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

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).

Automated summary

This study describes a method to manipulate the expression of m6A demethylases in HIV-1 producer cells, allowing for the production of HIV-1 with altered m6A levels. The RNA from purified virions is analyzed to determine the m6A levels, which are then used in downstream assays to investigate the function of m6A modification of viral RNA.