Phosphorylation Status of Tyrosine 78 Residue Regulates the Nuclear Export and Ubiquitination of Influenza A Virus Nucleoprotein

Liang Cui,Wenxian Yang,Jing Li,Xiaoyuan Bai,George Fu Gao,Minghui Li,Weinan Zheng,Wenjun Liu,Lei Sun,Wenhui Fan

doi:10.3389/fmicb.2019.01816

Abstract

Phosphorylation and dephosphorylation of nucleoprotein (NP) play significant roles in the life cycle of influenza A virus (IAV), and the biological functions of each phosphorylation site on NP are not exactly the same in controlling viral replication. Here, we identified tyrosine 78 residue (Y78) of NP as a novel phosphorylation site by mass spectrometry. Y78 is highly conserved, and the constant NP phosphorylation mimicked by Y78E delayed NP nuclear export through reducing the binding of NP to the cellular export receptor CRM1, and impaired virus growth. Furthermore, the tyrosine kinase inhibitors Dasatinib and AG490 reduced Y78 phosphorylation and accelerated NP nuclear export, suggesting that the Janus and Src kinases-catalyzed Y78 phosphorylation regulated NP nuclear export during viral replication. More importantly, we found that the NP phosphorylation could suppress NP ubiquitination via weakening the interaction between NP and E3 ubiquitin ligase TRIM22, which demonstrated a cross-talk between the phosphorylation and ubiquitination of NP. This study suggests that the phosphorylation status of Y78 regulates IAV replication by inhibiting the nuclear export and ubiquitination of NP. Overall, these findings shed new light on the biological roles of NP phosphorylation, especially its negative role in NP ubiquitination.

Highlights

Influenza A virus (IAV) is an enveloped virus belonging to the Orthomyxoviridae family (Kendal and Harmon, 1988)
To identify the phosphorylation site of IAV NP, 293 T cells were infected with the influenza virus A/WSN/1933 (H1N1) (WSN) and NP was purified by protein G agarose beads pre-bound to rabbit anti-NP polyclonal antibody
We found that the phosphorylation level of NP Y78F mutant was greatly decreased compared with that of WT NP (Figure 1C), demonstrating that NP Y78 could be phosphorylated during virus infection

Summary

Introduction

Influenza A virus (IAV) is an enveloped virus belonging to the Orthomyxoviridae family (Kendal and Harmon, 1988). Each vRNA coated with multiple nucleoprotein (NP) molecules and the viral polymerase (PB2, PB1, and PA proteins) form a viral ribonucleoprotein (vRNP) complex (Zheng and Tao, 2013). Influenza A virions contain eight vRNPs which form the minimal functional units for viral. VRNPs are transcribed into viral mRNAs for the production of viral proteins and replicated into full-length complementary genomic RNA (cRNA) for amplification of vRNA and generation of progeny vRNPs. In the late phase of infection, progeny vRNPs are transported from the nucleus to plasma membrane with the assistance of the M1 and NEP proteins by CRM1-dependent nuclear export pathway (Whittaker et al, 1996a; Boulo et al, 2007; Eisfeld et al, 2015; Stevaert and Naesens, 2016)

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