Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication.

Wei Luo ,Qibing Li,Yuan Zhou,Guangwen Wang,Yongping Jiang,Nan Sun,Pengfei Cui,Yuhui Zhao,Jie Zhang,Shanyu Huang,Li Jiang,Junping Li,Pucheng Chen,Chenchen Zhou,Guohua Deng,Zhigao Bu,Hualan Chen,Liang Liu ,Julia Yu Fong Chang ,Ping Zhu ,Chengjun Liu

doi:10.1371/journal.ppat.1006851

Wei Luo , Qibing Li + Show 19 more

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https://doi.org/10.1371/journal.ppat.1006851

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Abstract

Transcription and replication of the influenza A virus (IAV) genome occur in the nucleus of infected cells and are carried out by the viral ribonucleoprotein complex (vRNP). As a major component of the vRNP complex, the viral nucleoprotein (NP) mediates the nuclear import of the vRNP complex via its nuclear localization signals (NLSs). Clearly, an effective way for the host to antagonize IAV infection would be by targeting vRNP nuclear import. Here, we identified phospholipid scramblase 1 (PLSCR1) as a binding partner of NP by using a yeast two-hybrid (Y2H) screen. The interaction between NP and PLSCR1 in mammalian cells was demonstrated by using co-immunoprecipitation and pull-down assays. We found that the stable overexpression of PLSCR1 suppressed the nuclear import of NP, hindered the virus life cycle, and significantly inhibited the replication of various influenza subtypes. In contrast, siRNA knockdown or CRISPR/Cas9 knockout of PLSCR1 increased virus propagation. Further analysis indicated that the inhibitory effect of PLSCR1 on the nuclear import of NP was not caused by affecting the phosphorylation status of NP or by stimulating the interferon (IFN) pathways. Instead, PLSCR1 was found to form a trimeric complex with NP and members of the importin α family, which inhibited the incorporation of importin β, a key mediator of the classical nuclear import pathway, into the complex, thus impairing the nuclear import of NP and suppressing virus replication. Our results demonstrate that PLSCR1 negatively regulates virus replication by interacting with NP in the cytoplasm and preventing its nuclear import.

Highlights

Influenza A virus (IAV), a single-stranded, negative-sense RNA virus with an eight-segmented genome, is the causative agent of influenza in many animal species, including humans
Influenza viral RNA is encapsidated by three polymerase proteins and the NP protein to form the viral ribonucleoprotein (vRNP) complex, which is transported to the nucleus of infected cells for viral transcription and replication
The active nuclear import of the vRNP complex is mediated by the interaction between NP and importin α through the nuclear import pathway

Summary

Introduction

Influenza A virus (IAV), a single-stranded, negative-sense RNA virus with an eight-segmented genome, is the causative agent of influenza in many animal species, including humans. A prominent feature of the IAV life cycle is that the transcription and replication of the viral genome occur in the nucleus of infected cells [6, 7]. NP relies on the classical nuclear import pathway to enter the nucleus of infected cells. In this pathway, importin α functions as an adaptor by recognizing NLS sequences in cargo proteins and associating with the importin β receptor [13, 14]. Previous studies have shown that the nuclear import of vRNP and newly synthesized NP to the nucleus of infected cells is a crucial step in the IAV life cycle [12, 18]. The detailed mechanism that regulates the migration of vRNP complexes and newly produced NP into the nucleus remains obscure, and the identification of the potential host factors involved is not yet complete

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