116 results on '"John W. Schoggins"'
Search Results
2. Development of a mutant aerosolized ACE2 that neutralizes SARS-CoV-2 in vivo
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Daniel L. Kober, Marley C. Caballero Van Dyke, Jennifer L. Eitson, Ian N. Boys, Matthew B. McDougal, Daniel M. Rosenbaum, and John W. Schoggins
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SARS-CoV-2 ,ACE2 ,receptors ,receptor-ligand interaction ,antiviral agents ,mutational studies ,Microbiology ,QR1-502 - Abstract
ABSTRACT The rapid evolution of SARS-CoV-2 variants highlights the need for new therapies to prevent disease spread. SARS-CoV-2, like SARS-CoV-1, uses the human cell surface protein angiotensin-converting enzyme 2 (ACE2) as its native receptor. Here, we design and characterize a mutant ACE2 that enables rapid affinity purification of a dimeric protein by altering the active site to prevent autoproteolytic digestion of a C-terminal His10 epitope tag. In cultured cells, mutant ACE2 competitively inhibits lentiviral vectors pseudotyped with spikes from multiple SARS-CoV-2 variants and infectious SARS-CoV-2. Moreover, the protein can be nebulized and retains virus-binding properties. We developed a system for the delivery of aerosolized ACE2 to K18-hACE2 mice and demonstrated protection by our modified ACE2 when delivered as a prophylactic agent. These results show proof-of-concept for an aerosolized delivery method to evaluate anti-SARS-CoV-2 agents in vivo and suggest a new tool in the ongoing fight against SARS-CoV-2 and other ACE2-dependent viruses.IMPORTANCEThe rapid evolution of SARS-CoV-2 variants poses a challenge for immune recognition and antibody therapies. However, the virus is constrained by the requirement that it recognizes a human host receptor protein. A recombinant ACE2 could protect against SARS-CoV-2 infection by functioning as a soluble decoy receptor. We designed a mutant version of ACE2 with impaired catalytic activity to enable the purification of the protein using a single affinity purification step. This protein can be nebulized and retains the ability to bind the relevant domains from SARS-CoV-1 and SARS-CoV-2. Moreover, this protein inhibits viral infection against a panel of coronaviruses in cells. Finally, we developed an aerosolized delivery system for animal studies and show the modified ACE2 offers protection in an animal model of COVID-19. These results show proof-of-concept for an aerosolized delivery method to evaluate anti-SARS-CoV-2 agents in vivo and suggest a new tool in the ongoing fight against SARS-CoV-2.
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- 2024
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3. Interferon signaling drives epithelial metabolic reprogramming to promote secondary bacterial infection
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Grace P. Carreno-Florez, Brian R. Kocak, Matthew R. Hendricks, Jeffrey A. Melvin, Katrina B. Mar, Jessica Kosanovich, Rachel L. Cumberland, Greg M. Delgoffe, Sruti Shiva, Kerry M. Empey, John W. Schoggins, and Jennifer M. Bomberger
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Published
- 2023
4. FDA approved drugs with antiviral activity against SARS-CoV-2: From structure-based repurposing to host-specific mechanisms
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Mahmoud S. Ahmed, Ayman B. Farag, Ian N. Boys, Ping Wang, Ivan Menendez-Montes, Ngoc Uyen Nhi Nguyen, Jennifer L. Eitson, Maikke B. Ohlson, Wenchun Fan, Matthew B. McDougal, Katrina Mar, Suwannee Thet, Francisco Ortiz, Soo Young Kim, Ashley Solmonson, Noelle S. Williams, Andrew Lemoff, Ralph J. DeBerardinis, John W. Schoggins, and Hesham A. Sadek
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SARS-CoV-2 ,Structure-based drug repurposing ,Purine Metabolism ,Therapeutics. Pharmacology ,RM1-950 - Abstract
The continuing heavy toll of the COVID-19 pandemic necessitates development of therapeutic options. We adopted structure-based drug repurposing to screen FDA-approved drugs for inhibitory effects against main protease enzyme (Mpro) substrate-binding pocket of SARS-CoV-2 for non-covalent and covalent binding. Top candidates were screened against infectious SARS-CoV-2 in a cell-based viral replication assay. Promising candidates included atovaquone, mebendazole, ouabain, dronedarone, and entacapone, although atovaquone and mebendazole were the only two candidates with IC50s that fall within their therapeutic plasma concentration. Additionally, we performed Mpro assays on the top hits, which demonstrated inhibition of Mpro by dronedarone (IC50 18 µM), mebendazole (IC50 19 µM) and entacapone (IC50 9 µM). Atovaquone showed only modest Mpro inhibition, and thus we explored other potential mechanisms. Although atovaquone is Dihydroorotate dehydrogenase (DHODH) inhibitor, we did not observe inhibition of DHODH at the respective SARS-CoV-2 IC50. Metabolomic profiling of atovaquone treated cells showed dysregulation of purine metabolism pathway metabolite, where ecto-5′-nucleotidase (NT5E) was downregulated by atovaquone at concentrations equivalent to its antiviral IC50. Atovaquone and mebendazole are promising candidates with SARS-CoV-2 antiviral activity. While mebendazole does appear to target Mpro, atovaquone may inhibit SARS-CoV-2 viral replication by targeting host purine metabolism.
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- 2023
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5. Genome-Scale CRISPR Screening Reveals Host Factors Required for Ribosome Formation and Viral Replication
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Maikke B. Ohlson, Jennifer L. Eitson, Alexandra I. Wells, Ashwani Kumar, Seoyeon Jang, Chunyang Ni, Chao Xing, Michael Buszczak, and John W. Schoggins
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RNA virus ,flavivirus ,ribosomes ,translation ,virus-host interactions ,Microbiology ,QR1-502 - Abstract
ABSTRACT Viruses are known to co-opt host machinery for translation initiation, but less is known about which host factors are required for the formation of ribosomes used to synthesize viral proteins. Using a loss-of-function CRISPR screen, we show that synthesis of a flavivirus-encoded fluorescent reporter depends on multiple host factors, including several 60S ribosome biogenesis proteins. Viral phenotyping revealed that two of these factors, SBDS, a known ribosome biogenesis factor, and the relatively uncharacterized protein SPATA5, were broadly required for replication of flaviviruses, coronaviruses, alphaviruses, paramyxoviruses, an enterovirus, and a poxvirus. Mechanistic studies revealed that loss of SPATA5 caused defects in rRNA processing and ribosome assembly, suggesting that this human protein may be a functional ortholog of yeast Drg1. These studies implicate specific ribosome biogenesis proteins as viral host dependency factors that are required for synthesis of virally encoded protein and accordingly, optimal viral replication. IMPORTANCE Viruses are well known for their ability to co-opt host ribosomes to synthesize viral proteins. The specific factors involved in translation of viral RNAs are not fully described. In this study, we implemented a unique genome-scale CRISPR screen to identify previously uncharacterized host factors that are important for the synthesis of virally encoded protein. We found that multiple genes involved in 60S ribosome biogenesis were required for viral RNA translation. Loss of these factors severely impaired viral replication. Mechanistic studies on the AAA ATPase SPATA5 indicate that this host factor is required for a late step in ribosome formation. These findings reveal insight into the identity and function of specific ribosome biogenesis proteins that are critical for viral infections.
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- 2023
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6. CRISPR screening reveals a dependency on ribosome recycling for efficient SARS-CoV-2 programmed ribosomal frameshifting and viral replication
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Frederick Rehfeld, Jennifer L. Eitson, Maikke B. Ohlson, Tsung-Cheng Chang, John W. Schoggins, and Joshua T. Mendell
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CP: Microbiology ,CP: Molecular biology ,Biology (General) ,QH301-705.5 - Abstract
Summary: During translation of the genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus in the COVID-19 pandemic, host ribosomes undergo programmed ribosomal frameshifting (PRF) at a conserved structural element. Although PRF is essential for coronavirus replication, host factors that regulate this process have not yet been identified. Here we perform genome-wide CRISPR-Cas9 knockout screens to identify regulators of SARS-CoV-2 PRF. These screens reveal that loss of ribosome recycling factors markedly decreases frameshifting efficiency and impairs SARS-CoV-2 viral replication. Mutational studies support a model wherein efficient removal of ribosomal subunits at the ORF1a stop codon is required for frameshifting of trailing ribosomes. This dependency upon ribosome recycling is not observed with other non-pathogenic human betacoronaviruses and is likely due to the unique position of the ORF1a stop codon in the SARS clade of coronaviruses. These findings therefore uncover host factors that support efficient SARS-CoV-2 translation and replication.
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- 2023
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7. Atovaquone for treatment of COVID-19: A prospective randomized, double-blind, placebo-controlled clinical trial
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Mamta K. Jain, James A. De Lemos, Darren K. McGuire, Colby. Ayers, Jennifer L. Eitson, Claudia L. Sanchez, Dena Kamel, Jessica A. Meisner, Emilia V. Thomas, Anita A. Hegde, Satish Mocherla, Joslyn K. Strebe, Xilong Li, Noelle S. Williams, Chao Xing, Mahmoud S. Ahmed, Ping Wang, Hesham A. Sadek, and John W. Schoggins
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COVID-19 ,clinical trial ,atovaquone ,placebo controlled ,double blind ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Background: An in silico screen was performed to identify FDA approved drugs that inhibit SARS-CoV-2 main protease (Mpro), followed by in vitro viral replication assays, and in vivo pharmacokinetic studies in mice. These studies identified atovaquone as a promising candidate for inhibiting viral replication.Methods: A 2-center, randomized, double-blind, placebo-controlled trial was performed among patients hospitalized with COVID-19 infection. Enrolled patients were randomized 2:1 to atovaquone 1500 mg BID versus matched placebo. Patients received standard of care treatment including remdesivir, dexamethasone, or convalescent plasma as deemed necessary by the treating team. Saliva was collected at baseline and twice per day for up to 10 days for RNA extraction for SARS-CoV-2 viral load measurement by quantitative reverse-transcriptase PCR. The primary outcome was the between group difference in log-transformed viral load (copies/mL) using a generalized linear mixed-effect models of repeated measures from all samples.Results: Of the 61 patients enrolled; 41 received atovaquone and 19 received placebo. Overall, the population was predominately male (63%) and Hispanic (70%), with a mean age of 51 years, enrolled a mean of 5 days from symptom onset. The log10 viral load was 5.25 copies/mL vs. 4.79 copies/mL at baseline in the atovaquone vs. placebo group. Change in viral load did not differ over time between the atovaquone plus standard of care arm versus the placebo plus standard of care arm. Pharmacokinetic (PK) studies of atovaquone plasma concentration demonstrated a wide variation in atovaquone levels, with an inverse correlation between BMI and atovaquone levels, (Rho −0.45, p = 0.02). In post hoc analysis, an inverse correlation was observed between atovaquone levels and viral load (Rho −0.54, p = 0.005).Conclusion: In this prospective, randomized, placebo-controlled trial, atovaquone did not demonstrate evidence of enhanced SARS-CoV-2 viral clearance compared with placebo. However, based on the observed inverse correlation between atovaquone levels and viral load, additional PK-guided studies may be warranted to examine the antiviral effect of atovaquone in COVID-19 patients.
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- 2022
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8. Antithetic effect of interferon-α on cell-free and cell-to-cell HIV-1 infection
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Ryuichi Kumata, Shoya Iwanami, Katrina B. Mar, Yusuke Kakizoe, Naoko Misawa, Shinji Nakaoka, Yoshio Koyanagi, Alan S. Perelson, John W. Schoggins, Shingo Iwami, and Kei Sato
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Biology (General) ,QH301-705.5 - Abstract
In HIV-1-infected individuals, transmitted/founder (TF) virus contributes to establish new infection and expands during the acute phase of infection, while chronic control (CC) virus emerges during the chronic phase of infection. TF viruses are more resistant to interferon-alpha (IFN-α)-mediated antiviral effects than CC virus, however, its virological relevance in infected individuals remains unclear. Here we perform an experimental-mathematical investigation and reveal that IFN-α strongly inhibits cell-to-cell infection by CC virus but only weakly affects that by TF virus. Surprisingly, IFN-α enhances cell-free infection of HIV-1, particularly that of CC virus, in a virus-cell density-dependent manner. We further demonstrate that LY6E, an IFN-stimulated gene, can contribute to the density-dependent enhancement of cell-free HIV-1 infection. Altogether, our findings suggest that the major difference between TF and CC viruses can be explained by their resistance to IFN-α-mediated inhibition of cell-to-cell infection and their sensitivity to IFN-α-mediated enhancement of cell-free infection. Author summary HIV-1 experiences a strong bottleneck during transmission, and only the virus(es) with higher resistance to the host’s innate immunity, interferon (IFN), can be successfully transmitted. Because the IFN resistance tends to be disappeared during infection in infected individuals, this phenotype would be crucial for human-to-human transmission. By combining mathematical modeling with well-designed time-series viral infection experiments, we investigated the difference on the IFN resistance of two types of HIV-1, which were respectively isolated at the acute and chronic phases of infection, and classified it into two virus transmission modes, cell-free and cell-to-cell infections. We found that IFN suppresses HIV-1 cell-to-cell infection, but surprisingly, promotes cell-free infection. Moreover, the virus isolated during chronic infection is more sensitive to the IFN-mediated promoting effect than that isolated during acute infection. Our results suggest that HIV-1 selects different strategies to adapt to different host environments. We further provide an insight how viruses evolve to counteract or hijack the host immunity.
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- 2022
9. Identification of interferon-stimulated genes that attenuate Ebola virus infection
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Makoto Kuroda, Peter J. Halfmann, Lindsay Hill-Batorski, Makoto Ozawa, Tiago J. S. Lopes, Gabriele Neumann, John W. Schoggins, Charles M. Rice, and Yoshihiro Kawaoka
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Science - Abstract
Here, Kuroda et al. screen a library of nearly 400 interferon-stimulated genes (ISGs) and identify several ISGs that inhibit Ebola virus entry, viral transcription/replication, or virion formation. The study provides insights into interactions between Ebola and the host cells.
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- 2020
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10. A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating
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Harish N. Ramanathan, Shuo Zhang, Florian Douam, Katrina B. Mar, Jinhong Chang, Priscilla L. Yang, John W. Schoggins, Alexander Ploss, and Brett D. Lindenbach
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flavivirus ,nucleocapsid ,uncoating ,viral entry ,Microbiology ,QR1-502 - Abstract
ABSTRACT While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry. IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.
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- 2020
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11. LY6E mediates an evolutionarily conserved enhancement of virus infection by targeting a late entry step
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Katrina B. Mar, Nicholas R. Rinkenberger, Ian N. Boys, Jennifer L. Eitson, Matthew B. McDougal, R. Blake Richardson, and John W. Schoggins
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Science - Abstract
The interferon-induced gene LY6E increases virus infection, but the underlying mechanism is poorly understood. Here, Mar et al. show that LY6E enhances uncoating of influenza A virus after endosomal escape and that viral enhancement by LY6E is conserved across evolution.
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- 2018
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12. Western Zika Virus in Human Fetal Neural Progenitors Persists Long Term with Partial Cytopathic and Limited Immunogenic Effects
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Natasha W. Hanners, Jennifer L. Eitson, Noriyoshi Usui, R. Blake Richardson, Eric M. Wexler, Genevieve Konopka, and John W. Schoggins
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Biology (General) ,QH301-705.5 - Abstract
The recent Zika virus (ZIKV) outbreak in the Western hemisphere is associated with severe pathology in newborns, including microcephaly and brain damage. The mechanisms underlying these outcomes are under intense investigation. Here, we show that a 2015 ZIKV isolate replicates in multiple cell types, including primary human fetal neural progenitors (hNPs). In immortalized cells, ZIKV is cytopathic and grossly rearranges endoplasmic reticulum membranes similar to other flaviviruses. In hNPs, ZIKV infection has a partial cytopathic phase characterized by cell rounding, pyknosis, and activation of caspase 3. Despite notable cell death, ZIKV did not activate a cytokine response in hNPs. This lack of cell intrinsic immunity to ZIKV is consistent with our observation that virus replication persists in hNPs for at least 28 days. These findings, supported by published fetal neuropathology, establish a proof-of-concept that neural progenitors in the developing human fetus can be direct targets of detrimental ZIKV-induced pathology.
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- 2016
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13. A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating
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Shuo Zhang, Harish N. Ramanathan, Florian Douam, Katrina B. Mar, Jinhong Chang, Priscilla L. Yang, John W. Schoggins, Alexander Ploss, and Brett D. Lindenbach
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flavivirus ,nucleocapsid ,uncoating ,viral entry ,ubiquitin ,VCP/p97 ,General Works - Abstract
Flaviviruses are enveloped, arthropod-borne, positive-strand RNA viruses that cause significant human disease. While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We recently developed a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter to quantitively monitor the translation of incoming virus particle-delivered genomes. We validated that viral gene expression can be neutralized by YFV-specific antisera and requires known pathways of flavivirus entry; however, as expected, gene expression from the defective reporter virus was insensitive to a small molecule inhibitor of YFV RNA replication. The initial round of viral gene expression was also shown to require: (i) cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur, and (ii) valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large macromolecular complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry.
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- 2020
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14. Mucolipin-2 Cation Channel Increases Trafficking Efficiency of Endocytosed Viruses
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Nicholas Rinkenberger and John W. Schoggins
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endocytosis ,flavivirus ,influenza ,ion channels ,vesicular trafficking ,virus entry ,Microbiology ,QR1-502 - Abstract
ABSTRACT Receptor-mediated endocytosis is a cellular process commonly hijacked by viruses to enter cells. The stages of entry are well described for certain viruses, but the host factors that mediate each step are less well characterized. We previously identified endosomal cation channel mucolipin-2 (MCOLN2) as a host factor that promotes viral infection. Here, we assign a role for MCOLN2 in modulating viral entry. We show that MCOLN2 specifically promotes viral vesicular trafficking and subsequent escape from endosomal compartments. This mechanism requires channel activity, occurs independently of antiviral signaling, and broadly applies to enveloped RNA viruses that require transport to late endosomes for infection, including influenza A virus, yellow fever virus, and Zika virus. We further identify a rare allelic variant of human MCOLN2 that has a loss-of-function phenotype with respect to viral enhancement. These findings establish a mechanistic link between an endosomal cation channel and late stages of viral entry. IMPORTANCE Viruses must co-opt cellular processes to complete their life cycle. To enter cells, viruses frequently take advantage of cellular receptor-mediated endocytosis pathways. A growing number of host proteins are implicated in these viral uptake pathways. Here, we describe a new role for the gated cation channel MCOLN2 in viral entry. This endosomal protein modulates viral entry by enhancing the efficiency of viral trafficking through the endosomal system. Thus, MCOLN2-mediated enhancement of infection may represent a key vulnerability in the viral life cycle that could be targeted for therapeutic intervention.
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- 2018
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15. Recent advances in antiviral interferon-stimulated gene biology [version 1; referees: 2 approved]
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John W. Schoggins
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Medicine ,Science - Abstract
The interferon response protects cells from invading viral pathogens by transcriptionally inducing the expression of interferon-stimulated genes (ISGs), some of which encode effectors with varied antiviral functions. As screening technologies improve and mouse model development quickens, more ISGs are continually being identified, characterized mechanistically, and evaluated for protective roles in vivo. This review highlights selected recent findings of ISG effectors that contribute to our understanding of the interferon antiviral response.
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- 2018
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16. A Multi-Omics Approach Reveals Features That Permit Robust and Widespread Regulation of IFN-Inducible Antiviral Effectors
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Loránd Göczi, Mária Csumita, Attila Horváth, Gergely Nagy, Szilárd Póliska, Matteo Pigni, Christoph Thelemann, Bence Dániel, Hamidreza Mianesaz, Tamás Varga, Kaushik Sen, Sunil K. Raghav, John W. Schoggins, Laszlo Nagy, Hans Acha-Orbea, Felix Meissner, Walter Reith, and Lajos Széles
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Antiviral Restriction Factors ,Mice ,Immunology ,Animals ,Immunology and Allergy ,Interferons ,Nucleotide Motifs ,Promoter Regions, Genetic ,Response Elements ,Chromatin - Abstract
The antiviral state, an initial line of defense against viral infection, is established by a set of IFN-stimulated genes (ISGs) encoding antiviral effector proteins. The effector ISGs are transcriptionally regulated by type I IFNs mainly via activation of IFN-stimulated gene factor 3 (ISGF3). In this study, the regulatory elements of effector ISGs were characterized to determine the (epi)genetic features that enable their robust induction by type I IFNs in multiple cell types. We determined the location of regulatory elements, the DNA motifs, the occupancy of ISGF3 subunits (IRF9, STAT1, and STAT2) and other transcription factors, and the chromatin accessibility of 37 effector ISGs in murine dendritic cells. The IFN-stimulated response element (ISRE) and its tripartite version occurred most frequently in the regulatory elements of effector ISGs than in any other tested ISG subsets. Chromatin accessibility at their promoter regions was similar to most other ISGs but higher than at the promoters of inflammation-related cytokines, which were used as a reference gene set. Most effector ISGs (81.1%) had at least one ISGF3 binding region proximal to the transcription start site (TSS), and only a subset of effector ISGs (24.3%) was associated with three or more ISGF3 binding regions. The IRF9 signals were typically higher, and ISRE motifs were “stronger” (more similar to the canonical sequence) in TSS-proximal versus TSS-distal regulatory regions. Moreover, most TSS-proximal regulatory regions were accessible before stimulation in multiple cell types. Our results indicate that “strong” ISRE motifs and universally accessible promoter regions that permit robust, widespread induction are characteristic features of effector ISGs.
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- 2022
17. Interferon inhibits a model RNA virus via a limited set of inducible effector genes
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Matthew B. McDougal, Anthony M. De Maria, Maikke B. Ohlson, Ashwani Kumar, Chao Xing, and John W. Schoggins
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Article - Abstract
Interferons control viral infection by inducing the expression of antiviral effector proteins encoded by interferon-stimulated genes (ISGs). The field has mostly focused on identifying individual antiviral ISG effectors and defining their mechanisms of action. However, fundamental gaps in knowledge about the interferon response remain. For example, it is not known how many ISGs are required to protect cells from a particular virus, though it is theorized that numerous ISGs act in concert to achieve viral inhibition. Here, we used CRISPR-based loss-of-function screens to identify a markedly limited set of ISGs that confer interferon-mediated suppression of a model alphavirus, Venezuelan equine encephalitis virus (VEEV). We show via combinatorial gene targeting that three antiviral effectors – ZAP, IFIT3, and IFIT1 – together constitute the majority of interferon-mediated restriction of VEEV, while accounting for less than 0.5% of the interferon-induced transcriptome. Together, our data suggests a refined model of the antiviral interferon response in which a small subset of “dominant” ISGs may confer the bulk of the inhibition of a given virus.
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- 2023
18. LY6E protects mice from pathogenic effects of murine coronavirus and SARS-CoV-2
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Katrina B. Mar, Marley C. Van Dyke, Alexandra H. Lopez, Jennifer L. Eitson, Wenchun Fan, Natasha W. Hanners, Bret M. Evers, John M. Shelton, and John W. Schoggins
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Article - Abstract
LY6E is an antiviral protein that inhibits coronavirus entry. Its expression in immune cells allows mice to control murine coronavirus infection. However, it is not known which immune cell subsets mediate this control or whether LY6E protects mice from SARS-CoV-2. In this study, we used tissue-specific Cre recombinase expression to ablateLy6ein distinct immune compartments or in all epiblast-derived cells, and bone marrow chimeras to target Ly6e in a subset of radioresistant cells. Mice lackingLy6einLyz2-expressing cells and radioresistantVav1-expressing cells were more susceptible to lethal murine coronavirus infection. Mice lackingLy6eglobally developed clinical disease when challenged with the Gamma (P.1) variant of SARS-CoV-2. By contrast, wildtype mice and mice lacking type I and type III interferon signaling had no clinical symptoms after SARS-CoV-2 infection. Transcriptomic profiling of lungs from SARS-CoV-2-infected wildtype andLy6eknockout mice revealed a striking reduction of secretory cell-associated genes in infected knockout mice, includingMuc5b, an airway mucin-encoding gene that may protect against SARS-CoV-2-inflicted respiratory disease. Collectively, our study reveals distinct cellular compartments in which Ly6e confers cell intrinsic antiviral effects, thereby conferring resistance to disease caused by murine coronavirus and SARS-CoV-2.
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- 2023
19. Author response: A concerted mechanism involving ACAT and SREBPs by which oxysterols deplete accessible cholesterol to restrict microbial infection
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Kristen A Johnson, David B Heisler, Duo H Ma, Maikke B Ohlson, Lishu Zhang, Michelle Tran, Chase D Corley, Michael E Abrams, Jeffrey G McDonald, John W Schoggins, Neal M Alto, and Arun Radhakrishnan
- Published
- 2023
20. Genome-scale CRISPR screening reveals host factors required for ribosome formation and viral replication
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Maikke B. Ohlson, Ashwani Kumar, Jennifer L. Eitson, Seoyeon Jang, Chunyang Ni, Chao Xing, Michael Buszczak, and John W. Schoggins
- Abstract
Viruses are known to co-opt host machinery for translation initiation, but less is known about which host factors are required for the formation of ribosomes used to synthesize viral proteins. Using a loss of function CRISPR screen, we show that synthesis of a flavivirus encoded reporter depends on multiple host factors, including several 60S ribosome biogenesis proteins. Viral phenotyping revealed that two of these factors, SBDS, a known ribosome biogenesis factor, and the relatively uncharacterized protein SPATA5, were broadly required for replication of flaviviruses, coronaviruses, alphaviruses, paramyxoviruses, an enterovirus, and a poxvirus. Mechanistic studies revealed that loss of SPATA5 caused defects in ribosomal RNA processing and ribosome assembly, suggesting that this human protein may be a functional ortholog of yeastDrg1. These studies implicate specific ribosome biogenesis proteins as viral host dependency factors that are required for synthesis of virally encoded protein and accordingly, optimal viral replication.
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- 2022
21. A Concerted Mechanism Involving ACAT and SREBPs By which Oxysterols Deplete Accessible Cholesterol To Restrict Microbial Infection
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Kristen A Johnson, David B Heisler, Duo H Ma, Maikke B Ohlson, Lishu Zhang, Michelle Tran, Chase D Corley, Michael E Abrams, Jeffrey G McDonald, John W Schoggins, Neal M Alto, and Arun Radhakrishnan
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General Immunology and Microbiology ,General Neuroscience ,General Medicine ,General Biochemistry, Genetics and Molecular Biology - Abstract
Most of the cholesterol in the plasma membranes (PMs) of animal cells is sequestered through interactions with phospholipids and transmembrane domains of proteins. However, as cholesterol concentration rises above the PM’s sequestration capacity, a new pool of cholesterol, called accessible cholesterol, emerges. The transport of accessible cholesterol between the PM and the endoplasmic reticulum (ER) is critical to maintain cholesterol homeostasis. This pathway has also been implicated in the suppression of both bacterial and viral pathogens by immunomodulatory oxysterols. Here, we describe a mechanism of depletion of accessible cholesterol from PMs by the oxysterol 25-hydroxycholesterol (25HC). We show that 25HC-mediated activation of acyl coenzyme A: cholesterol acyltransferase (ACAT) in the ER creates an imbalance in the equilibrium distribution of accessible cholesterol between the ER and PM. This imbalance triggers the rapid internalization of accessible cholesterol from the PM, and this depletion is sustained for long periods of time through 25HC-mediated suppression of SREBPs and continued activation of ACAT. In support of a physiological role for this mechanism, 25HC failed to suppress Zika virus and human coronavirus infection in ACAT-deficient cells, and Listeria monocytogenes infection in ACAT-deficient cells and mice. We propose that selective depletion of accessible PM cholesterol triggered by ACAT activation and sustained through SREBP suppression underpins the immunological activities of 25HC and a functionally related class of oxysterols.
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- 2022
22. Editor's evaluation: Human DUX4 and mouse Dux interact with STAT1 and broadly inhibit interferon-stimulated gene induction
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John W Schoggins
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- 2022
23. Enterovirus 3C Protease Cleaves TRIM7 To Dampen Its Antiviral Activity
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Wenchun Fan, Matthew B. McDougal, and John W. Schoggins
- Subjects
Glutamine ,Ubiquitin-Protein Ligases ,Immunology ,3C Viral Proteases ,Microbiology ,Virus-Cell Interactions ,Tripartite Motif Proteins ,Virology ,Insect Science ,Host-Pathogen Interactions ,Enterovirus Infections ,Animals ,Histidine ,Phascolarctidae ,Enterovirus - Abstract
Mammalian TRIM7 is an antiviral protein that inhibits multiple human enteroviruses by degrading the viral 2BC protein. Whether TRIM7 is reciprocally targeted by enteroviruses is not known. Here, we report that the 3C protease (3Cpro) from two enteroviruses, coxsackievirus B3 (CVB3) and poliovirus, targets TRIM7 for cleavage. CVB3 3Cpro cleaves TRIM7 at glutamine 24 (Q24), resulting in a truncated TRIM7 that fails to inhibit CVB3 due to dampened E3 ubiquitin ligase activity. TRIM7 Q24 is highly conserved across mammals, except in marsupials, which instead have a naturally occurring histidine (H24) that is not subject to 3Cpro cleavage. Marsupials also express two isoforms of TRIM7, and the two proteins from koalas have distinct antiviral activities. The longer isoform contains an additional exon due to alternate splice site usage. This additional exon contains a unique 3Cpro cleavage site, suggesting that certain enteroviruses may have evolved to target marsupial TRIM7 even if the canonical Q24 is missing. Combined with computational analyses indicating that TRIM7 is rapidly evolving, our data raise the possibility that TRIM7 may be targeted by enterovirus evasion strategies and that evolution of TRIM7 across mammals may have conferred unique antiviral properties. IMPORTANCE Enteroviruses are significant human pathogens that cause viral myocarditis, pancreatitis, and meningitis. Knowing how the host controls these viruses and how the viruses may evade host restriction is important for understanding fundamental concepts in antiviral immunity and for informing potential therapeutic interventions. In this study, we demonstrate that coxsackievirus B3 uses its virally encoded protease to target the host antiviral protein TRIM7 for cleavage, suggesting a potential mechanism of viral immune evasion. We additionally show that TRIM7 has evolved in certain mammalian lineages to express protein variants with distinct antiviral activities and susceptibilities to viral protease-mediated cleavage.
- Published
- 2022
24. The mechanism of RNA capping by SARS-CoV-2
- Author
-
Gina J. Park, Adam Osinski, Genaro Hernandez, Jennifer L. Eitson, Abir Majumdar, Marco Tonelli, Katie Henzler-Wildman, Krzysztof Pawłowski, Zhe Chen, Yang Li, John W. Schoggins, and Vincent S. Tagliabracci
- Subjects
RNA Caps ,Multidisciplinary ,SARS-CoV-2 ,COVID-19 ,Methyltransferases ,RNA-Dependent RNA Polymerase ,Antiviral Agents ,Guanosine Diphosphate ,Nucleotidyltransferases ,COVID-19 Drug Treatment ,Viral Proteins ,Protein Domains ,Catalytic Domain ,Humans ,RNA, Viral - Abstract
The RNA genome of SARS-CoV-2 contains a 5′ cap that facilitates the translation of viral proteins, protection from exonucleases and evasion of the host immune response1–4. How this cap is made in SARS-CoV-2 is not completely understood. Here we reconstitute the N7- and 2′-O-methylated SARS-CoV-2 RNA cap (7MeGpppA2′-O-Me) using virally encoded non-structural proteins (nsps). We show that the kinase-like nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain5 of nsp12 transfers the RNA to the amino terminus of nsp9, forming a covalent RNA–protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers the RNA to GDP, forming the core cap structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication–transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N terminus of nsp9 and the kinase-like active-site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.
- Published
- 2022
25. Identification of interferon-stimulated genes that attenuate Ebola virus infection
- Author
-
Gabriele Neumann, John W. Schoggins, Charles M. Rice, Peter Halfmann, Makoto Ozawa, Lindsay Hill-Batorski, Makoto Kuroda, Tiago J. S. Lopes, and Yoshihiro Kawaoka
- Subjects
0301 basic medicine ,viruses ,Fluorescent Antibody Technique ,Gene Expression ,General Physics and Astronomy ,Virus Replication ,medicine.disease_cause ,Ebola virus ,Interferon ,Chlorocebus aethiops ,lcsh:Science ,Multidisciplinary ,Reverse Transcriptase Polymerase Chain Reaction ,virus diseases ,Ebolavirus ,Protein Binding ,medicine.drug ,Cell Survival ,Viral protein ,Science ,Blotting, Western ,030106 microbiology ,Biology ,Virus-host interactions ,Article ,General Biochemistry, Genetics and Molecular Biology ,Virus ,Viral Proteins ,03 medical and health sciences ,Viral entry ,medicine ,Animals ,Humans ,Immunoprecipitation ,Vero Cells ,Gene ,Outbreak ,General Chemistry ,Virus Internalization ,Virology ,Cytoskeletal Proteins ,HEK293 Cells ,030104 developmental biology ,Vero cell ,lcsh:Q ,Interferons ,Restriction factors ,Carrier Proteins ,HeLa Cells - Abstract
The West Africa Ebola outbreak was the largest outbreak ever recorded, with over 28,000 reported infections; this devastating epidemic emphasized the need to understand the mechanisms to counteract virus infection. Here, we screen a library of nearly 400 interferon-stimulated genes (ISGs) against a biologically contained Ebola virus and identify several ISGs not previously known to affect Ebola virus infection. Overexpression of the top ten ISGs attenuates virus titers by up to 1000-fold. Mechanistic studies demonstrate that three ISGs interfere with virus entry, six affect viral transcription/replication, and two inhibit virion formation and budding. A comprehensive study of one ISG (CCDC92) that shows anti-Ebola activity in our screen reveals that CCDC92 can inhibit viral transcription and the formation of complete virions via an interaction with the viral protein NP. Our findings provide insights into Ebola virus infection that could be exploited for the development of therapeutics against this virus., Here, Kuroda et al. screen a library of nearly 400 interferon-stimulated genes (ISGs) and identify several ISGs that inhibit Ebola virus entry, viral transcription/replication, or virion formation. The study provides insights into interactions between Ebola and the host cells.
- Published
- 2020
26. Editor's evaluation: Brucella activates the host RIDD pathway to subvert BLOS1-directed immune defense
- Author
-
John W Schoggins
- Published
- 2021
27. Overexpression screen of interferon-stimulated genes identifies RARRES3 as a restrictor of Toxoplasma gondii infection
- Author
-
Nicholas Rinkenberger, Michael E Abrams, Sumit K Matta, John W Schoggins, Neal M Alto, and L David Sibley
- Subjects
Microbiology and Infectious Disease ,General Immunology and Microbiology ,QH301-705.5 ,Science ,General Neuroscience ,General Medicine ,growth restriction ,General Biochemistry, Genetics and Molecular Biology ,egress ,cell death ,interferon gamma ,parasitic diseases ,Medicine ,Animals ,Humans ,Parasites ,Other ,Biology (General) ,Toxoplasma ,intracellular parasite ,Research Article - Abstract
Toxoplasma gondiiis an important human pathogen infecting an estimated one in three people worldwide. The cytokine interferon gamma (IFNγ) is induced during infection and is critical for restrictingT. gondiigrowth in human cells. Growth restriction is presumed to be due to the induction of interferon-stimulated genes (ISGs) that are upregulated to protect the host from infection. Although there are hundreds of ISGs induced by IFNγ, their individual roles in restricting parasite growth in human cells remain somewhat elusive. To address this deficiency, we screened a library of 414 IFNγ induced ISGs to identify factors that impactT. gondiiinfection in human cells. In addition to IRF1, which likely acts through the induction of numerous downstream genes, we identified RARRES3 as a single factor that restrictsT. gondiiinfection by inducing premature egress of the parasite in multiple human cell lines. Overall, while we successfully identified a novel IFNγ induced factor restrictingT. gondiiinfection, the limited number of ISGs capable of restrictingT. gondiiinfection when individually expressed suggests that IFNγ-mediated immunity toT. gondiiinfection is a complex, multifactorial process.
- Published
- 2021
28. Author response: Overexpression screen of interferon-stimulated genes identifies RARRES3 as a restrictor of Toxoplasma gondii infection
- Author
-
Nicholas Rinkenberger, Michael E Abrams, Sumit K Matta, John W Schoggins, Neal M Alto, and L David Sibley
- Published
- 2021
29. Author response: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway
- Author
-
Shahanshah Khan, Mahnoush S Shafiei, Christopher Longoria, John W Schoggins, Rashmin C Savani, and Hasan Zaki
- Published
- 2021
30. Defective viral RNA sensing linked to severe COVID-19
- Author
-
John W. Schoggins
- Subjects
2019-20 coronavirus outbreak ,Multidisciplinary ,Coronavirus disease 2019 (COVID-19) ,SARS-CoV-2 ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,fungi ,food and beverages ,COVID-19 ,Humans ,RNA, Viral ,Viral rna ,Biology ,Virology - Abstract
Genetic variation in a sensor of double-stranded RNA can exacerbate COVID-19
- Published
- 2021
31. Shiftless inhibits flavivirus replication in vitro and is neuroprotective in a mouse model of Zika virus pathogenesis
- Author
-
Natasha W. Hanners, Katrina B. Mar, Ian N. Boys, Jennifer L. Eitson, Pamela C. De La Cruz-Rivera, R. Blake Richardson, Wenchun Fan, Mary Wight-Carter, and John W. Schoggins
- Subjects
Male ,Mice, Knockout ,Multidisciplinary ,Zika Virus Infection ,viruses ,Flavivirus ,RNA-Binding Proteins ,Zika Virus ,Biological Sciences ,Virus Replication ,Cell Line ,Flavivirus Infections ,Mice, Inbred C57BL ,Disease Models, Animal ,Mice ,Neuroprotective Agents ,Cytopathogenic Effect, Viral ,Animals ,Disease Susceptibility - Abstract
Flaviviruses such as Zika virus and West Nile virus have the potential to cause severe neuropathology if they invade the central nervous system. The type I interferon response is well characterized as contributing to control of flavivirus-induced neuropathogenesis. However, the interferon-stimulated gene (ISG) effectors that confer these neuroprotective effects are less well studied. Here, we used an ISG expression screen to identify Shiftless (SHFL, C19orf66) as a potent inhibitor of diverse positive-stranded RNA viruses, including multiple members of the Flaviviridae (Zika, West Nile, dengue, yellow fever, and hepatitis C viruses). In cultured cells, SHFL functions as a viral RNA-binding protein that inhibits viral replication at a step after primary translation of the incoming genome. The murine ortholog, Shfl, is expressed constitutively in multiple tissues, including the central nervous system. In a mouse model of Zika virus infection, Shfl(−/−) knockout mice exhibit reduced survival, exacerbated neuropathological outcomes, and increased viral replication in the brain and spinal cord. These studies demonstrate that Shfl is an important antiviral effector that contributes to host protection from Zika virus infection and virus-induced neuropathological disease.
- Published
- 2021
32. Identification of Atovaquone and Mebendazole as Repurposed Drugs with Antiviral Activity against SARS-CoV-2 (Version 6)
- Author
-
Mahmoud Ahmed, Ayman Farag, Ian N. Boys, Ping Wang, Jennifer L. Eitson, Maikke B. Ohlson, Wenchun Fan, Matthew B. McDougal, Ivan Menendez-Montez, Ngoc Uyen Nhi Nguyen, Uyen Nhi Nguyen, Katrina Mar, Francisco Ortiz, Soo Young Kim, Noelle Williams, Andrew Lemoff lemoff, Ralph DeBerardinis, John W. Schoggins, and Hesham Sadek
- Abstract
Given the continuing heavy toll of the COVID-19 pandemic, therapeutic options for treatment are urgently needed. Here, we adopted a repositioning approach using in silico molecular modeling to screen FDA-approved drugs with established safety profiles for potential inhibitory effects against SARS-CoV-2. We used structure-based drug design to screen more than 2000 FDA approved drugs against SARS-CoV-2 main protease enzyme (Mpro) substrate-binding pocket. We additionally screened the top hits from both sites for potential covalent binding via nucleophilic thiol attack of Cys 145. High-scoring candidates were then screened for antiviral activity against infectious SARS-CoV-2 in a cell-based viral replication assay, and counter screened for toxicity. Promising candidates included atovaquone, mebendazole, ouabain, dronedarone, and entacapone, although atovaquone and mebendazole were the only two candidates with IC50s that fall within their therapeutic plasma concentration. In addition, we performed Mpro assays on the top hits, which demonstrated inhibition of Mpro by dronedarone (IC50 18 M), mebendazole (IC50 19 M) and entacapone (IC50 9 M). Atovaquone showed only modest Mpro inhibition, and thus we explored other potential antiviral mechanisms. Although atovaquone is a known DHODH inhibitor, we did not observe inhibition of DHODH by atovaquone at concentrations relevant to the SARS-CoV-2 IC50. Interestingly, metabolomic profiling of atovaquone treated cells demonstrated marked dysregulation of metabolites in the purine metabolism pathway. In summary, a number of our top hits from the in-silico screen demonstrated Mpro inhibitory activity associated with antiviral effects. Atovaquone and mebendazole are the most promising candidates targeting SARS-CoV-2 from our screen, however atovaquone did not significantly inhibit Mpro at therapeutically meaningful concentrations but may inhibit SARS-CoV-2 viral replication by targeting host purine metabolism.
- Published
- 2021
33. Over-expression Screen of Interferon-Stimulated Genes Identifies RARRES3 as a Restrictor ofToxoplasma gondiiInfection
- Author
-
Michael E. Abrams, Neal M. Alto, Sumit K. Matta, John W. Schoggins, L. David Sibley, and Nicholas Rinkenberger
- Subjects
medicine.medical_treatment ,Toxoplasma gondii ,Biology ,biology.organism_classification ,Virology ,Cytokine ,IRF1 ,Downregulation and upregulation ,Interferon ,Immunity ,parasitic diseases ,medicine ,Interferon gamma ,Gene ,medicine.drug - Abstract
Toxoplasma gondiiis an important human pathogen infecting an estimated 1 in 3 people worldwide. The cytokine interferon gamma (IFNγ) is induced during infection and is critical for restrictingT. gondiigrowth in human cells. Growth restriction is presumed to be due to the induction interferon stimulated genes (ISGs) that are upregulated to protect the host from infection. Although there are hundreds of ISGs induced by IFNγ, their individual roles in restricting parasite growth in human cells remain somewhat elusive. To address this deficiency, we screened a library of 414 IFNγ induced ISGs to identify factors that impactT. gondiiinfection in human cells. In addition to IRF1, which likely acts through induction of numerous downstream genes, we identified RARRES3 as a single factor that restrictsT. gondiiinfection by inducing premature egress of the parasite in multiple human cell lines. Overall, while we successfully identified a novel IFNγ induced factor restrictingT. gondiiinfection, the limited number of ISGs capable of restrictingT. gondiiinfection when individually expressed suggests that IFNγ mediated immunity toT. gondiiinfection is a complex, multifactorial process.
- Published
- 2021
34. Identification of Atovaquone and Mebendazole as Repurposed Drugs with Antiviral Activity against SARS-CoV-2 (Version 5)
- Author
-
Mahmoud Ahmed, Ayman Farag, Ping Wang, Ian N. Boys, Jennifer L. Eitson, Maikke B. Ohlson, Wenchun Fan, Matthew B. McDougal, John W. Schoggins, and Hesham Sadek
- Abstract
Given the continuing heavy toll of the COVID-19 pandemic, therapeutic options for treatment are urgently needed. Here, we adopted a repositioning approach using in silico molecular modeling to screen FDA-approved drugs with established safety profiles for potential inhibitory effects against SARS-CoV-2. We used structure-based drug design to screen more than 2000 FDA approved drugs against SARS-CoV-2 main protease enzyme (Mpro) substrate-binding pocket. We additionally screened the top hits from both sites for potential covalent binding via nucleophilic thiol attack of Cys 145. High-scoring candidates were then screened for antiviral activity against infectious SARS-CoV-2 in a cell-based viral replication assay, and counter screened for toxicity. Promising candidates included atovaquone, mebendazole, ouabain, dronedarone, and entacapone, although atovaquone and mebendazole were the only two candidates with IC50s that fall within their therapeutic plasma concentration. In addition, we performed Mpro assays on the top hits, which demonstrated inhibition of Mpro by dronedarone (IC50 18 M), mebendazole (IC50 19 M) and entacapone (IC50 9 M). Atovaquone showed only modest Mpro inhibition, and thus we explored other potential antiviral mechanisms. Although atovaquone is a known DHODH inhibitor, we did not observe inhibition of DHODH by atovaquone at concentrations relevant to the SARS-CoV-2 IC50. Interestingly, metabolomic profiling of atovaquone treated cells demonstrated marked dysregulation of metabolites in the purine metabolism pathway. In summary, a number of our top hits from the in-silico screen demonstrated Mpro inhibitory activity associated with antiviral effects. Atovaquone and mebendazole are the most promising candidates targeting SARS-CoV-2 from our screen, however atovaquone did not significantly inhibit Mpro at therapeutically meaningful concentrations but may inhibit SARS-CoV-2 viral replication by targeting host purine metabolism.
- Published
- 2021
35. Identification of Atovaquone as and Mebendazole as Repurposed Drugs with Antiviral Activity against SARS-CoV-2 (Version 5)
- Author
-
Hesham A. Sadek, Matthew B. McDougal, John W. Schoggins, Ian N. Boys, Ayman B. Farag, Mahmoud S. Ahmed, Jennifer L. Eitson, Maikke B. Ohlson, Ping Wang, and Wenchun Fan
- Subjects
Drug ,Protease ,Chemistry ,media_common.quotation_subject ,medicine.medical_treatment ,Mebendazole ,Pharmacology ,Viral replication ,medicine ,Entacapone ,Purine metabolism ,IC50 ,Atovaquone ,medicine.drug ,media_common - Abstract
Given the continuing heavy toll of the COVID-19 pandemic, therapeutic options for treatment are urgently needed. Here, we adopted a repositioning approach using in silico molecular modeling to screen FDA-approved drugs with established safety profiles for potential inhibitory effects against SARS-CoV-2. We used structure-based drug design to screen more than 2000 FDA approved drugs against SARS-CoV-2 main protease enzyme (Mpro) substrate-binding pocket. We additionally screened the top hits from both sites for potential covalent binding via nucleophilic thiol attack of Cys 145. High-scoring candidates were then screened for antiviral activity against infectious SARS-CoV-2 in a cell-based viral replication assay, and counter screened for toxicity. Promising candidates included atovaquone, mebendazole, ouabain, dronedarone, and entacapone, although atovaquone and mebendazole were the only two candidates with IC50s that fall within their therapeutic plasma concentration. In addition, we performed Mpro assays on the top hits, which demonstrated inhibition of Mpro by dronedarone (IC50 18 M), mebendazole (IC50 19 M) and entacapone (IC50 9 M). Atovaquone showed only modest Mpro inhibition, and thus we explored other potential antiviral mechanisms. Although atovaquone is a known DHODH inhibitor, we did not observe inhibition of DHODH by atovaquone at concentrations relevant to the SARS-CoV-2 IC50. Interestingly, metabolomic profiling of atovaquone treated cells demonstrated marked dysregulation of metabolites in the purine metabolism pathway. In summary, a number of our top hits from the in-silico screen demonstrated Mpro inhibitory activity associated with antiviral effects. Atovaquone and mebendazole are the most promising candidates targeting SARS-CoV-2 from our screen, however atovaquone did not significantly inhibit Mpro at therapeutically meaningful concentrations but may inhibit SARS-CoV-2 viral replication by targeting host purine metabolism.
- Published
- 2021
36. In-Depth Evaluation of a Case of Presumed Myocarditis After the Second Dose of COVID-19 mRNA Vaccine
- Author
-
Imran Hitto, Mark H. Drazner, Quan Zhen Li, James A. de Lemos, Benjamin Greenberg, Madhusudhanan Narasimhan, John W. Schoggins, Kiran Batra, Amit Khera, Xuan Jiang, Carol L. Croft, Chengsong Zhu, Lenin Mahimainathan, Justin L. Grodin, Franklin Fuda, James B Cutrell, Sean J. Morrison, Pradeep P.A. Mammen, and Alagarraju Muthukumar
- Subjects
Male ,2019-20 coronavirus outbreak ,Myocarditis ,COVID-19 Vaccines ,Coronavirus disease 2019 (COVID-19) ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Immunization, Secondary ,Physiology (medical) ,vaccine ,Medicine ,Humans ,Cases and Traces ,Messenger RNA ,business.industry ,SARS-CoV-2 ,COVID-19 ,Middle Aged ,medicine.disease ,Virology ,ComputingMethodologies_DOCUMENTANDTEXTPROCESSING ,Cardiovascular Case Series ,Cardiology and Cardiovascular Medicine ,business ,COVID-19 vaccine ,2019-nCoV Vaccine mRNA-1273 - Abstract
Supplemental Digital Content is available in the text.
- Published
- 2021
37. Comparative analysis of viral entry for Asian and African lineages of Zika virus
- Author
-
John W. Schoggins and Nicholas Rinkenberger
- Subjects
Infectivity ,0303 health sciences ,Asia ,Lineage (genetic) ,biology ,Zika Virus Infection ,030302 biochemistry & molecular biology ,Endosomes ,Zika Virus ,Virus Internalization ,biology.organism_classification ,Virology ,Endocytosis ,Article ,Zika virus ,03 medical and health sciences ,Emerging pathogen ,Flavivirus ,Viral entry ,Africa ,Humans ,030304 developmental biology ,Immune activation - Abstract
Zika virus (ZIKV) is an emerging pathogen with global health and economic impacts. ZIKV circulates as two major lineages, Asian or African. The Asian lineage has recently been associated with significant disease in humans. Numerous studies have revealed differences between African and Asian ZIKV strains with respect to cellular infectivity, pathogenesis, and immune activation. Less is known about the mechanism of ZIKV entry and whether viral entry differs between strains. Here, we characterized ZIKV entry with two Asian and two African strains. All viruses exhibited a requirement for clathrin-mediated endocytosis and Rab5a function. Additionally, all ZIKV strains tested were sensitive to pH in the range of 6.5-6.1 and were reliant on endosomal acidification for infection. Finally, we provide direct evidence that ZIKV primarily fuses with late endosomes. These findings contribute new insight into the ZIKV entry process and suggest that divergent ZIKV strains enter cells in a highly conserved manner.
- Published
- 2019
38. A NIK-SIX signaling axis controls inflammation by targeted silencing of noncanonical NF-κB
- Author
-
Sofya S. Perelman, Neal M. Alto, John W. Schoggins, Mohammed Kanchwala, Katrina B. Mar, Chao Xing, Natasha W. Hanners, and Zixu Liu
- Subjects
Male ,Antineoplastic Agents ,Nerve Tissue Proteins ,Biology ,Protein Serine-Threonine Kinases ,Article ,Shigella flexneri ,03 medical and health sciences ,MAP3K14 ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Carcinoma, Non-Small-Cell Lung ,Cell Line, Tumor ,Gene silencing ,Animals ,Humans ,Promoter Regions, Genetic ,Transcription factor ,030304 developmental biology ,Homeodomain Proteins ,Inflammation ,0303 health sciences ,Multidisciplinary ,Kinase ,RELB ,Transcription Factor RelB ,NF-kappa B ,Transcription Factor RelA ,Promoter ,NF-κB ,Fibroblasts ,Listeria monocytogenes ,Cell biology ,HEK293 Cells ,Proteasome ,chemistry ,Gene Expression Regulation ,030220 oncology & carcinogenesis ,Female ,Signal Transduction - Abstract
The non-canonical NF-κB signalling cascade is essential for lymphoid organogenesis, B cell maturation, osteoclast differentiation, and inflammation in mammals1,2; dysfunction of this system is associated with human diseases, including immunological disorders and cancer3–6. Although expression of NF-κB-inducing kinase (NIK, also known as MAP3K14) is the rate-limiting step in non-canonical NF-κB pathway activation2,7, the mechanisms by which transcriptional responses are regulated remain largely unknown. Here we show that the sine oculis homeobox (SIX) homologue family transcription factors SIX1 and SIX2 are integral components of the non-canonical NF-κB signalling cascade. The developmentally silenced SIX proteins are reactivated in differentiated macrophages by NIK-mediated suppression of the ubiquitin proteasome pathway. Consequently, SIX1 and SIX2 target a subset of inflammatory gene promoters and directly inhibit the trans-activation function of the transcription factors RELA and RELB in a negative feedback circuit. In support of a physiologically pivotal role for SIX proteins in host immunity, a human SIX1 transgene suppressed inflammation and promoted the recovery of mice from endotoxic shock. In addition, SIX1 and SIX2 protected RAS/P53-driven non-small-cell lung carcinomas from inflammatory cell death induced by SMAC-mimetic chemotherapeutic agents (small-molecule activators of the non-canonical NF-κB pathway). Our findings identify a NIK–SIX signalling axis that fine-tunes inflammatory gene expression programs under both physiological and pathological conditions. Non-canonical NF-κB is regulated by a negative feedback loop involving NIK-mediated activation of SIX1 and SIX2.
- Published
- 2019
39. Evolution of the interferon response: lessons from ISGs of diverse mammals
- Author
-
Matthew B McDougal, Ian N Boys, Pamela De La Cruz-Rivera, and John W Schoggins
- Subjects
Mammals ,Virus Diseases ,Virology ,Viruses ,Animals ,Humans ,Interferons ,Antiviral Agents - Abstract
The vertebrate interferon (IFN) response controls viral infections by inducing hundreds of interferon-stimulated genes (ISGs), many of which encode 'restriction factors' that uniquely target certain viruses. ISG studies have historically had a human-centric focus, which is justified because these natural defense mechanisms might be leveraged to treat human viral disease. However, certain mammals are reservoirs for zoonotic viruses that can 'spill over' into humans. Additionally, restriction factors have prominent roles in the ongoing evolutionary genetic conflicts between viruses and their hosts. Thus, there is a growing need to understand antiviral IFN/ISG responses in other species, particularly in known reservoirs of zoonotic viruses. This review focuses on functional and evolutionary insight into antiviral IFN responses that have been obtained from studying non-model mammalian species.
- Published
- 2022
40. SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway
- Author
-
Khan S, John W. Schoggins, Shafiei Ms, Longoria C, Zaki H, and Savani Rc
- Subjects
Chemokine ,QH301-705.5 ,Science ,Inflammation ,spike protein ,Article ,General Biochemistry, Genetics and Molecular Biology ,Proinflammatory cytokine ,Mice ,Immunology and Inflammation ,medicine ,Animals ,Humans ,TLR2 ,Biology (General) ,General Immunology and Microbiology ,biology ,Chemistry ,SARS-CoV-2 ,General Neuroscience ,NF-kappa B ,COVID-19 ,General Medicine ,medicine.disease ,Toll-Like Receptor 2 ,Cell biology ,CXCL1 ,CXCL2 ,HEK293 Cells ,A549 Cells ,inflammation ,Spike Glycoprotein, Coronavirus ,cytokine storm ,Viruses ,biology.protein ,Medicine ,Tumor necrosis factor alpha ,medicine.symptom ,Cytokine storm ,Signal Transduction ,Research Article - Abstract
The pathogenesis of COVID-19 is associated with a hyperinflammatory response; however, the precise mechanism of SARS-CoV-2-induced inflammation is poorly understood. Here, we investigated direct inflammatory functions of major structural proteins of SARS-CoV-2. We observed that spike (S) protein potently induced inflammatory cytokines and chemokines, including IL-6, IL-1β, TNFα, CXCL1, CXCL2, and CCL2, but not IFNs in human and mouse macrophages. No such inflammatory response was observed in response to membrane (M), envelope (E), and nucleocapsid (N) proteins. When stimulated with extracellular S protein, human and mouse lung epithelial cells also produced inflammatory cytokines and chemokines. Interestingly, epithelial cells expressing S protein intracellularly were non-inflammatory, but elicited an inflammatory response in macrophages when co-cultured. Biochemical studies revealed that S protein triggers inflammation via activation of the NF-κB pathway in a MyD88-dependent manner. Further, such an activation of the NF-κB pathway was abrogated in Tlr2-deficient macrophages. Consistently, administration of S protein-induced IL-6, TNF-α, and IL-1β in wild-type, but not Tlr2-deficient mice. Notably, upon recognition of S protein, TLR2 dimerizes with TLR1 or TLR6 to activate the NF-κB pathway. Taken together, these data reveal a mechanism for the cytokine storm during SARS-CoV-2 infection and suggest that TLR2 could be a potential therapeutic target for COVID-19.
- Published
- 2021
41. Laboratory Action Plan for Emerging SARS-CoV-2 Variants
- Author
-
Jason Y. Park, Jeffrey A. SoRelle, Laura M. Filkins, and John W. Schoggins
- Subjects
Biochemistry, medical ,2019-20 coronavirus outbreak ,Opinion ,Coronavirus disease 2019 (COVID-19) ,SARS-CoV-2 ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Biochemistry (medical) ,Clinical Biochemistry ,COVID-19 ,Biology ,Virology ,COVID-19 Serological Testing ,Coronavirus Disease 2019 ,Action plan ,Humans ,VUI-202012/01 ,Laboratories ,B.1.1.7 ,VOC-202012/01 - Published
- 2021
42. Sorting nexin 5 mediates virus-induced autophagy and immunity
- Author
-
Elizabeth R. Aguilera, Xuewu Zhang, Liwei Wang, Sandra L. Schmid, Nicholas T. Ktistakis, Bo Ci, Ramnik J. Xavier, John W. Schoggins, Julie K. Pfeiffer, Xiaonan Dong, Boris Simonetti, Madhura Bhave, Yuting Yang, R. Blake Richardson, Peter J. Cullen, Xiao Zang, Y. Zhao, Paul A. Gleeson, Guanghua Xiao, Li Yu, Yi Chun Kuo, Lin Zhong, Yang Xie, Zhongju Zou, Beth Levine, and Rui Zhong
- Subjects
Male ,Small interfering RNA ,Endosome ,Vesicular Transport Proteins ,Lipid kinase activity ,Autophagy-Related Proteins ,Endosomes ,In Vitro Techniques ,Biology ,Cell Line ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Interferon ,Autophagy ,medicine ,Animals ,Humans ,RNA, Small Interfering ,Sorting Nexins ,PI3K/AKT/mTOR pathway ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Class III Phosphatidylinositol 3-Kinases ,Cell biology ,Mice, Inbred C57BL ,Sorting nexin ,Viruses ,Beclin-1 ,Female ,030217 neurology & neurosurgery ,medicine.drug - Abstract
Autophagy, a process of degradation that occurs via the lysosomal pathway, has an essential role in multiple aspects of immunity, including immune system development, regulation of innate and adaptive immune and inflammatory responses, selective degradation of intracellular microorganisms, and host protection against infectious diseases1,2. Autophagy is known to be induced by stimuli such as nutrient deprivation and suppression of mTOR, but little is known about how autophagosomal biogenesis is initiated in mammalian cells in response to viral infection. Here, using genome-wide short interfering RNA screens, we find that the endosomal protein sorting nexin 5 (SNX5)3,4 is essential for virus-induced, but not for basal, stress- or endosome-induced, autophagy. We show that SNX5 deletion increases cellular susceptibility to viral infection in vitro, and that Snx5 knockout in mice enhances lethality after infection with several human viruses. Mechanistically, SNX5 interacts with beclin 1 and ATG14-containing class III phosphatidylinositol-3-kinase (PI3KC3) complex 1 (PI3KC3-C1), increases the lipid kinase activity of purified PI3KC3-C1, and is required for endosomal generation of phosphatidylinositol-3-phosphate (PtdIns(3)P) and recruitment of the PtdIns(3)P-binding protein WIPI2 to virion-containing endosomes. These findings identify a context- and organelle-specific mechanism-SNX5-dependent PI3KC3-C1 activation at endosomes-for initiation of autophagy during viral infection.
- Published
- 2020
43. Anterograde transneuronal tracing and genetic control with engineered yellow fever vaccine YFV-17D
- Author
-
Rachel Arano, Da-Ting Lin, John W. Schoggins, Yerim Kim, So Jung Oh, Heankel Cantu Oliveros, Todd F. Roberts, Jun Guo, Elizabeth Li, Jennifer L. Eitson, Yi Luo, Wenqin Du, Yuh-Tarng Chen, Wei Xu, and Ying Li
- Subjects
Mutant ,Yellow fever vaccine ,Neuronal toxicity ,Biology ,Antibodies, Viral ,Vaccines, Attenuated ,Biochemistry ,Mice ,Open Reading Frames ,Vaccine Development ,Yellow Fever ,medicine ,Animals ,Humans ,Transneuronal tracing ,Molecular Biology ,Gene ,Fluorescent Dyes ,Neurons ,Attenuated vaccine ,Effector ,Yellow Fever Vaccine ,Brain ,Cell Biology ,Dependovirus ,Cell biology ,Electrophysiology ,HEK293 Cells ,Mutation ,Axoplasmic transport ,Biotechnology ,medicine.drug - Abstract
Transneuronal viruses are powerful tools for tracing neuronal circuits or delivering genes to specific neurons in the brain. While there are multiple retrograde viruses, few anterograde viruses are available. Further, available anterograde viruses often have limitations such as retrograde transport, high neuronal toxicity or weak signals. We developed an anterograde viral system based on a live attenuated vaccine for yellow fever-YFV-17D. Replication- or packaging-deficient mutants of YFV-17D can be reconstituted in the brain, leading to efficient synapse-specific and anterograde-only transneuronal spreading, which can be controlled to achieve either monosynaptic or polysynaptic tracing. Moreover, inducible transient replication of YFV-17D mutant is sufficient to induce permanent transneuronal genetic modifications without causing neuronal toxicity. The engineered YFV-17D systems can be used to express fluorescent markers, sensors or effectors in downstream neurons, thus providing versatile tools for mapping and functionally controlling neuronal circuits.
- Published
- 2020
44. TRIM7 inhibits enterovirus replication and promotes emergence of a viral variant with increased pathogenicity
- Author
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John M. Shelton, Wenchun Fan, Bret M. Evers, Milo M. Lin, Ilona K. Gaszek, Katrina B. Mar, Levent Sari, Qiang Cheng, Mary Wight-Carter, Daniel J. Siegwart, and John W. Schoggins
- Subjects
Proteasome Endopeptidase Complex ,Viral pathogenesis ,viruses ,Ubiquitin-Protein Ligases ,Coxsackievirus ,Virus Replication ,General Biochemistry, Genetics and Molecular Biology ,Virus ,Article ,Cell Line ,Tripartite Motif Proteins ,03 medical and health sciences ,Viral Proteins ,0302 clinical medicine ,Interferon ,medicine ,Animals ,Humans ,030304 developmental biology ,Enterovirus ,Adenosine Triphosphatases ,Inflammation ,0303 health sciences ,biology ,Effector ,Ubiquitin ,biology.organism_classification ,Virology ,Ubiquitin ligase ,Mice, Inbred C57BL ,Viral replication ,Viral evolution ,Mutation ,Proteolysis ,biology.protein ,RNA, Viral ,Female ,030217 neurology & neurosurgery ,medicine.drug ,Protein Binding - Abstract
To control viral infection, vertebrates rely on both inducible interferon responses and less well characterized cell intrinsic responses comprised of “at the ready” antiviral effector proteins. Here, we show that E3 ubiquitin ligase TRIM7 is a cell intrinsic antiviral effector that restricts multiple human enteroviruses by targeting viral 2BC, a membrane remodeling protein, for ubiquitination and proteasome-dependent degradation. Selective pressure exerted by TRIM7 results in emergence of a TRIM7-resistant coxsackievirus with a single point mutation in the viral 2C ATPase/helicase. In cultured cells, the mutation helps the virus evade TRIM7, but impairs optimal viral replication, and this correlates with a hyperactive and structurally plastic 2C ATPase. Unexpectedly, the TRIM7-resistant virus has a replication advantage in mice and causes lethal pancreatitis. These findings reveal a unique mechanism for targeting enterovirus replication and provide molecular insight into the benefits and trade-offs of viral evolution imposed by a host restriction factor.
- Published
- 2020
45. Identification of Atovaquone, Ouabain and Mebendazole as FDA Approved Drugs Tar-geting SARS-CoV-2 (Version 4)
- Author
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Ian N. Boys, Mahmoud S. Ahmed, Wenchun Fan, Maikke B. Ohlson, Matthew B. McDougal, Hesham A. Sadek, Jennifer L. Eitson, John W. Schoggins, Ping Wang, and Ayman B. Farag
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,Drug ,Protease ,Chemistry ,medicine.medical_treatment ,media_common.quotation_subject ,Mebendazole ,Pharmacology ,medicine.disease_cause ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Enzyme ,Viral replication ,Docking (molecular) ,030220 oncology & carcinogenesis ,medicine ,Atovaquone ,medicine.drug ,Coronavirus ,media_common - Abstract
The newly emerged coronavirus, SARS-CoV-2, and the resulting COVID-19 disease, has spread swiftly across the globe since its initial detection in December 2019. Given the heavy toll of this pandemic, therapeutic options for treatment are urgently needed. Here, we adopted a repositioning approach using in-silico molecular modeling to screen FDA-approved drugs with established safety profiles for potential inhibitory effects against SARS-CoV-2. We used structure-based drug design to screen more than 2000 FDA approved drugs against SARS-CoV-2 main protease enzyme (Mpro) substrate-binding pocket, focusing on two potential sites (central and terminal sites) to identify hits based on their binding energies, binding modes, interacting amino acids, and therapeutic indications. We additionally screened the top hits from both sites for potential covalent binding via nucleophilic thiol attack of Cys 145. High-scoring candidates were then screened for antiviral activity against infectious SARS-CoV-2 in a cell-based viral replication assay, and counterscreened for toxicity. Atovaquone, Mebendazole, and Ouabain exhibited antiviral efficacy with IC50s well within their respective therapeutic plasma concentrations (low nanomolar to low micromolar range), and limited toxic effects. Notably, all three were predicted in docking studies to covalently bind SARS-CoV-2 Mpro, underscoring the utility of this in-silico approach for identifying putative antivirals for repurposing. These results do not confirm efficacy in animal models or in humans, but rather serve as a starting point for testing the antiviral potential of select FDA-approved drugs, either individually or in combination.
- Published
- 2020
46. A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating
- Author
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Brett D. Lindenbach, John W. Schoggins, Shuo Zhang, Harish N. Ramanathan, Jinhong Chang, Priscilla L. Yang, Katrina B. Mar, Alexander Ploss, and Florian Douam
- Subjects
Valosin-containing protein ,viruses ,nucleocapsid ,lcsh:A ,Genome, Viral ,Dengue virus ,Kidney ,Virus Replication ,medicine.disease_cause ,Microbiology ,Virus ,Cell Line ,Host-Microbe Biology ,Mice ,flavivirus ,Genes, Reporter ,Valosin Containing Protein ,Viral entry ,Cricetinae ,Virus Uncoating ,Virology ,ubiquitin ,medicine ,Animals ,Humans ,biology ,VCP/p97 ,RNA ,Virus Internalization ,biology.organism_classification ,QR1-502 ,Mice, Inbred C57BL ,Flavivirus ,HEK293 Cells ,Capsid ,biology.protein ,Female ,viral entry ,uncoating ,lcsh:General Works ,Yellow fever virus ,RNA transfection ,HeLa Cells ,Research Article - Abstract
Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97., While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry.
- Published
- 2020
47. LY6E impairs coronavirus fusion and confers immune control of viral disease
- Author
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Gert Zimmer, Stephanie Pfaender, Daniel Todt, Wenchun Fan, Tom Gallagher, Eleftherios Michailidis, Hans Heinrich Hoffmann, Katrina B. Mar, Annika Kratzel, Philip V'kovski, Nadine Ebert, Matthew B. McDougal, Ronald Dijkman, Natasha W. Hanners, Mohsan Saeed, Volker Thiel, Jenna N. Kelly, Mary Wight-Carter, Charles M. Rice, Hannah Kleine-Weber, Dagny Hirt, Ian N. Boys, Hanspeter Stalder, Eike Steinmann, Markus Hoffmann, John W. Schoggins, and Stefan Pöhlmann
- Subjects
Male ,viruses ,Viral pathogenesis ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Pathogenesis ,Mice ,0302 clinical medicine ,Coronavirus ,Mice, Knockout ,0303 health sciences ,virus diseases ,respiratory system ,3. Good health ,Haematopoiesis ,Severe acute respiratory syndrome-related coronavirus ,030220 oncology & carcinogenesis ,Antigens, Surface ,Knockout mouse ,Middle East Respiratory Syndrome Coronavirus ,Female ,Angiotensin-Converting Enzyme 2 ,Viral disease ,Coronavirus Infections ,Viral load ,Microbiology (medical) ,Middle East respiratory syndrome coronavirus ,Pneumonia, Viral ,Immunology ,Peptidyl-Dipeptidase A ,Biology ,GPI-Linked Proteins ,Microbiology ,Article ,Betacoronavirus ,03 medical and health sciences ,Immune system ,Antigen ,Genetics ,medicine ,Animals ,Pandemics ,030304 developmental biology ,Innate immune system ,SARS-CoV-2 ,COVID-19 ,Cell Biology ,Virus Internalization ,biochemical phenomena, metabolism, and nutrition ,medicine.disease ,Virology ,respiratory tract diseases ,Mice, Inbred C57BL ,Middle East respiratory syndrome - Abstract
Zoonotic coronaviruses (CoVs) are significant threats to global health, as exemplified by the recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1. Host immune responses to CoV are complex and regulated in part through antiviral interferons. However, the interferon-stimulated gene products that inhibit CoV are not well characterized2. Here, we show that interferon-inducible lymphocyte antigen 6 complex, locus E (LY6E) potently restricts cellular infection by multiple CoVs, including SARS-CoV, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in hematopoietic cells were highly susceptible to murine CoV infection. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic and splenic immune cells and reduction in global antiviral gene pathways. Accordingly, we found that Ly6e directly protects primary B cells and dendritic cells from murine CoV infection. Our results demonstrate that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo, knowledge that could help inform strategies to combat infection by emerging CoV.
- Published
- 2020
48. The immune vulnerability landscape of the 2019 Novel Coronavirus, SARS-CoV-2
- Author
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Yunguan Wang, Lenny Moise, He Zhang, Yang Xie, Xue Xiao, Xiaowei Zhan, James Zhu, John W. Schoggins, Jiwoong Kim, Shuang Jiang, Lin Xu, Tao Wang, Ran Chen, Danni Luo, Guanghua Xiao, Andres H. Gutierrz, and Anne S. De Groot
- Subjects
B cell ,SARS-CoV-2 ,viruses ,T cell ,Immunogenicity ,virus diseases ,immunogenicity ,biochemical phenomena, metabolism, and nutrition ,Biology ,Virology ,Article ,Virus ,Epitope ,respiratory tract diseases ,medicine.anatomical_structure ,Immune system ,Immunity ,medicine ,mutation ,skin and connective tissue diseases ,CD8 - Abstract
The outbreak of the 2019 Novel Coronavirus (SARS-CoV-2) rapidly spread from Wuhan, China to more than 150 countries, areas, or territories, causing staggering numbers of infections and deaths. In this study, bioinformatics analyses were performed on 5,568 complete genomes of SARS-CoV-2 virus to predict the T cell and B cell immunogenic epitopes of all viral proteins, which formed a systematic immune vulnerability landscape of SARS-CoV-2. The immune vulnerability and genetic variation profiles of SARS-CoV were compared with those of SARS-CoV and MERS-CoV. In addition, a web portal was developed to broadly share the data and results as a resource for the research community. Using this resource, we showed that genetic variations in SARS-CoV-2 are associated with loss of B cell immunogenicity, an increase in CD4+ T cell immunogenicity, and a minimum loss in CD8+ T cell immunogenicity, indicating the existence of a curious correlation between SARS-CoV-2 genetic evolutions and the immunity pressure from the host. Overall, we present an immunological resource for SARS-CoV-2 that could promote both therapeutic/vaccine development and mechanistic research.
- Published
- 2020
49. Oxysterols provide innate immunity to bacterial infection by mobilizing cell surface accessible cholesterol
- Author
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John W. Schoggins, Bonne M. Thompson, Katrina B. Mar, Jeffrey G. McDonald, Kristen A. Johnson, Neal M. Alto, Sofya S. Perelman, Shreya Endapally, Arun Radhakrishnan, Li shu Zhang, and Michael E. Abrams
- Subjects
Microbiology (medical) ,Models, Molecular ,Oxysterol ,Listeria ,Immunology ,Sterol O-acyltransferase ,Molecular Conformation ,Applied Microbiology and Biotechnology ,Microbiology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Structure-Activity Relationship ,Immunity ,Genetics ,medicine ,Humans ,030304 developmental biology ,0303 health sciences ,Innate immune system ,Bacteria ,Molecular Structure ,030306 microbiology ,Chemistry ,Cholesterol ,Cell Membrane ,Inflammasome ,Epithelial Cells ,Cell Biology ,Bacterial Infections ,Oxysterols ,Immunity, Innate ,Cell biology ,Cytokines ,lipids (amino acids, peptides, and proteins) ,Interferons ,Shigella ,Sphingomyelin ,Cholesterol storage ,medicine.drug ,Sterol O-Acyltransferase - Abstract
Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene that converts cholesterol to the oxysterol 25-hydroxycholesterol (25HC). Circulating 25HC modulates essential immunological processes including antiviral immunity, inflammasome activation and antibody class switching; and dysregulation of CH25H may contribute to chronic inflammatory disease and cancer. Although 25HC is a potent regulator of cholesterol storage, uptake, efflux and biosynthesis, how these metabolic activities reprogram the immunological state of target cells remains poorly understood. Here, we used recently designed toxin-based biosensors that discriminate between distinct pools of plasma membrane cholesterol to elucidate how 25HC prevents Listeria monocytogenes from traversing the plasma membrane of infected host cells. The 25HC-mediated activation of acyl-CoA:cholesterol acyltransferase (ACAT) triggered rapid internalization of a biochemically defined fraction of cholesterol, termed 'accessible' cholesterol, from the plasma membrane while having little effect on cholesterol in complexes with sphingomyelin. We show that evolutionarily distinct bacterial species, L. monocytogenes and Shigella flexneri, exploit the accessible pool of cholesterol for infection and that acute mobilization of this pool by oxysterols confers immunity to these pathogens. The significance of this signal-mediated membrane remodelling pathway probably extends beyond host defence systems, as several other biologically active oxysterols also mobilize accessible cholesterol through an ACAT-dependent mechanism.
- Published
- 2020
50. A CRISPR screen identifies IFI6 as an ER-resident interferon effector that blocks flavivirus replication
- Author
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Katrina B. Mar, Jennifer L. Eitson, Ashwani Kumar, John W. Schoggins, Ian N. Boys, Genevieve Konopka, Connor Douglas, Matthew B. McDougal, Pamela C. De La Cruz-Rivera, Chao Xing, R. Blake Richardson, and Maikke B. Ohlson
- Subjects
0301 basic medicine ,Microbiology (medical) ,viruses ,Immunology ,Dengue virus ,medicine.disease_cause ,Endoplasmic Reticulum ,Virus Replication ,Applied Microbiology and Biotechnology ,Microbiology ,Antiviral Agents ,Virus ,Article ,Mitochondrial Proteins ,03 medical and health sciences ,Flaviviridae ,Gene Knockout Techniques ,Species Specificity ,Interferon ,Yellow Fever ,medicine ,Genetics ,Animals ,Humans ,Clustered Regularly Interspaced Short Palindromic Repeats ,Endoplasmic Reticulum Chaperone BiP ,Heat-Shock Proteins ,biology ,Effector ,Endoplasmic reticulum ,Interferon-alpha ,Cell Biology ,biology.organism_classification ,3. Good health ,Cell biology ,Flavivirus ,030104 developmental biology ,Viral replication ,Yellow fever virus ,medicine.drug ,Genome-Wide Association Study ,Protein Binding - Abstract
The endoplasmic reticulum (ER) is an architecturally diverse organelle that serves as a membrane source for the replication of multiple viruses. Flaviviruses, including yellow fever virus, West Nile virus, dengue virus and Zika virus, induce unique single-membrane ER invaginations that house the viral replication machinery1. Whether this virus-induced ER remodelling is vulnerable to antiviral pathways is unknown. Here, we show that flavivirus replication at the ER is targeted by the interferon (IFN) response. Through genome-scale CRISPR screening, we uncovered an antiviral mechanism mediated by a functional gene pairing between IFI6 (encoding IFN-α-inducible protein 6), an IFN-stimulated gene cloned over 30 years ago2, and HSPA5, which encodes the ER-resident heat shock protein 70 chaperone BiP. We reveal that IFI6 is an ER-localized integral membrane effector that is stabilized through interactions with BiP. Mechanistically, IFI6 prophylactically protects uninfected cells by preventing the formation of virus-induced ER membrane invaginations. Notably, IFI6 has little effect on other mammalian RNA viruses, including the related Flaviviridae family member hepatitis C virus, which replicates in double-membrane vesicles that protrude outwards from the ER. These findings support a model in which the IFN response is armed with a membrane-targeted effector that discriminately blocks the establishment of virus-specific ER microenvironments that are required for replication.
- Published
- 2018
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