Study on NS mRNA splicing and nuclear export mediated by N(6)-methyladenosine and NS1 in influenza A virus

Abstract

The NS segment of influenza A virus (IAV) expresses two key proteins: non-structural protein 1 (NS1) and nuclear export protein (NEP). NS1, a non-essential protein, plays a crucial role in various functions during the virus life cycle, including modulation of RNA processing, evasion of antiviral responses, and influencing of host apoptosis, while the timing of NEP expression is vital for regulating virus replication efficiency in infected cells. Among these functions, the investigation into how NS1 controls its own mRNA splicing and nuclear exportation has yielded different findings over the years. Some studies suggest that NS1 exerts a negative self-regulatory effect on its splicing, while others present evidence that NS1 does not regulate its own mRNA splicing. This study aims to explore the molecular mechanisms of NS mRNA splicing and nuclear export during IAV replication. In this study, a NS1-null mutant virus was generated to exclusively transcribe full-length NS mRNA without producing functional NS1 protein (WSN/Ans1). Comparative analysis between WSN/WT and WSN/Ans1 revealed that NS1 negatively affects its own splicing but has a positive impact on NS mRNA nuclear exportation. However, additional host factors are required to carry out these functions effectively. In this study, I uncovered an interaction partner of NS mRNA, YTHDC1, which is an N6-methyladenosine (m6A) reader known to influence RNA splicing. These findings suggest the potential of m6A modification on NS mRNA processing. Methylation stands out as one of the most prevalent RNA modifications in eukaryotic systems and is known to influence viral functions during infections. However, the regulatory role of m6A in IAV remains poorly understood. This study identified an m6A modification site (A385) within the IAV NS segment through specialized MeRIP-qRT-PCR and direct RNA sequencing techniques. Evolutionary analysis revealed the conservation of this site across all human and avian IAV strains. By reducing m6A modification on NS mRNA through the A385C mutation, I uncovered the inhibitory effect of m6A modification on splicing while promoting NS mRNA nuclear export, similar to NS1. Notably, the regulatory role of m6A modification in NS mRNA processing is consistent across various strains in both human and non-human originated IAV. In this process, the nuclear m6A reader YTHDC1 was found to read the m6A site on NS mRNA, then inhibited mRNA splicing by impeding SRSF3 binding to NS mRNA. Furthermore, it was found in this study that the m6A addition on NS mRNA was controlled by the NS1 protein. In conclusion, the findings presented here emphasized that NS1 controls its own mRNA splicing and nuclear export via m6A modification. The conservation of the regulatory m6A site (A385) highlights its significance as a key target for future antiviral interventions.