Influenza A virus infection of HEK293 cells

Influenza A virus (IAV) is a segmented negative-sense RNA virus and is the cause of major global epidemics and pandemics. TRIM25 E3 ubiquitin ligase is a new RNA-binding protein. We present new evidence that TRIM25 restricts IAV by directly binding to and destabilising its mRNAs. Finally, we show that direct tethering of TRIM25 to RNA is sufficient to downregulate the targeted RNA. In summary, our results uncover a novel mechanism that TRIM25 uses to inhibit IAV infection and regulate RNA metabolism. The wild type (PRwt) and NS1 RNA-binding mutant R38A/K41A (PR8mut) of the IAV H1N1 A/Puerto Rico/8/1934 strain were generated by reverse genetics using pDUAL plasmids encoding all 8 segments of the viral genome and were used for infections. HEK293 TRIM25-knockout cells (HEK-KO) with integrated T7-tagged codon-optimised wild-type TRIM25 (HEK-WT) or TRIM25dRBD (lacking RNA-binding residues 470-508 located in the PRY-SPRY domain; HEK-RBD) were infected with the virus. RNA was extracted from HEK293 cells and subjected to RNA-seq and sequencing of TRIM25-associated RNAs (CLIP-seq). CLIP-seq protocol details: The protocol was adapted from Moore et al., 2014. Two P100 dishes per sample with HEK-WT, HEK-RBD and HEK-KO cells were infected with the WT and NS1 mutant of IAV for 1 or 6 hours at an MOI of 5. After UV crosslinking, cells were lysed with passive lysis buffer. Anti-T7 antibody was coupled to Dynabeads. Following immunoprecipitation, the RNA was dephosphorylated, the 3-prime linker (NAGATCGGAAGAGCACACGTCTG) was ligated and radiolabeled and the 5-prime linker (HEK-RBD: NNNCACTAGCN, HEK-WT: NNNGTGAGCN, HEK-KO: NNNAGAGCN) was ligated on the beads. The RNA was reverse transcribed, amplified by PCR using LA Takara Taq, and subjected to single-end stranded sequencing with 50 nt reads. Raw sequence data was de-multiplexed. RNA-seq protocol details: HEK-WT and HEK-RBD cells were infected with the NS1 IAV mutant for 6 hours at an MOI of 5. Total RNA from cells was extracted with Trizol. After rRNA depletion (NEBNext rRNA Depletion Kit) and stranded RNA sequencing library preparation (NEBNext Ultra II Directional RNA Library Prep Kit), samples (n=3) were subjected to paired-end stranded sequencing with 150 nt reads. Raw sequence data was de-multiplexed. Overall design: an examination of viral RNA with RNA-seq and CLIP-seq analyses.