Functional Analysis of GRSF1 in the Nuclear Export and Translation of Influenza A Virus mRNAs

Influenza A viruses (IAV) utilize host proteins throughout their life cycle to infect and replicate in their hosts. We previously showed that host adaptive mutations in avian IAV PA help recruit host protein G-Rich RNA Sequence Binding Factor 1 (GRSF1) to the nucleoprotein (NP) 5' untranslated region (UTR), leading to the enhanced nuclear export and translation of NP mRNA. In this study, we evaluated the impact of GRSF1 in the viral life cycle. We rescued and characterized a 2009 pH1N1 virus with a mutated GRSF1 binding site in the 5' UTR of NP mRNA. Mutant viral growth was attenuated relative to pH1N1 wild-type (WT) in mammalian cells. We observed a specific reduction in the NP protein production and cytosolic accumulation of NP mRNAs, indicating a critical role of GRSF1 in the nuclear export of IAV NP mRNAs. Further, in vitro-transcribed mutated NP mRNA was translated less efficiently than WT NP mRNA in transfected cells. Together, these findings show that GRSF1 binding is important for both mRNA nuclear export and translation and affects overall IAV growth. Enhanced association of GRSF1 to NP mRNA by PA mutations leads to rapid virus growth, which could be a key process of mammalian host adaptation of IAV.