Protocol for the generation of HIV-1 genomic RNA with altered levels of N6-methyladenosine

Stacia Phillips; Alice Baek; Sanggu Kim; Shuliang Chen; Li Wu

doi:10.1016/j.xpro.2022.101616

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Protocol for the generation of HIV-1 genomic RNA with altered levels of N6-methyladenosine

Journal article

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Protocol for the generation of HIV-1 genomic RNA with altered levels of N6-methyladenosine

Stacia Phillips, Alice Baek, Sanggu Kim, Shuliang Chen and Li Wu

STAR protocols, Vol.3(3), p.101616

09/16/2022

DOI: 10.1016/j.xpro.2022.101616

PMCID: PMC9389413

PMID: 35990737

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9389413View

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Abstract

N6-methyladenosine (m6A) modification of human immunodeficiency virus type 1 (HIV-1) RNA plays a critical role in regulating viral replication and evasion of innate immunity. Here, we describe a protocol for the production of HIV-1 with altered m6A levels by manipulating the expression of m6A demethylases in HIV-1 producer cells. RNA from purified virions is analyzed by northern blot and dot blot for m6A levels prior to use in downstream assays to determine the function of m6A modification of viral RNA. For complete details on the use and execution of this protocol, please refer to Chen et al. (2021). [Display omitted] •Procedures to generate m6A eraser knockout cells•Procedures to generate m6A eraser overexpressing cells•Protocols for HIV-1 production using m6A eraser modulated cells•Protocols for analysis of HIV-1 RNA and detecting m6A levels Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. N6-methyladenosine (m6A) modification of human immunodeficiency virus type 1 (HIV-1) RNA plays a critical role in regulating viral replication and evasion of innate immunity. Here, we describe a protocol for the production of HIV-1 with altered m6A levels by manipulating the expression of m6A demethylases in HIV-1 producer cells. RNA from purified virions is analyzed by northern blot and dot blot for m6A levels prior to use in downstream assays to determine the function of m6A modification of viral RNA.

Microbiology

Molecular biology

Details

Title: Subtitle: Protocol for the generation of HIV-1 genomic RNA with altered levels of N6-methyladenosine
Creators: Stacia Phillips - University of Iowa
Alice Baek - The Ohio State University
Sanggu Kim - The Ohio State University
Shuliang Chen - Wuhan University
Li Wu - Department of Microbiology and Immunology, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242, USA
Resource Type: Journal article
Publication Details: STAR protocols, Vol.3(3), p.101616
DOI: 10.1016/j.xpro.2022.101616
PMID: 35990737
PMCID: PMC9389413
NLM abbreviation: STAR Protoc
ISSN: 2666-1667
eISSN: 2666-1667
Publisher: Elsevier Inc
Grant note: DOI: 10.13039/100000002, name: National Institutes of Health, award: AI141495, AI150343, AI159546, AI169659, CA086862-22S1, HG010108, HG010318; DOI: 10.13039/501100001809, name: National Natural Science Foundation of China, award: 82172258
Language: English
Date published: 09/16/2022
Academic Unit: Microbiology and Immunology
Record Identifier: 9984297331902771

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