Journal: Frontiers in Microbiology

Article Title: Pseudorabies virus exploits N 6 -methyladenosine modification to promote viral replication

doi: 10.3389/fmicb.2023.1087484

Figure Lengend Snippet: PRV infection affected m 6 A level and expression of m 6 A regulators in PK15 cells. (A) Total RNA was extracted from PRV-infected and uninfected PK15 cells at different time periods, and the m 6 A level of RNA was quantified by ELISA. (B) PK15 cells were infected with PRV for 12 and 24 h. m 6 A regulators were assessed by immunoblotting analysis. β-actin was used as a loading control. (C) RT-qPCR analysis was used to evaluate the mRNA levels of m 6 A regulators at different times of PRV infection. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: According to the manufacturer′s protocol, the intact mRNA in the sample was captured with Seq-StarTM poly(A) mRNA Isolation Kit (AS-MB-006-01/02, ArrayStar, Rockville, MD, United States), and then the isolated mRNA was chemically fragmented into 300-nucleotide-long fragments by incubation in the fragmentation buffer (10 mM Zn 2+ and 10 mM Tris-HCl, pH 7.0).

Techniques: Infection, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: Pseudorabies virus exploits N 6 -methyladenosine modification to promote viral replication

doi: 10.3389/fmicb.2023.1087484

Figure Lengend Snippet: Schematic representation of m 6 A regulation of PRV replication. Upon viral infection, virions first attach to the host cell surface, subsequently enter the cell, and finally the viral genome is released into the host cell nucleus. In the nucleus, the methyltransferases METTL3/14 co-induce the methylation of multiple viral mRNAs, whereas the demethylases FTO and ALKBH5 regulate the demethylation process. The methylation of viral mRNA promotes its own nuclear export. In the cytoplasm, YTHDF1 and YTHDF3 synergistically promote mRNA stability and translation, and YTHDF3 cooperates with YTHDF2 to promote mRNA degradation. Ultimately, the expression of PRV proteins is promoted by the cooperation of YTHDF1/2/3, and these products are transported back into the nucleus, where they complete the viral nucleocapsid assembly and eventually release more viral particles.

Article Snippet: According to the manufacturer′s protocol, the intact mRNA in the sample was captured with Seq-StarTM poly(A) mRNA Isolation Kit (AS-MB-006-01/02, ArrayStar, Rockville, MD, United States), and then the isolated mRNA was chemically fragmented into 300-nucleotide-long fragments by incubation in the fragmentation buffer (10 mM Zn 2+ and 10 mM Tris-HCl, pH 7.0).

Techniques: Infection, Methylation, Expressing