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Article ; Online: Mammalian Reoviruses: Propagation, Quantification, and Storage.

Coombs, Kevin M

Current protocols

2022 Volume 3, Issue 4, Page(s) e716

Abstract: Mammalian reoviruses are pathogens that cause gastrointestinal and respiratory infections. In humans, the mammalian reoviruses usually cause mild or subclinical disease, and they are ubiquitous, with most people mounting immunity at a young age. ... ...

Abstract	Mammalian reoviruses are pathogens that cause gastrointestinal and respiratory infections. In humans, the mammalian reoviruses usually cause mild or subclinical disease, and they are ubiquitous, with most people mounting immunity at a young age. Reoviruses are prototypic representations of the Reoviridae family, which contains many highly pathogenic viruses. This article describes techniques for culturing mouse fibroblast L929 cell lines, the preferred cell line in which most mammalian reovirus studies take place. In addition, mammalian reovirus propagation, quantification, purification, and storage are described. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Propagation of mammalian reoviruses in cell culture from virus stocks Alternate Protocol 1: Large-scale propagation (and purification) of mammalian reoviruses in cell culture from virus stocks Basic Protocol 2: Quantification of mammalian reoviruses by plaque assay with neutral red staining Alternate Protocol 2: Quantification of mammalian reoviruses by plaque assay with crystal violet staining Basic Protocol 3: Storage of mammalian reoviruses Support Protocol 1: Growth and maintenance of mouse L929 cells Support Protocol 2: Plating L929 cells.
MeSH term(s)	Humans ; Animals ; Mice ; Reoviridae ; Cell Line ; Orthoreovirus ; Orthoreovirus, Mammalian ; Cell Culture Techniques/methods ; Mammals
Language	English
Publishing date	2022-10-25
Publishing country	United States
Document type	Journal Article
ISSN	2691-1299
ISSN (online)	2691-1299
DOI	10.1002/cpz1.716
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Chloride Intracellular Channel Protein 1 (CLIC1) Is a Critical Host Cellular Factor for Influenza A Virus Replication.

Rashid, Mahamud-Ur / Coombs, Kevin M

Viruses

2024 Volume 16, Issue 1

Abstract: 1) Background: Influenza A Virus (IAV) uses host cellular proteins during replication in host cells. IAV infection causes elevated expression of chloride intracellular channel protein 1 (CLIC1) in lung epithelial cells, but the importance of this ... ...

Abstract	(1) Background: Influenza A Virus (IAV) uses host cellular proteins during replication in host cells. IAV infection causes elevated expression of chloride intracellular channel protein 1 (CLIC1) in lung epithelial cells, but the importance of this protein in IAV replication is unknown. (2) In this study, we determined the role of CLIC1 in IAV replication by investigating the effects of CLIC1 knockdown (KD) on IAV viral protein translation, genomic RNA transcription, and host cellular proteome dysregulation. (3) Results: CLIC1 KD in A549 human lung epithelial cells resulted in a significant decrease in progeny supernatant IAV, but virus protein expression was unaffected. However, a significantly larger number of viral RNAs accumulated in CLIC1 KD cells. Treatment with a CLIC1 inhibitor also caused a significant reduction in IAV replication, suggesting that CLIC1 is an important host factor in IAV replication. SomaScan
MeSH term(s)	Humans ; Chloride Channels/genetics ; Influenza A virus/physiology ; Influenza, Human ; RNA, Viral ; Virus Replication
Chemical Substances	Chloride Channels ; CLIC1 protein, human ; RNA, Viral
Language	English
Publishing date	2024-01-16
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v16010129
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Article ; Online: Update on Proteomic approaches to uncovering virus-induced protein alterations and virus -host protein interactions during the progression of viral infection.

Coombs, Kevin M

Expert review of proteomics

2020 Volume 17, Issue 7-8, Page(s) 513–532

Abstract: Introduction: Viruses induce profound changes in the cells they infect. Understanding these perturbations will assist in designing better therapeutics to combat viral infection. System-based proteomic assays now provide unprecedented opportunity to ... ...

Abstract	Introduction: Viruses induce profound changes in the cells they infect. Understanding these perturbations will assist in designing better therapeutics to combat viral infection. System-based proteomic assays now provide unprecedented opportunity to monitor large numbers of cellular proteins. Areas covered: This review will describe various quantitative and functional mass spectrometry-based methods, and complementary non-mass spectrometry-based methods, such as aptamer profiling and proximity extension assays, and examples of how each are used to delineate how viruses affect host cells, identify which viral proteins interact with which cellular proteins, and how these change during the course of a viral infection. PubMed was searched multiple times prior to manuscript submissions and revisions, using virus, viral, proteomics; in combination with each keyword. The most recent examples of published works from each search were then analyzed. Expert opinion: There has been exponential growth in numbers and types of proteomic analyses in recent years. Continued development of reagents that allow increased multiplexing and deeper proteomic probing of the cell, at quantitative and functional levels, enhancements that target more important protein modifications, and improved bioinformatics software tools and pathway prediction algorithms will accelerate this growth and usher in a new era of host proteome understanding.
MeSH term(s)	Chromatography, Liquid ; Computational Biology ; Host-Pathogen Interactions/genetics ; Humans ; Mass Spectrometry ; Proteome/genetics ; Proteomics ; Software ; Viral Proteins/genetics ; Viral Proteins/isolation & purification ; Virus Diseases/genetics ; Virus Diseases/pathology ; Virus Diseases/virology
Chemical Substances	Proteome ; Viral Proteins
Keywords	covid19
Language	English
Publishing date	2020-09-21
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2299100-1
ISSN	1744-8387 ; 1478-9450
ISSN (online)	1744-8387
ISSN	1478-9450
DOI	10.1080/14789450.2020.1821656
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Article: Update on Proteomic approaches to uncovering virus-induced protein alterations and virus -host protein interactions during the progression of viral infection

Coombs, Kevin M

Expert Rev Proteomics

Abstract: INTRODUCTION: Viruses induce profound changes in the cells they infect. Understanding these perturbations will assist in designing better therapeutics to combat viral infection. System-based proteomic assays now provide unprecedented opportunity to ... ...

Abstract	INTRODUCTION: Viruses induce profound changes in the cells they infect. Understanding these perturbations will assist in designing better therapeutics to combat viral infection. System-based proteomic assays now provide unprecedented opportunity to monitor large numbers of cellular proteins. AREAS COVERED: This review will describe various quantitative and functional mass spectrometry-based methods, and complementary non-mass spectrometry-based methods, such as aptamer profiling and proximity extension assays, and examples of how each are used to delineate how viruses affect host cells, identify which viral proteins interact with which cellular proteins, and how these change during the course of a viral infection. PubMed was searched multiple times prior to manuscript submissions and revisions, using virus, viral, proteomics; in combination with each keyword. The most recent examples of published works from each search were then analyzed. EXPERT OPINION: There has been exponential growth in numbers and types of proteomic analyses in recent years. Continued development of reagents that allow increased multiplexing and deeper proteomic probing of the cell, at quantitative and functional levels, enhancements that target more important protein modifications, and improved bioinformatics software tools and pathway prediction algorithms will accelerate this growth and usher in a new era of host proteome understanding.
Keywords	covid19
Publisher	WHO
Document type	Article
Note	WHO #Covidence: #759817
Database	COVID19

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Article ; Online: HLA-A, HSPA5, IGFBP5 and PSMA2 Are Restriction Factors for Zika Virus Growth in Astrocytic Cells.

Sher, Affan A / Lao, Ying Tenny / Coombs, Kevin M

Viruses

2022 Volume 15, Issue 1

Abstract: 1) Background: Zika virus (ZIKV), an arbo-flavivirus, is transmitted ... ...

Abstract	(1) Background: Zika virus (ZIKV), an arbo-flavivirus, is transmitted via
MeSH term(s)	Animals ; Female ; Humans ; Pregnancy ; Astrocytes ; Guillain-Barre Syndrome/epidemiology ; HLA-A Antigens ; Virus Replication ; Zika Virus/physiology ; Zika Virus Infection
Chemical Substances	HLA-A Antigens ; HSPA5 protein, human ; IGFBP5 protein, human ; PSMA2 protein, human
Language	English
Publishing date	2022-12-29
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v15010097
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Article ; Online: Influenza A Virus Uses PSMA2 for Downregulation of the NRF2-Mediated Oxidative Stress Response.

Rashid, Mahamud-Ur / Gao, Ang / Coombs, Kevin M

Journal of virology

2022 Volume 96, Issue 5, Page(s) e0199021

Abstract: Influenza A virus (IAV), an obligatory intracellular parasite, uses host cellular molecules to complete its replication cycle and suppress immune responses. Proteasome subunit alpha type 2 (PSMA2) is a cellular protein highly expressed in IAV-infected ... ...

Abstract	Influenza A virus (IAV), an obligatory intracellular parasite, uses host cellular molecules to complete its replication cycle and suppress immune responses. Proteasome subunit alpha type 2 (PSMA2) is a cellular protein highly expressed in IAV-infected human lung epithelial A549 cells. PSMA2 is part of the 20S proteasome complex that degrades or recycles defective proteins and involves proteolytic modification of many cellular regulatory proteins. However, the role of PSMA2 in IAV replication is not well understood. In this study, PSMA2 knockdown (KD) in A549 cells caused a significant reduction in extracellular progeny IAV, but intracellular viral protein translation and viral RNA transcription were not affected. This indicates that PSMA2 is a critical host factor for IAV maturation. To better understand the interplay between PSMA2 KD and IAV infection at the proteomic level, we used the SomaScan 1.3K version, which measures 1,307 proteins to analyze alterations induced by these treatments. We found seven cellular signaling pathways, including phospholipase C signaling, Pak signaling, and nuclear factor erythroid 2p45-related factor 2 (NRF2)-mediated oxidative stress response signaling, that were inhibited by IAV infection but significantly activated by PSMA2 KD. Further analysis of NRF2-mediated oxidative stress response signaling indicated IAV inhibits accumulation of reactive oxygen species (ROS), but ROS levels significantly increased during IAV infection in PSMA2 KD cells. However, IAV infection caused significantly higher NFR2 nuclear translocation that was inhibited in PSMA2 KD cells. This indicates that PSMA2 is required for NRF2-mediated ROS neutralization and that IAV uses PSMA2 to escape viral clearance via the NRF2-mediated cellular oxidative response.
MeSH term(s)	Down-Regulation ; Host-Pathogen Interactions/genetics ; Humans ; Immune Evasion/genetics ; Influenza A virus/genetics ; Influenza A virus/immunology ; Influenza, Human/immunology ; Influenza, Human/virology ; NF-E2-Related Factor 2/genetics ; Oxidative Stress ; Proteasome Endopeptidase Complex/genetics ; Proteasome Endopeptidase Complex/metabolism ; Proteomics ; Reactive Oxygen Species/metabolism ; Virus Replication/genetics
Chemical Substances	NF-E2-Related Factor 2 ; Reactive Oxygen Species ; Proteasome Endopeptidase Complex (EC 3.4.25.1)
Language	English
Publishing date	2022-01-12
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	80174-4
ISSN	1098-5514 ; 0022-538X
ISSN (online)	1098-5514
ISSN	0022-538X
DOI	10.1128/jvi.01990-21
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Article ; Online: ZIKV Infection Induces DNA Damage Response and Alters the Proteome of Gastrointestinal Cells.

Glover, Kathleen / Coombs, Kevin M

Viruses

2020 Volume 12, Issue 7

Abstract: The zika virus (ZIKV) is a neurotropic virus that causes congenital abnormalities in babies when they are infected in utero. Some studies have reported these congenital abnormalities result from ZIKV attacking neural progenitor cells within the brain ... ...

Abstract	The zika virus (ZIKV) is a neurotropic virus that causes congenital abnormalities in babies when they are infected in utero. Some studies have reported these congenital abnormalities result from ZIKV attacking neural progenitor cells within the brain which differentiate into neurons, oligodendrocytes, and astrocytes. Each of these glial cells play important roles during development of the fetal brain. In addition to ZIKV-induced congenital abnormalities, infected patients experience gastrointestinal complications. There are presently no reports investigating the role of this virus at the proteomic level in gastrointestinal associated cells, so we conducted an in vitro proteomic study of ZIKV-induced changes in Caco-2, a colon-derived human cell line which is known to be permissive to ZIKV infection. We used SomaScan, a new aptamer-based proteomic tool to identify host proteins that are dysregulated during ZIKV infection at 12, 24, and 48 h post-infection. Bioinformatic analyses predicted that dysregulation of differentially-regulated host proteins results in various gastrointestinal diseases. Validation of the clinical relevance of these promising protein targets will add to the existing knowledge of ZIKV biology. These potential proteins may be useful targets towards the development of therapeutic interventions.
MeSH term(s)	Caco-2 Cells/metabolism ; Caco-2 Cells/virology ; DNA Damage ; Gastrointestinal Tract/metabolism ; Gastrointestinal Tract/virology ; Gene Expression Regulation, Viral ; Humans ; Proteome/metabolism ; Zika Virus Infection/metabolism ; Zika Virus Infection/pathology
Chemical Substances	Proteome
Language	English
Publishing date	2020-07-17
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v12070771
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Article ; Online: Zika Virus Infection of Sertoli Cells Alters Protein Expression Involved in Activated Immune and Antiviral Response Pathways, Carbohydrate Metabolism and Cardiovascular Disease.

Rashid, Mahamud-Ur / Lao, Ying / Spicer, Victor / Coombs, Kevin M

Viruses

2022 Volume 14, Issue 2

Abstract: Zika virus (ZIKV), a re-emerging virus, causes congenital brain abnormalities and Guillain-Barré syndrome. It is mainly transmitted by Aedes mosquitoes, but infections are also linked to sexual transmissions. Infectious ZIKV has been isolated, and viral ... ...

Abstract	Zika virus (ZIKV), a re-emerging virus, causes congenital brain abnormalities and Guillain-Barré syndrome. It is mainly transmitted by Aedes mosquitoes, but infections are also linked to sexual transmissions. Infectious ZIKV has been isolated, and viral RNA has been detected in semen over a year after the onset of initial symptoms, but the mode of long-term persistence is not yet understood. ZIKV can proliferate in human Sertoli cells (HSerC) for several weeks in vitro, suggesting that it might be a reservoir for persistent ZIKV infection. This study determined proteomic changes in HSerC during ZIKV infections by TMT-mass spectrometry analysis. Levels of 4416 unique Sertoli cell proteins were significantly altered at 3, 5, and 7 days after ZIKV infection. The significantly altered proteins include enzymes, transcription regulators, transporters, kinases, peptidases, transmembrane receptors, cytokines, ion channels, and growth factors. Many of these proteins are involved in pathways associated with antiviral response, antigen presentation, and immune cell activation. Several immune response pathway proteins were significantly activated during infection, e.g., interferon signaling, T cell receptor signaling, IL-8 signaling, and Th1 signaling. The altered protein levels were linked to predicted activation of immune response in HSerC, which was predicted to suppress ZIKV infection. ZIKV infection also affected the levels of critical regulators of gluconeogenesis and glycolysis pathways such as phosphoglycerate mutase, phosphoglycerate kinase, and enolase. Interestingly, many significantly altered proteins were associated with cardiac hypertrophy, which may induce heart failure in infected patients. In summary, our research contributes to a better understanding of ZIKV replication dynamics and infection in Sertoli cells.
MeSH term(s)	Carbohydrate Metabolism/immunology ; Cardiovascular Diseases/immunology ; Disease Transmission, Infectious ; Humans ; Male ; Protein Processing, Post-Translational ; Proteomics ; RNA, Viral/genetics ; Semen/virology ; Sertoli Cells/immunology ; Sertoli Cells/virology ; Virus Replication ; Zika Virus/isolation & purification ; Zika Virus Infection/immunology ; Zika Virus Infection/transmission
Chemical Substances	RNA, Viral
Language	English
Publishing date	2022-02-11
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v14020377
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Serum-reduced media impacts on cell viability and protein expression in human lung epithelial cells.

Rashid, Mahamud-Ur / Coombs, Kevin M

Journal of cellular physiology

2018 Volume 234, Issue 6, Page(s) 7718–7724

Abstract: Serum starvation is a widely used condition in molecular biology experiments. Opti-MEM is a serum-reduced media used during transfection of genetic molecules into mammalian cells. However, the impact of such media on cell viability and protein synthesis ... ...

Abstract	Serum starvation is a widely used condition in molecular biology experiments. Opti-MEM is a serum-reduced media used during transfection of genetic molecules into mammalian cells. However, the impact of such media on cell viability and protein synthesis is unknown. A549 human lung epithelial cell viability and morphology were adversely affected by growing in Opti-MEM. The cellular protein levels of chloride intracellular channel protein 1, proteasome subunit alpha Type 2, and heat shock 70 kDa protein 5 were dysregulated in A549 cells after growing in serum-reduced media. Small interfering RNA transfection was done in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, and knockdown efficacy was determined compared with Opti-MEM. Similar amounts of knockdown of the target proteins were achieved in DMEM, and cell viability was higher compared with Opti-MEM after transfection. Careful consideration of the impact of Opti-MEM media during the culture or transfection is important for experimental design and results interpretation.
MeSH term(s)	A549 Cells ; Cell Count/methods ; Cell Differentiation/physiology ; Cell Proliferation/physiology ; Cell Survival/physiology ; Cells, Cultured/metabolism ; Culture Media ; Epithelial Cells/cytology ; Humans ; Lung/cytology
Chemical Substances	Culture Media
Language	English
Publishing date	2018-12-04
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	3116-1
ISSN	1097-4652 ; 0021-9541
ISSN (online)	1097-4652
ISSN	0021-9541
DOI	10.1002/jcp.27890
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Article: Zika Virus Infection of Sertoli Cells Alters Protein Expression Involved in Activated Immune and Antiviral Response Pathways, Carbohydrate Metabolism and Cardiovascular Disease

Rashid, Mahamud-ur / Lao, Ying / Spicer, Victor / Coombs, Kevin M.

Viruses. 2022 Feb. 11, v. 14, no. 2

2022

Abstract: Zika virus (ZIKV), a re-emerging virus, causes congenital brain abnormalities and Guillain–Barré syndrome. It is mainly transmitted by Aedes mosquitoes, but infections are also linked to sexual transmissions. Infectious ZIKV has been isolated, and viral ... ...

Abstract	Zika virus (ZIKV), a re-emerging virus, causes congenital brain abnormalities and Guillain–Barré syndrome. It is mainly transmitted by Aedes mosquitoes, but infections are also linked to sexual transmissions. Infectious ZIKV has been isolated, and viral RNA has been detected in semen over a year after the onset of initial symptoms, but the mode of long-term persistence is not yet understood. ZIKV can proliferate in human Sertoli cells (HSerC) for several weeks in vitro, suggesting that it might be a reservoir for persistent ZIKV infection. This study determined proteomic changes in HSerC during ZIKV infections by TMT-mass spectrometry analysis. Levels of 4416 unique Sertoli cell proteins were significantly altered at 3, 5, and 7 days after ZIKV infection. The significantly altered proteins include enzymes, transcription regulators, transporters, kinases, peptidases, transmembrane receptors, cytokines, ion channels, and growth factors. Many of these proteins are involved in pathways associated with antiviral response, antigen presentation, and immune cell activation. Several immune response pathway proteins were significantly activated during infection, e.g., interferon signaling, T cell receptor signaling, IL-8 signaling, and Th1 signaling. The altered protein levels were linked to predicted activation of immune response in HSerC, which was predicted to suppress ZIKV infection. ZIKV infection also affected the levels of critical regulators of gluconeogenesis and glycolysis pathways such as phosphoglycerate mutase, phosphoglycerate kinase, and enolase. Interestingly, many significantly altered proteins were associated with cardiac hypertrophy, which may induce heart failure in infected patients. In summary, our research contributes to a better understanding of ZIKV replication dynamics and infection in Sertoli cells.
Keywords	Aedes ; RNA ; Sertoli cells ; T-lymphocytes ; Zika virus ; antigen presentation ; brain ; gluconeogenesis ; glycolysis ; heart failure ; humans ; hypertrophy ; immune response ; interferons ; interleukin-8 ; peptidases ; phosphoglycerate kinase ; phosphopyruvate hydratase ; protein synthesis ; proteomics ; semen ; spectroscopy ; viruses
Language	English
Dates of publication	2022-0211
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2516098-9
ISSN	1999-4915
ISSN	1999-4915
DOI	10.3390/v14020377
Database	NAL-Catalogue (AGRICOLA)

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