polymerase complex identified as hits several compounds with an amino-tetrahydrocarbazole scaffold. This scaffold was structurally similar to GSK983, a compound previously described as having broad-spectrum antiviral activity due to its impairing de novo pyrimidine biosynthesis through inhibition of dihydroorotate dehydrogenase (DHODH). We generated compound SW835, the racemic version of GSK983 and demonstrated that SW835 and brequinar, another DHODH inhibitor, potently inhibit the MG assay and the replication of EBOV, vesicular stomatitis virus (VSV) and Zika (ZIKV) in vitro. Nucleoside and deoxynucleoside supplementation studies demonstrated that depletion of pyrimidine pools contributes to antiviral activity of these compounds. As reported for other DHODH inhibitors, SW835 and brequinar also induced expression of interferon stimulated genes (ISGs). ISG induction was demonstrated to occur without production of IFNα/β and independently of the IFNα receptor and was not blocked by EBOV-encoded suppressors of IFN signaling pathways. Furthermore, we demonstrated that transcription factor IRF1 is required for this ISG induction, and that IRF1 induction requires the DNA damage response kinase ATM. Therefore, de novo pyrimidine biosynthesis is critical for the replication of EBOV and other RNA viruses and inhibition of this pathway activates an ATM and IRF1-dependent innate immune response that subverts EBOV immune evasion functions.
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http://dx.doi.org/10.1016/j.antiviral.2018.08.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436837PMC
October 2018

Conservation of Structure and Immune Antagonist Functions of Filoviral VP35 Homologs Present in Microbat Genomes.

Cell Rep 2018 07;24(4):861-872.e6

Center for Microbial Pathogenesis, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA 30303, USA. Electronic address:

Non-retroviral integrated RNA viral sequences (NIRVs) potentially encoding ∼280 amino acid homologs to filovirus VP35 proteins are present across the Myotis genus of bats. These are estimated to have been maintained for ∼18 million years, indicating their co-option. To address the reasons for co-option, 16 Myotis VP35s were characterized in comparison to VP35s from the extant filoviruses Ebola virus and Marburg virus, in which VP35s play critical roles in immune evasion and RNA synthesis. The Myotis VP35s demonstrated a conserved suppression of innate immune signaling, albeit with reduced potency, in either human or Myotis cells. Their attenuation reflects a lack of dsRNA binding that in the filoviral VP35s correlates with potent suppression of interferon responses. Despite divergent function, evolution has preserved in Myotis the structure of the filoviral VP35s, indicating that this structure is critical for co-opted function, possibly as a regulator of innate immune signaling.
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http://dx.doi.org/10.1016/j.celrep.2018.06.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474348PMC
July 2018

The Antiviral Drug Arbidol Inhibits Zika Virus.

Sci Rep 2018 06 12;8(1):8989. Epub 2018 Jun 12.

Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA.

There are many emerging and re-emerging globally prevalent viruses for which there are no licensed vaccines or antiviral medicines. Arbidol (ARB, umifenovir), used clinically for decades in several countries as an anti-influenza virus drug, inhibits many other viruses. In the current study, we show that ARB inhibits six different isolates of Zika virus (ZIKV), including African and Asian lineage viruses in multiple cell lines and primary human vaginal and cervical epithelial cells. ARB protects against ZIKV-induced cytopathic effects. Time of addition studies indicate that ARB is most effective at suppressing ZIKV when added to cells prior to infection. Moreover, ARB inhibits pseudoviruses expressing the ZIKV Envelope glycoprotein. Thus, ARB, a broadly acting anti-viral agent with a well-established safety profile, inhibits ZIKV, likely by blocking viral entry.
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http://dx.doi.org/10.1038/s41598-018-27224-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997637PMC
June 2018

Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly.

Cell 2018 02;172(5):966-978.e12

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We generated a three-dimensional structure of the eNP NC-like assembly at 5.8 Å using electron cryo-microscopy and identified a new regulatory role for eNP helices α22-α23. Biochemical, biophysical, and mutational analyses revealed that inter-eNP contacts within α22-α23 are critical for viral NC assembly and regulate viral RNA synthesis. These observations suggest that the N terminus and α22-α23 of eNP function as context-dependent regulatory modules (CDRMs). Our current study provides a framework for a structural mechanism for NC-like assembly and a new therapeutic target.
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http://dx.doi.org/10.1016/j.cell.2018.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973842PMC
February 2018

A high throughput screen identifies benzoquinoline compounds as inhibitors of Ebola virus replication.

Antiviral Res 2018 02 30;150:193-201. Epub 2017 Dec 30.

Center of Microbial Pathogenesis, Institute of Biomedical Sciences, Georgia State University, Atlanta, GA, USA. Electronic address:

Ebola virus (EBOV) is an enveloped negative-sense, single-stranded RNA virus of the filovirus family that causes severe disease in humans. Approved therapies for EBOV disease are lacking. EBOV RNA synthesis is carried out by a virus-encoded complex with RNA-dependent RNA polymerase activity that is required for viral propagation. This complex and its activities are therefore potential antiviral targets. To identify potential lead inhibitors of EBOV RNA synthesis, a library of small molecule compounds was screened against a previously established assay of EBOV RNA synthesis, the EBOV minigenome assay (MGA), in 384 well microplate format. The screen identified 56 hits that inhibited EBOV MGA activity by more than 70% while exhibiting less than 20% cell cytotoxicity. Inhibitory chemical scaffolds included angelicin derivatives, derivatives of the antiviral compound GSK983 and benzoquinolines. Structure-activity relationship (SAR) studies of the benzoquinoline scaffold produced ∼50 analogs and led to identification of an optimized compound, SW456, with a submicromolar IC in the EBOV MGA and antiviral activity against infectious EBOV in cell culture. The compound was also active against a MGA for another deadly filovirus, Marburg virus. It also exhibited antiviral activity towards a negative-sense RNA virus from the rhabdovirus family, vesicular stomatitis virus, and a positive-sense RNA virus, Zika virus. Overall, these data demonstrate the potential of the EBOV MGA to identify anti-EBOV compounds and identifies the benzoquinoline series as a broad-spectrum antiviral lead.
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http://dx.doi.org/10.1016/j.antiviral.2017.12.019DOI Listing
February 2018

The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication.

J Virol 2017 09 24;91(18). Epub 2017 Aug 24.

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA

Ebola virus (EBOV), a member of the family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection. EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35 protein contributes to pathogenesis, because it serves as an essential cofactor of the viral polymerase as well as a potent antagonist of innate immunity. However, how VP35 function is regulated by host cellular factors is poorly understood. Here, we report that the host E3-ubiquitin ligase TRIM6 promotes VP35 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM6, as a potential target in the development of antiviral drugs against EBOV.
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http://dx.doi.org/10.1128/JVI.00833-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5571272PMC
September 2017

Structural basis for human respiratory syncytial virus NS1-mediated modulation of host responses.

Nat Microbiol 2017 Jun 30;2:17101. Epub 2017 Jun 30.

Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri 63110, USA.

Human respiratory syncytial virus (hRSV) is a major cause of morbidity and mortality in the paediatric, elderly and immune-compromised populations. A gap in our understanding of hRSV disease pathology is the interplay between virally encoded immune antagonists and host components that limit hRSV replication. hRSV encodes for non-structural (NS) proteins that are important immune antagonists; however, the role of these proteins in viral pathogenesis is incompletely understood. Here, we report the crystal structure of hRSV NS1 protein, which suggests that NS1 is a structural paralogue of hRSV matrix (M) protein. Comparative analysis of the shared structural fold with M revealed regions unique to NS1. Studies on NS1 wild type or mutant alone or in recombinant RSVs demonstrate that structural regions unique to NS1 contribute to modulation of host responses, including inhibition of type I interferon responses, suppression of dendritic cell maturation and promotion of inflammatory responses. Transcriptional profiles of A549 cells infected with recombinant RSVs show significant differences in multiple host pathways, suggesting that NS1 may have a greater role in regulating host responses than previously appreciated. These results provide a framework to target NS1 for therapeutic development to limit hRSV-associated morbidity and mortality.
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http://dx.doi.org/10.1038/nmicrobiol.2017.101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739881PMC
June 2017

The Ebola virus VP35 protein binds viral immunostimulatory and host RNAs identified through deep sequencing.

PLoS One 2017 21;12(6):e0178717. Epub 2017 Jun 21.

Virology Group, J. Craig Venter Institute, Rockville, Maryland, United States of America.

Ebola virus and Marburg virus are members of the Filovirdae family and causative agents of hemorrhagic fever with high fatality rates in humans. Filovirus virulence is partially attributed to the VP35 protein, a well-characterized inhibitor of the RIG-I-like receptor pathway that triggers the antiviral interferon (IFN) response. Prior work demonstrates the ability of VP35 to block potent RIG-I activators, such as Sendai virus (SeV), and this IFN-antagonist activity is directly correlated with its ability to bind RNA. Several structural studies demonstrate that VP35 binds short synthetic dsRNAs; yet, there are no data that identify viral immunostimulatory RNAs (isRNA) or host RNAs bound to VP35 in cells. Utilizing a SeV infection model, we demonstrate that both viral isRNA and host RNAs are bound to Ebola and Marburg VP35s in cells. By deep sequencing the purified VP35-bound RNA, we identified the SeV copy-back defective interfering (DI) RNA, previously identified as a robust RIG-I activator, as the isRNA bound by multiple filovirus VP35 proteins, including the VP35 protein from the West African outbreak strain (Makona EBOV). Moreover, RNAs isolated from a VP35 RNA-binding mutant were not immunostimulatory and did not include the SeV DI RNA. Strikingly, an analysis of host RNAs bound by wild-type, but not mutant, VP35 revealed that select host RNAs are preferentially bound by VP35 in cell culture. Taken together, these data support a model in which VP35 sequesters isRNA in virus-infected cells to avert RIG-I like receptor (RLR) activation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178717PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5479518PMC
October 2017

Ebola virus VP30 and nucleoprotein interactions modulate viral RNA synthesis.

Nat Commun 2017 06 8;8:15576. Epub 2017 Jun 8.

Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri 63105, USA.

Ebola virus (EBOV) is an enveloped negative-sense RNA virus that causes sporadic outbreaks with high case fatality rates. Ebola viral protein 30 (eVP30) plays a critical role in EBOV transcription initiation at the nucleoprotein (eNP) gene, with additional roles in the replication cycle such as viral assembly. However, the mechanistic basis for how eVP30 functions during the virus replication cycle is currently unclear. Here we define a key interaction between eVP30 and a peptide derived from eNP that is important to facilitate interactions leading to the recognition of the RNA template. We present crystal structures of the eVP30 C-terminus in complex with this eNP peptide. Functional analyses of the eVP30-eNP interface identify residues that are critical for viral RNA synthesis. Altogether, these results support a model where the eVP30-eNP interaction plays a critical role in transcription initiation and provides a novel target for the development of antiviral therapy.
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http://dx.doi.org/10.1038/ncomms15576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472179PMC
June 2017

Assays to Measure Suppression of Type I Interferon Responses by Filovirus VP35 Proteins.

Methods Mol Biol 2017 ;1628:133-142

Center for Microbial Pathogenesis, Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA.

Innate immunity is the first line of defense against virus infections and is marked by production of type I interferons (IFN), a family of cytokines that includes IFN-β and several IFN-αs. For the filoviruses and many other RNA viruses that replicate in the cytoplasm, the RIG-I-like pattern recognition receptors (RLRs) are potential triggers of IFN production. To counteract such innate antiviral responses, many viruses encode proteins that antagonize RLR signaling. Ebola virus (EBOV) and other filoviruses produce VP35 proteins that block IFN induction via RLR signaling. We describe here cell-based reporter gene assays that quantify the IFN-antagonist function of filovirus VP35 proteins by assessing activation of the IFN-β promoter.
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http://dx.doi.org/10.1007/978-1-4939-7116-9_10DOI Listing
March 2018

Topoisomerase II Inhibitors Induce DNA Damage-Dependent Interferon Responses Circumventing Ebola Virus Immune Evasion.

mBio 2017 04 4;8(2). Epub 2017 Apr 4.

Center for Microbial Pathogenesis, Institute for Biomedical Sciences, Georgia State University, Atlanta, Georgia, USA

Ebola virus (EBOV) protein VP35 inhibits production of interferon alpha/beta (IFN) by blocking RIG-I-like receptor signaling pathways, thereby promoting virus replication and pathogenesis. A high-throughput screening assay, developed to identify compounds that either inhibit or bypass VP35 IFN-antagonist function, identified five DNA intercalators as reproducible hits from a library of bioactive compounds. Four, including doxorubicin and daunorubicin, are anthracycline antibiotics that inhibit topoisomerase II and are used clinically as chemotherapeutic drugs. These compounds were demonstrated to induce IFN responses in an ATM kinase-dependent manner and to also trigger the DNA-sensing cGAS-STING pathway of IFN induction. These compounds also suppress EBOV replication and induce IFN in the presence of IFN-antagonist proteins from multiple negative-sense RNA viruses. These findings provide new insights into signaling pathways activated by important chemotherapy drugs and identify a novel therapeutic approach for IFN induction that may be exploited to inhibit RNA virus replication. Ebola virus and other emerging RNA viruses are significant but unpredictable public health threats. Therapeutic approaches with broad-spectrum activity could provide an attractive response to such infections. We describe a novel assay that can identify small molecules that overcome Ebola virus-encoded innate immune evasion mechanisms. This assay identified as hits cancer chemotherapeutic drugs, including doxorubicin. Follow-up studies provide new insight into how doxorubicin induces interferon (IFN) responses, revealing activation of both the DNA damage response kinase ATM and the DNA sensor cGAS and its partner signaling protein STING. The studies further demonstrate that the ATM and cGAS-STING pathways of IFN induction are a point of vulnerability not only for Ebola virus but for other RNA viruses as well, because viral innate immune antagonists consistently fail to block these signals. These studies thereby define a novel avenue for therapeutic intervention against emerging RNA viruses.
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http://dx.doi.org/10.1128/mBio.00368-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380843PMC
April 2017

Dengue virus NS2B protein targets cGAS for degradation and prevents mitochondrial DNA sensing during infection.

Nat Microbiol 2017 Mar 27;2:17037. Epub 2017 Mar 27.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York 10029-6574, USA.

During the last few decades, the global incidence of dengue virus (DENV) has increased dramatically, and it is now endemic in more than 100 countries. To establish a productive infection in humans, DENV uses different strategies to inhibit or avoid the host innate immune system. Several DENV proteins have been shown to strategically target crucial components of the type I interferon system. Here, we report that the DENV NS2B protease cofactor targets the DNA sensor cyclic GMP-AMP synthase (cGAS) for lysosomal degradation to avoid the detection of mitochondrial DNA during infection. Such degradation subsequently results in the inhibition of type I interferon production in the infected cell. Our data demonstrate a mechanism by which cGAS senses cellular damage upon DENV infection.
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http://dx.doi.org/10.1038/nmicrobiol.2017.37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457382PMC
March 2017

Differential Regulation of Interferon Responses by Ebola and Marburg Virus VP35 Proteins.

Cell Rep 2016 Feb 11;14(7):1632-1640. Epub 2016 Feb 11.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

Suppression of innate immune responses during filoviral infection contributes to disease severity. Ebola (EBOV) and Marburg (MARV) viruses each encode a VP35 protein that suppresses RIG-I-like receptor signaling and interferon-α/β (IFN-α/β) production by several mechanisms, including direct binding to double stranded RNA (dsRNA). Here, we demonstrate that in cell culture, MARV infection results in a greater upregulation of IFN responses as compared to EBOV infection. This correlates with differences in the efficiencies by which EBOV and MARV VP35s antagonize RIG-I signaling. Furthermore, structural and biochemical studies suggest that differential recognition of RNA elements by the respective VP35 C-terminal IFN inhibitory domain (IID) rather than affinity for RNA by the respective VP35s is critical for this observation. Our studies reveal functional differences in EBOV versus MARV VP35 RNA binding that result in unexpected differences in the host response to deadly viral pathogens.
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http://dx.doi.org/10.1016/j.celrep.2016.01.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767585PMC
February 2016

An Intrinsically Disordered Peptide from Ebola Virus VP35 Controls Viral RNA Synthesis by Modulating Nucleoprotein-RNA Interactions.

Cell Rep 2015 Apr 9;11(3):376-89. Epub 2015 Apr 9.

Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA. Electronic address:

During viral RNA synthesis, Ebola virus (EBOV) nucleoprotein (NP) alternates between an RNA-template-bound form and a template-free form to provide the viral polymerase access to the RNA template. In addition, newly synthesized NP must be prevented from indiscriminately binding to noncognate RNAs. Here, we investigate the molecular bases for these critical processes. We identify an intrinsically disordered peptide derived from EBOV VP35 (NPBP, residues 20-48) that binds NP with high affinity and specificity, inhibits NP oligomerization, and releases RNA from NP-RNA complexes in vitro. The structure of the NPBP/ΔNPNTD complex, solved to 3.7 Å resolution, reveals how NPBP peptide occludes a large surface area that is important for NP-NP and NP-RNA interactions and for viral RNA synthesis. Together, our results identify a highly conserved viral interface that is important for EBOV replication and can be targeted for therapeutic development.
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http://dx.doi.org/10.1016/j.celrep.2015.03.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599368PMC
April 2015

Infectious disease. Life-threatening influenza and impaired interferon amplification in human IRF7 deficiency.

Science 2015 Apr 26;348(6233):448-53. Epub 2015 Mar 26.

St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY, USA. Laboratory of Human Genetics of Infectious Diseases, Necker Branch, INSERM UMR1163, Paris, France. University Paris Descartes, Imagine Institute, Paris, France.

Severe influenza disease strikes otherwise healthy children and remains unexplained. We report compound heterozygous null mutations in IRF7, which encodes the transcription factor interferon regulatory factor 7, in an otherwise healthy child who suffered life-threatening influenza during primary infection. In response to influenza virus, the patient's leukocytes and plasmacytoid dendritic cells produced very little type I and III interferons (IFNs). Moreover, the patient's dermal fibroblasts and induced pluripotent stem cell (iPSC)-derived pulmonary epithelial cells produced reduced amounts of type I IFN and displayed increased influenza virus replication. These findings suggest that IRF7-dependent amplification of type I and III IFNs is required for protection against primary infection by influenza virus in humans. They also show that severe influenza may result from single-gene inborn errors of immunity.
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http://dx.doi.org/10.1126/science.aaa1578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4431581PMC
April 2015

Ebola virus VP35 interaction with dynein LC8 regulates viral RNA synthesis.

J Virol 2015 May 4;89(9):5148-53. Epub 2015 Mar 4.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA

Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.
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http://dx.doi.org/10.1128/JVI.03652-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403485PMC
May 2015

In silico derived small molecules bind the filovirus VP35 protein and inhibit its polymerase cofactor activity.

J Mol Biol 2014 May 1;426(10):2045-58. Epub 2014 Feb 1.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

The Ebola virus (EBOV) genome only encodes a single viral polypeptide with enzymatic activity, the viral large (L) RNA-dependent RNA polymerase protein. However, currently, there is limited information about the L protein, which has hampered the development of antivirals. Therefore, antifiloviral therapeutic efforts must include additional targets such as protein-protein interfaces. Viral protein 35 (VP35) is multifunctional and plays important roles in viral pathogenesis, including viral mRNA synthesis and replication of the negative-sense RNA viral genome. Previous studies revealed that mutation of key basic residues within the VP35 interferon inhibitory domain (IID) results in significant EBOV attenuation, both in vitro and in vivo. In the current study, we use an experimental pipeline that includes structure-based in silico screening and biochemical and structural characterization, along with medicinal chemistry, to identify and characterize small molecules that target a binding pocket within VP35. NMR mapping experiments and high-resolution x-ray crystal structures show that select small molecules bind to a region of VP35 IID that is important for replication complex formation through interactions with the viral nucleoprotein (NP). We also tested select compounds for their ability to inhibit VP35 IID-NP interactions in vitro as well as VP35 function in a minigenome assay and EBOV replication. These results confirm the ability of compounds identified in this study to inhibit VP35-NP interactions in vitro and to impair viral replication in cell-based assays. These studies provide an initial framework to guide development of antifiloviral compounds against filoviral VP35 proteins.
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http://dx.doi.org/10.1016/j.jmb.2014.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4163021PMC
May 2014

Development of RNA aptamers targeting Ebola virus VP35.

Biochemistry 2013 Nov 14;52(47):8406-19. Epub 2013 Nov 14.

Department of Pathology and Immunology, Washington University School of Medicine , St. Louis, Missouri 63110, United States.

Viral protein 35 (VP35), encoded by filoviruses, is a multifunctional dsRNA binding protein that plays important roles in viral replication, innate immune evasion, and pathogenesis. The multifunctional nature of these proteins also presents opportunities to develop countermeasures that target distinct functional regions. However, functional validation and the establishment of therapeutic approaches toward such multifunctional proteins, particularly for nonenzymatic targets, are often challenging. Our previous work on filoviral VP35 proteins defined conserved basic residues located within its C-terminal dsRNA binding interferon (IFN) inhibitory domain (IID) as important for VP35 mediated IFN antagonism and viral polymerase cofactor functions. In the current study, we used a combination of structural and functional data to determine regions of Ebola virus (EBOV) VP35 (eVP35) to target for aptamer selection using SELEX. Select aptamers, representing, two distinct classes, were further characterized based on their interaction properties to eVP35 IID. These results revealed that these aptamers bind to distinct regions of eVP35 IID with high affinity (10-50 nM) and specificity. These aptamers can compete with dsRNA for binding to eVP35 and disrupt the eVP35-nucleoprotein (NP) interaction. Consistent with the ability to antagonize the eVP35-NP interaction, select aptamers can inhibit the function of the EBOV polymerase complex reconstituted by the expression of select viral proteins. Taken together, our results support the identification of two aptamers that bind filoviral VP35 proteins with high affinity and specificity and have the capacity to potentially function as filoviral VP35 protein inhibitors.
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http://dx.doi.org/10.1021/bi400704dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3909728PMC
November 2013

Mutual antagonism between the Ebola virus VP35 protein and the RIG-I activator PACT determines infection outcome.

Cell Host Microbe 2013 Jul;14(1):74-84

Department of Microbiology, Icahn School of Medicine at Mount Sinai School, New York, NY 10029, USA.

The cytoplasmic pattern recognition receptor RIG-I is activated by viral RNA and induces type I IFN responses to control viral replication. The cellular dsRNA binding protein PACT can also activate RIG-I. To counteract innate antiviral responses, some viruses, including Ebola virus (EBOV), encode proteins that antagonize RIG-I signaling. Here, we show that EBOV VP35 inhibits PACT-induced RIG-I ATPase activity in a dose-dependent manner. The interaction of PACT with RIG-I is disrupted by wild-type VP35, but not by VP35 mutants that are unable to bind PACT. In addition, PACT-VP35 interaction impairs the association between VP35 and the viral polymerase, thereby diminishing viral RNA synthesis and modulating EBOV replication. PACT-deficient cells are defective in IFN induction and are insensitive to VP35 function. These data support a model in which the VP35-PACT interaction is mutually antagonistic and plays a fundamental role in determining the outcome of EBOV infection.
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http://dx.doi.org/10.1016/j.chom.2013.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3875338PMC
July 2013

The V protein of mumps virus plays a critical role in pathogenesis.

J Virol 2012 Feb 16;86(3):1768-76. Epub 2011 Nov 16.

Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA.

Mumps virus (MuV) causes an acute infection in humans characterized by a wide array of symptoms ranging from relatively mild manifestations, such as parotitis, to more-severe complications, such as meningitis and encephalitis. Widespread mumps vaccination has reduced mumps incidence dramatically; however, outbreaks still occur in vaccinated populations. The V protein of MuV, when expressed in cell culture, blocks interferon (IFN) expression and signaling and interleukin-6 (IL-6) signaling. In this work, we generated a recombinant MuV incapable of expressing the V protein (rMuVΔV). The rescued MuV was derived from a clinical wild-type isolate from a recent outbreak in the United States (MuV(Iowa/US/06), G genotype). Analysis of the virus confirmed the roles of V protein in blocking IFN expression and signaling and IL-6 signaling. We also found that the rMuV(Iowa/US/06)ΔV virus induced high levels of IL-6 expression in vitro, suggesting that V plays a role in reducing IL-6 expression. In vivo, the rMuV(Iowa/US/06)ΔV virus was highly attenuated, indicating that the V protein plays an essential role in viral virulence.
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http://dx.doi.org/10.1128/JVI.06019-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3264346PMC
February 2012

Identification of a phosphorylation site within the P protein important for mRNA transcription and growth of parainfluenza virus 5.

J Virol 2011 Aug 15;85(16):8376-85. Epub 2011 Jun 15.

Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.

The viral RNA-dependent RNA polymerase (vRdRp) of paramyxovirus consists of the large (L) protein and the phosphoprotein (P). P is heavily phosphorylated, and it is thought that the phosphorylation of P plays a role in regulating viral RNA synthesis. However, no phosphorylation site within the P protein in paramyxovirus has been identified as playing a positive role in viral RNA synthesis in virus infection. Using mass spectrometry analysis, the threonine residue at position 286 of P of parainfluenza virus 5 (PIV5) was found phosphorylated. Mutation of T286 to alanine (T286A), aspartic acid (T286D), or glutamic acid (T286E) reduced minigenome activity. Recombinant virus containing a mutation at the T286 position (rPIV5-P-T286A) grew slower than wild-type virus; viral mRNA synthesis and protein expression of rPIV5-P-T286A were delayed. Biochemical studies showed that the binding of NP or L protein with the P mutants or tetramer formation by the mutant P proteins was unaltered from that for wild-type P. While we failed to rescue rPIV5-P-T286E virus, several revertant viruses were obtained. All non-wild-type revertants had mutations at T286 and showed defects in both minigenome activity and viral growth. This is the first time that a phosphorylation site within the P protein in paramyxovirus has been found to play a positive role in viral mRNA synthesis and virus growth.
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http://dx.doi.org/10.1128/JVI.00618-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147951PMC
August 2011

Activation of IFN-β expression by a viral mRNA through RNase L and MDA5.

Proc Natl Acad Sci U S A 2011 Feb 18;108(5):2118-23. Epub 2011 Jan 18.

Department of Infectious Diseases, University of Georgia, Athens, GA 30602, USA.

IFNs play a critical role in innate immunity against viral infections. Melanoma differentiation-associated protein 5 (MDA5), an RNA helicase, is a key component in activating the expression of type I IFNs in response to certain types of viral infection. MDA5 senses noncellular RNA and triggers the signaling cascade that leads to IFN production. Synthetic double-stranded RNAs are known activators of MDA5. Natural single-stranded RNAs have not been reported to activate MDA5, however. We have serendipitously identified a viral mRNA from parainfluenza virus 5 (PIV5) that activates IFN expression through MDA5. We provide evidence that the signaling pathway includes the antiviral enzyme RNase L. The L mRNA of PIV5 activated expression of IFN-β. We have mapped the RNA to a region of 430 nucleotides within the L mRNA of PIV5. Our results indicate that a viral mRNA, with 5'-cap and 3'-poly (A), can activate IFN expression through an RNase L-MDA5 pathway.
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http://dx.doi.org/10.1073/pnas.1012409108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033319PMC
February 2011

PLK1 down-regulates parainfluenza virus 5 gene expression.

PLoS Pathog 2009 Jul 24;5(7):e1000525. Epub 2009 Jul 24.

Intercollege Graduate Program in Cell and Developmental Biology, Pennsylvania State University, University Park, Pennsylvania, USA.

The paramyxoviruses are a family of negative-sense RNA viruses that includes many important human and animal pathogens. Paramyxovirus RNA synthesis requires the viral phosphoprotein (P) and the large (L) protein. Phosphorylation of P is thought to regulate viral gene expression, though direct proof remains elusive. Recently, we reported that phosphorylation of a specific residue (Ser157) of the P protein of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, correlates with decreased viral gene expression and cytokine expression in infected cells. Here, we show that: Polo-like kinase 1 (PLK1), a serine/theronine kinase that plays a critical role in regulating the cell cycle, interacts with PIV5 P through the S157 residue; PLK1 inhibition increases viral gene expression; PLK1 over-expression inhibits viral gene expression; and PLK1 directly phosphorylates P in vitro, indicating that PLK1 down-regulates viral gene expression by phosphorylating P. Furthermore, we have determined the PLK1 phosphorylation site on P and found that mutant recombinant PIV5 whose P proteins cannot either bind to or be phosphorylated by PLK1 have similar phenotypes. Increased viral gene expression in PIV5 with mutations in the PLK1 binding/phosphorylation sites correlates with increased induction of cell death and cytokine expression, suggesting that PIV5 limits its viral gene expression to avoid these host effects. It is possible that targeting PLK1 will enhance host innate immune responses, leading to a novel strategy of clearing paramyxovirus infections quickly.
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http://dx.doi.org/10.1371/journal.ppat.1000525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709441PMC
July 2009