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Article ; Online: Clip for studying protein-RNA interactions that regulate virus replication.

Shema Mugisha, Christian / Tenneti, Kasyap / Kutluay, Sebla B

2019 Volume 183, Page(s) 84–92

Abstract: Viral and cellular RNA-binding proteins regulate numerous key steps in the replication of diverse virus genera. Viruses efficiently co-opt the host cell machinery for purposes such as transcription, splicing and subcellular localization of viral genomes. ...

Abstract	Viral and cellular RNA-binding proteins regulate numerous key steps in the replication of diverse virus genera. Viruses efficiently co-opt the host cell machinery for purposes such as transcription, splicing and subcellular localization of viral genomes. Though viral RNAs often need to resemble cellular RNAs to effectively utilize the cellular machinery, they still retain unique sequence and structural features for recognition by viral proteins for processes such as RNA polymerization, RNA export and selective packaging into virus particles. While beneficial for virus replication, distinct features of viral nucleic acids can also be recognized as foreign by several host defense proteins. Development of the crosslinking immunoprecipitation coupled with sequencing (CLIP) approach has allowed the study of viral and cellular RNA binding proteins that regulate critical aspects of viral replication in unprecedented detail. By combining immunoprecipitation of covalently crosslinked protein-RNA complexes with high-throughput sequencing, CLIP provides a global account of RNA sequences bound by RNA-binding proteins of interest in physiological settings and at near-nucleotide resolution. Here, we describe the step-by-step application of the CLIP methodology within the context of two cellular splicing regulatory proteins, hnRNP A1 and hnRNP H1 that regulate HIV-1 splicing. In principle, this versatile protocol can be applied to many other viral and cellular RNA-binding proteins.
MeSH term(s)	Chromatin Immunoprecipitation Sequencing/methods ; HEK293 Cells ; HIV-1/genetics ; Heterogeneous Nuclear Ribonucleoprotein A1/genetics ; Heterogeneous Nuclear Ribonucleoprotein A1/metabolism ; Heterogeneous-Nuclear Ribonucleoprotein Group F-H/genetics ; Heterogeneous-Nuclear Ribonucleoprotein Group F-H/metabolism ; Humans ; RNA Splicing ; RNA, Viral/genetics ; RNA, Viral/metabolism ; Virus Replication
Chemical Substances	Heterogeneous Nuclear Ribonucleoprotein A1 ; Heterogeneous-Nuclear Ribonucleoprotein Group F-H ; RNA, Viral
Language	English
Publishing date	2019-11-22
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Review
ZDB-ID	1066584-5
ISSN	1095-9130 ; 1046-2023
ISSN (online)	1095-9130
ISSN	1046-2023
DOI	10.1016/j.ymeth.2019.11.011
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Emergence of Compensatory Mutations Reveals the Importance of Electrostatic Interactions between HIV-1 Integrase and Genomic RNA.

Shema Mugisha, Christian / Dinh, Tung / Kumar, Abhishek / Tenneti, Kasyap / Eschbach, Jenna E / Davis, Keanu / Gifford, Robert / Kvaratskhelia, Mamuka / Kutluay, Sebla B

mBio

2022 Volume 13, Issue 5, Page(s) e0043122

Abstract: HIV-1 integrase (IN) has a noncatalytic function in virion maturation through its binding to the viral RNA genome (gRNA). Class II IN substitutions inhibit IN-gRNA binding and result in the formation of virions with aberrant morphologies marked by ... ...

Abstract	HIV-1 integrase (IN) has a noncatalytic function in virion maturation through its binding to the viral RNA genome (gRNA). Class II IN substitutions inhibit IN-gRNA binding and result in the formation of virions with aberrant morphologies marked by mislocalization of the gRNA between the capsid lattice and the lipid envelope. These viruses are noninfectious due to a block at an early reverse transcription stage in target cells. HIV-1 IN utilizes basic residues within its C-terminal domain (CTD) to bind to the gRNA; however, the molecular nature of how these residues mediate gRNA binding and whether other regions of IN are involved remain unknown. To address this, we have isolated compensatory substitutions in the background of a class II IN mutant virus bearing R269A/K273A substitutions within the IN-CTD. We found that the nearby D256N and D270N compensatory substitutions restored the ability of IN to bind gRNA and led to the formation of mature infectious virions. Reinstating the local positive charge of the IN-CTD through individual D256R, D256K, D278R, and D279R substitutions was sufficient to specifically restore IN-gRNA binding and reverse transcription for the IN R269A/K273A as well as the IN R262A/R263A class II mutants. Structural modeling suggested that compensatory substitutions in the D256 residue created an additional interaction interface for gRNA binding, whereas other substitutions acted locally within the unstructured C-terminal tail of IN. Taken together, our findings highlight the essential role of CTD in gRNA binding and reveal the importance of pliable electrostatic interactions between the IN-CTD and the gRNA.
MeSH term(s)	RNA, Viral/genetics ; RNA, Viral/metabolism ; Virus Assembly/genetics ; Static Electricity ; Aspartic Acid/metabolism ; HIV-1/physiology ; Virion/genetics ; Virion/metabolism ; Mutation ; Genomics ; Lipids
Chemical Substances	p31 integrase protein, Human immunodeficiency virus 1 (YY6481J2FF) ; RNA, Viral ; Aspartic Acid (30KYC7MIAI) ; Lipids
Language	English
Publishing date	2022-08-17
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2557172-2
ISSN	2150-7511 ; 2161-2129
ISSN (online)	2150-7511
ISSN	2161-2129
DOI	10.1128/mbio.00431-22
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Clip for studying protein-RNA interactions that regulate virus replication

Shema Mugisha, Christian / Tenneti, Kasyap / Kutluay, Sebla B

Methods. 2019 Nov. 19,

2019

Abstract	Viral and cellular RNA-binding proteins regulate numerous key steps in the replication of diverse virus genera. Viruses efficiently co-opt the host cell machinery for purposes such as transcription, splicing and subcellular localization of viral genomes. Though viral RNAs often need to resemble cellular RNAs to effectively utilize the cellular machinery, they still retain unique sequence and structural features for recognition by viral proteins for processes such as RNA polymerization, RNA export and selective packaging into virus particles. While beneficial for virus replication, distinct features of viral nucleic acids can also be recognized as foreign by several host defense proteins. Development of the crosslinking immunoprecipitation coupled with sequencing (CLIP) approach has allowed the study of viral and cellular RNA binding proteins that regulate critical aspects of viral replication in unprecedented detail. By combining immunoprecipitation of covalently crosslinked protein-RNA complexes with high-throughput sequencing, CLIP provides a global account of RNA sequences bound by RNA-binding proteins of interest in physiological settings and at near-nucleotide resolution. Here, we describe the step-by-step application of the CLIP methodology within the context of two cellular splicing regulatory proteins, hnRNP A1 and hnRNP H1 that regulate HIV-1 splicing. In principle, this versatile protocol can be applied to many other viral and cellular RNA-binding proteins.
Keywords	Human immunodeficiency virus 1 ; RNA ; RNA transport ; RNA-binding proteins ; chemical bonding ; crosslinking ; genome ; high-throughput nucleotide sequencing ; nucleotide sequences ; packaging ; polymerization ; precipitin tests ; regulatory proteins ; transcription (genetics) ; viral proteins ; virion ; virus replication ; viruses
Language	English
Dates of publication	2019-1119
Publishing place	Elsevier Inc.
Document type	Article
Note	Pre-press version
ZDB-ID	1066584-5
ISSN	1095-9130 ; 1046-2023
ISSN (online)	1095-9130
ISSN	1046-2023
DOI	10.1016/j.ymeth.2019.11.011
Database	NAL-Catalogue (AGRICOLA)

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Article: A facile Q-RT-PCR assay for monitoring SARS-CoV-2 growth in cell culture.

Mugisha, Christian Shema / Vuong, Hung R / Puray-Chavez, Maritza / Kutluay, Sebla B

bioRxiv : the preprint server for biology

2020

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the ongoing COVID-19 pandemic, has infected millions within just a few months and is continuing to spread around the globe causing immense respiratory disease and ... ...

Abstract	Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the ongoing COVID-19 pandemic, has infected millions within just a few months and is continuing to spread around the globe causing immense respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here we developed a facile Q-RT-PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 replication kinetics from a small amount of cell culture supernatants. Using this assay, we screened the activities of a number of entry, SARS-CoV-2- and HIV-1-specific inhibitors in a proof of concept study. In line with previous studies which has shown that processing of the viral Spike protein by cellular proteases and endosomal fusion are required for entry, we found that E64D and apilimod potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that macropinocytosis inhibitor EIPA similarly decreased viral RNA in supernatants suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (an NNRTI), amprenavir (a protease inhibitor), and ALLINI-2 (an allosteric integrase inhibitor) modestly inhibited SARS-CoV-2 replication, albeit the IC
Keywords	covid19
Language	English
Publishing date	2020-06-28
Publishing country	United States
Document type	Preprint
DOI	10.1101/2020.06.26.174698
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: A facile Q-RT-PCR assay for monitoring SARS-CoV-2 growth in cell culture

Shema Mugisha, Christian / Vuong, Hung R. / Puray-Chavez, Maritza / Kutluay, Sebla Bulent

bioRxiv

Abstract	Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the ongoing COVID-19 pandemic, has infected millions within just a few months and is continuing to spread around the globe causing immense respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here we developed a facile Q-RT-PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 replication kinetics from a small amount of cell culture supernatants. Using this assay, we screened the activities of a number of entry, SARS-CoV-2- and HIV-1-specific inhibitors in a proof of concept study. In line with previous studies which has shown that processing of the viral Spike protein by cellular proteases and endosomal fusion are required for entry, we found that E64D and apilimod potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that macropinocytosis inhibitor EIPA similarly decreased viral RNA in supernatants suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (an NNRTI), amprenavir (a protease inhibitor), and ALLINI-2 (an allosteric integrase inhibitor) modestly inhibited SARS-CoV-2 replication, albeit the IC<sub>50</sub> values were much higher than that required for HIV-1. Taken together, this facile assay will undoubtedly expedite basic SARS-CoV-2 research, be amenable to mid-throughput screens to identify chemical inhibitors of SARS-CoV-2, and be applicable to a broad number of RNA and DNA viruses.
Keywords	covid19
Language	English
Publishing date	2020-06-28
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2020.06.26.174698
Database	COVID19

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Article ; Online: A Simplified Quantitative Real-Time PCR Assay for Monitoring SARS-CoV-2 Growth in Cell Culture.

Shema Mugisha, Christian / Vuong, Hung R / Puray-Chavez, Maritza / Bailey, Adam L / Fox, Julie M / Chen, Rita E / Wessel, Alex W / Scott, Jason M / Harastani, Houda H / Boon, Adrianus C M / Shin, Haina / Kutluay, Sebla B

mSphere

2020 Volume 5, Issue 5

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 ... ...

Abstract	Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth
MeSH term(s)	Antiviral Agents/pharmacology ; Betacoronavirus/drug effects ; Betacoronavirus/genetics ; Betacoronavirus/growth & development ; Betacoronavirus/pathogenicity ; COVID-19 ; Cell Culture Techniques/methods ; Coronavirus Infections/virology ; Pandemics ; Pneumonia, Viral/virology ; RNA, Viral/analysis ; RNA, Viral/isolation & purification ; Real-Time Polymerase Chain Reaction/methods ; SARS-CoV-2 ; Virus Replication/drug effects
Chemical Substances	Antiviral Agents ; RNA, Viral
Keywords	covid19
Language	English
Publishing date	2020-09-02
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	2379-5042
ISSN (online)	2379-5042
DOI	10.1128/mSphere.00658-20
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: CARD8 is an inflammasome sensor for HIV-1 protease activity.

Wang, Qiankun / Gao, Hongbo / Clark, Kolin M / Mugisha, Christian Shema / Davis, Keanu / Tang, Jack P / Harlan, Gray H / DeSelm, Carl J / Presti, Rachel M / Kutluay, Sebla B / Shan, Liang

Science (New York, N.Y.)

2021 Volume 371, Issue 6535

Abstract: HIV-1 has high mutation rates and exists as mutant swarms within the host. Rapid evolution of HIV-1 allows the virus to outpace the host immune system, leading to viral persistence. Approaches to targeting immutable components are needed to clear HIV-1 ... ...

Abstract	HIV-1 has high mutation rates and exists as mutant swarms within the host. Rapid evolution of HIV-1 allows the virus to outpace the host immune system, leading to viral persistence. Approaches to targeting immutable components are needed to clear HIV-1 infection. Here, we report that the caspase recruitment domain-containing protein 8 (CARD8) inflammasome senses HIV-1 protease activity. HIV-1 can evade CARD8 sensing because its protease remains inactive in infected cells before viral budding. Premature intracellular activation of the viral protease triggered CARD8 inflammasome-mediated pyroptosis of HIV-1-infected cells. This strategy led to the clearance of latent HIV-1 in patient CD4
MeSH term(s)	Alkynes/pharmacology ; Anti-HIV Agents/pharmacology ; Benzoxazines/pharmacology ; CARD Signaling Adaptor Proteins/chemistry ; CARD Signaling Adaptor Proteins/metabolism ; CD4-Positive T-Lymphocytes/physiology ; CD4-Positive T-Lymphocytes/virology ; Caspase 1/metabolism ; Cyclopropanes/pharmacology ; Enzyme Activation ; HIV Infections/drug therapy ; HIV Infections/virology ; HIV Protease/metabolism ; HIV-1/drug effects ; HIV-1/physiology ; Humans ; Inflammasomes/metabolism ; Macrophages/physiology ; Macrophages/virology ; Neoplasm Proteins/chemistry ; Neoplasm Proteins/metabolism ; Pyroptosis ; Reverse Transcriptase Inhibitors/pharmacology ; Rilpivirine/pharmacology ; THP-1 Cells ; Virus Latency
Chemical Substances	Alkynes ; Anti-HIV Agents ; Benzoxazines ; CARD Signaling Adaptor Proteins ; CARD8 protein, human ; Cyclopropanes ; Inflammasomes ; Neoplasm Proteins ; Reverse Transcriptase Inhibitors ; Caspase 1 (EC 3.4.22.36) ; HIV Protease (EC 3.4.23.-) ; p16 protease, Human immunodeficiency virus 1 (EC 3.4.23.-) ; Rilpivirine (FI96A8X663) ; efavirenz (JE6H2O27P8)
Language	English
Publishing date	2021-02-04
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	128410-1
ISSN	1095-9203 ; 0036-8075
ISSN (online)	1095-9203
ISSN	0036-8075
DOI	10.1126/science.abe1707
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Article ; Online: Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures.

Zafferani, Martina / Haddad, Christina / Luo, Le / Davila-Calderon, Jesse / Chiu, Liang-Yuan / Mugisha, Christian Shema / Monaghan, Adeline G / Kennedy, Andrew A / Yesselman, Joseph D / Gifford, Robert J / Tai, Andrew W / Kutluay, Sebla B / Li, Mei-Ling / Brewer, Gary / Tolbert, Blanton S / Hargrove, Amanda E

Science advances

2021 Volume 7, Issue 48, Page(s) eabl6096

Abstract: The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, emphasized the urgent need for development of novel antivirals. Small-molecule chemical probes offer both to reveal aspects of virus replication and to serve as leads for ... ...

Abstract	The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, emphasized the urgent need for development of novel antivirals. Small-molecule chemical probes offer both to reveal aspects of virus replication and to serve as leads for antiviral therapeutic development. Here, we report on the identification of amiloride-based small molecules that potently inhibit OC43 and SARS-CoV-2 replication through targeting of conserved structured elements within the viral 5′-end. Nuclear magnetic resonance–based structural studies revealed specific amiloride interactions with stem loops containing bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Amilorides represent the first antiviral small molecules that target RNA structures within the 5′ untranslated regions and proximal region of the CoV genomes. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNA–targeted antivirals.
Language	English
Publishing date	2021-11-26
Publishing country	United States
Document type	Journal Article
ZDB-ID	2810933-8
ISSN	2375-2548 ; 2375-2548
ISSN (online)	2375-2548
ISSN	2375-2548
DOI	10.1126/sciadv.abl6096
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Book ; Online: A simplified quantitative real-time PCR assay for monitoring SARS-CoV-2 growth in cell culture

Open Access Publications

2020

Abstract	Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth
Keywords	Antiviral Agents ; Betacoronavirus ; Cell Culture Techniques ; Coronavirus Infections ; Pandemics ; Pneumonia ; Viral ; RNA ; Real-Time Polymerase Chain Reaction ; Virus Replication ; covid19
Publishing date	2020-09-02T07:00:00Z
Publisher	Digital Commons@Becker
Publishing country	us
Document type	Book ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell.

Puray-Chavez, Maritza / LaPak, Kyle M / Schrank, Travis P / Elliott, Jennifer L / Bhatt, Dhaval P / Agajanian, Megan J / Jasuja, Ria / Lawson, Dana Q / Davis, Keanu / Rothlauf, Paul W / Jo, Heejoon / Lee, Nakyung / Tenneti, Kasyap / Eschbach, Jenna E / Mugisha, Christian Shema / Vuong, Hung R / Bailey, Adam L / Hayes, D Neil / Whelan, Sean P J /

Horani, Amjad / Brody, Steven L / Goldfarb, Dennis / Major, M Ben / Kutluay, Sebla B

bioRxiv : the preprint server for biology

2021

Abstract: ... ...

Abstract	Established
Language	English
Publishing date	2021-03-01
Publishing country	United States
Document type	Preprint
DOI	10.1101/2021.03.01.433431
Database	MEDical Literature Analysis and Retrieval System OnLINE

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