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

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.

Characterization of Influenza A Virus Infection in Mouse Pulmonary Stem/Progenitor Cells

“…Previous study has shown that druginduced NP aggregation in cytosol might block migration of NP into nucleus and subsequently impair virus replication (Kao et al, 2010). Host protein phospholipid scramblase 1 (PLSCR1) (Luo et al, 2018) and moloney leukemia virus 10 (MOV10) (Zhang et al, 2016) also have been reported can interact with importin α to inhibit viral vRNP imported into nucleus. Therefore, aggregation of NP in mPSCs Oct4+ might block vRNP nuclear import, then interfere with vRNA replication.…”

The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry

“…Cell viability assay. Cell viability was determined by using the CellTiter-Glo kit (Promega, Madison, WI) as described previously (65,67). Briefly, A549 cells seeded in opaque-walled 96-well plates containing 100 or 10 M 4-CMTB or Cmp58 were cultured for 24 h, and A549 cells transfected with different siRNAs at a concentration of 30 nM were cultured for 48 h. CellTiter-Glo reagent (100 l) was then added directly into each well to induce cell lysis on a shaker for 10 min before luminescence was measured with a GloMax 96 microplate luminometer (Promega).…”

Influenza virus matrix protein M1 interacts with SLD5 to block host cell cycle

“…The yeast two-hybrid screen to identify M1 interacting protein was performed as previously described (Luo et al, 2018). The yeast two-hybrid screen to identify M1 interacting protein was performed as previously described (Luo et al, 2018).…”

“…We used a Make Your Own "Mate&Plate™" Library System kit (Clontech) to construct the cDNA library from lung tissues of C57BL/6 mice. The yeast two-hybrid screen to identify M1 interacting protein was performed as previously described (Luo et al, 2018).…”