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. 2017 May 23;8(3):e00597-17.
doi: 10.1128/mBio.00597-17.

CNOT4-Mediated Ubiquitination of Influenza A Virus Nucleoprotein Promotes Viral RNA Replication

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Free PMC article

CNOT4-Mediated Ubiquitination of Influenza A Virus Nucleoprotein Promotes Viral RNA Replication

Yu-Chen Lin et al. mBio. .
Free PMC article

Abstract

Influenza A virus (IAV) RNA segments are individually packaged with viral nucleoprotein (NP) and RNA polymerases to form a viral ribonucleoprotein (vRNP) complex. We previously reported that NP is a monoubiquitinated protein which can be deubiquitinated by a cellular ubiquitin protease, USP11. In this study, we identified an E3 ubiquitin ligase, CNOT4 (Ccr4-Not transcription complex subunit 4), which can ubiquitinate NP. We found that the levels of viral RNA, protein, viral particles, and RNA polymerase activity in CNOT4 knockdown cells were lower than those in the control cells upon IAV infection. Conversely, overexpression of CNOT4 rescued viral RNP activity. In addition, CNOT4 interacted with the NP in the cell. An in vitro ubiquitination assay also showed that NP could be ubiquitinated by in vitro-translated CNOT4, but ubiquitination did not affect the protein stability of NP. Significantly, CNOT4 increased NP ubiquitination, whereas USP11 decreased it. Mass spectrometry analysis of ubiquitinated NP revealed multiple ubiquitination sites on the various lysine residues of NP. Three of these, K184, K227, and K273, are located on the RNA-binding groove of NP. Mutations of these sites to arginine reduced viral RNA replication. These results indicate that CNOT4 is a ubiquitin ligase of NP, and ubiquitination of NP plays a positive role in viral RNA replication.IMPORTANCE Influenza virus, particularly influenza A virus, causes severe and frequent outbreaks among human and avian species. Finding potential target sites for antiviral agents is of utmost importance from the public health point of view. We previously found that viral nucleoprotein (NP) is ubiquitinated, and ubiquitination enhances viral RNA replication. In this study, we found a cellular ubiquitin ligase, CNOT4, capable of ubiquitinating NP. The ubiquitination sites are scattered on the surface of the NP molecule, which is critical for RNA replication. CNOT4 and a ubiquitin protease, USP11, together regulate the extent of NP ubiquitination and thereby the efficiency of RNA replication. This study thus identifies a potential antiviral target site and reveals a novel posttranslational mechanism for regulating viral replication. This represents a novel finding in the literature of influenza virus research.

Keywords: CNOT4; IAV; NP; RNA replication; influenza A virus; nucleoprotein; ubiquitination.

Figures

FIG 1
FIG 1
Knockdown of CNOT4 inhibits IAV replication. (A) Immunofluorescence analysis of secondary RNAi screening. A549 cells were transduced with various shRNA-harboring lentiviruses that targeted the different genes identified in the primary pooled shRNA library screen (19). The transduced cells were then infected with WSN/33 virus at an MOI of 1. At 6 h p.i., the cells were subjected to immunofluorescence staining using NP antibody and DAPI. The intensity of NP-DAPI was used as an indicator and normalized to control shLacZ staining intensity to estimate the percentage of cells infected with IAV. (B and C) CNOT4 knockdown efficiency (B) and relative viral NP RNA levels (C) were determined by qRT-PCR, using RNA from the various CNOT4 knockdown A549 cells (shRNA clones CN1 to CN5) at 6 h p.i. (means ± SD, n = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001, based on one-way ANOVA with Dunnett’s multiple-comparison test. (D) Viral proteins were detected by immunoblotting at 6 h p.i. with an anti-NP antibody. Actin was used as an internal control. (E) Virus titers from progeny virus released into the medium were determined at 24 h p.i. via plaque assay on MDCK cells (means ± SD, n = 3). ***, P < 0.001, based on one-way ANOVA with Dunnett’s multiple-comparison test.
FIG 2
FIG 2
Viral RNP activity is correlated with the expression level of CNOT4. (A) Minireplicon assay results for RNP activity in CNOT4 knockdown cells. Two shCNOT4 clones, CN1 and CN3, and control shLacZ 293T cells were transfected with the plasmids for expression of viral PB1, PB2, PA, and NP and a reporter plasmid expressing the antisense luciferase gene as described elsewhere for the minireplicon assay (29). At 24 h posttransfection, luciferase activity was determined and normalized to the response of control shLacZ (means ± SD, n = 3). **, P < 0.01; ***, P < 0.001, by one-way ANOVA with Dunnett’s multiple-comparison test. (B) CNOT4 knockdown efficiency in 293T cells. (C) Overexpression of CNOT4 enhances viral RNP activity. CNOT4 knockdown (shRNA clones CN1 and CN3) or control cells (shLacZ) were transfected together with wild-type CNOT4 isoform e (CNe) or its wobble mutant (wCNe) and used for the minireplicon assay. Relative luciferase activities were measured as described for panel A (means ± SD, n = 3). ***, P < 0.001 (versus shLacZ); ##, P < 0.01; ###, P < 0.001 (versus shRNA clones CN1 or CN3), based on a one-way ANOVA with Tukey’s multiple-comparison test. (D) Interaction of CNOT4 with viral RNP components in an immunoprecipitation (IP) assay. 293T cells were cotransfected with Flag-tagged CNOT4 isoform e (CNe-Flag) and one of the HA-tagged viral proteins (PB1, PB2, PA, and NP). At 48 h posttransfection, cell lysates were precipitated using Flag-agarose. The proteins were visualized by Western blotting (IB) with anti-HA and anti-Flag antibodies.
FIG 3
FIG 3
CNOT4 enhances viral NP ubiquitination, whereas the deubiquitinase USP11 reduces it. (A) Knockdown/overexpression of CNOT4 correlates with viral NP ubiquitination. CNOT4 knockdown (shCNOT4 clones) or control shLacZ 293T cells were transfected with Ub-myc and NP-HA, with or without wild-type (CN) or wobble mutant (wCN) CNOT4e-Flag. Cells were subjected to immunoblotting (IB) using HA-agarose. The immunoprecipitated proteins were visualized with anti-HA and anti-myc antibodies. The solid arrowhead indicates the monoubiquitinated NP. The arrows indicate the protein bands cut out for mass spectrometry analysis (see Fig. 5). (B) CNOT4 and USP11 had opposing effects on viral NP ubiquitination. CNOT4 knockdown or control shLacZ 293T cells were cotransfected with NP-HA, Ub-myc, CNOT4e-Flag, and USP11, as indicated, and immunoblotted with HA-agarose. The immunoprecipitated proteins were visualized with anti-NP and anti-myc antibodies. The relative amounts of monoubiquitinated NP (arrow) in each sample are indicated below the gel.
FIG 4
FIG 4
Viral NP is ubiquitinated by CNOT4 in vitro, and its ubiquitination does not lead to NP degradation. (A) In vitro transcription/translation of NP, CNOT4, and p53. Plasmids carrying NP-HA, CNOT4e-Flag, or p53-HA open reading frames were incubated with TNT lysates at 30°C for 1 h to synthesize the target proteins. The samples were examined by Western immunoblotting (IB) using anti-HA antibody (for p53 and NP) or anti-Flag antibody (for CNe). The respective protein products are indicated by arrows. (B) In vitro ubiquitination assay results for NP with CNOT4 E3 ligase. In vitro-translated NP-HA and p53-HA TNT lysates were obtained as described for panel A and incubated using a ubiquitination kit with or without in vitro-translated CNOT4-Flag at 37°C for 1 h. The ubiquitinated proteins were then detected via immunoblotting using anti-HA antibody. Arrowheads indicate the main protein products without ubiquitination. Vertical lines indicate the ubiquitinated proteins. (C) NP protein stability assay. 293T cells were transfected with plasmids expressing NP-HA, with or without CNOT4e-Flag. At 24 h posttransfection, cells were treated with cycloheximide (50 μM) and MG132 (10 μM) for the indicated lengths of time before lysis. The relative amounts of NP in each sample are indicated below the gel.
FIG 5
FIG 5
MS/MS analysis of ubiquitinated NP revealed multiple ubiquitination sites. Tandem mass spectra of peptides derived from ubiquitinated NP show ubiquitin conjugation at different residues of ubiquitin with di-Gly modification. Ubiquitinated NP was separated as described for Fig. 3 and isolated using anti-HA–agarose from 293T cells transfected with NP-HA or Ub-myc, with or without CNOT4e-Flag. Protein samples were cut from the indicated protein bands (as for Fig. 3) and further purified (see Materials and Methods). Samples were analyzed by MS analysis. The Mascot search engine was used, with the criteria for searching ubiquitinated lysines marked as LeuArgGlyGly (KLRGG) or GlyGly (KGG). GG or LRGG modification was observed at the K31 (A), K77 (B), K87 (C), K91 (D), K113 (E), K184 (F), K227 (G), K229 (H), K273 (I), and K351 (J) residues.
FIG 6
FIG 6
Viral RNP activity of the lysine-to-arginine mutated NP in relationship to its structure; the illustration shows the importance of the lysine residues in the RNA-binding groove of NP. (A) RdRp activities of the various lysine-to-arginine mutants in the absence or presence of exogenous CNOT4. A minireplicon assay for different lysine mutants of NP was performed as described for Fig. 2. Luciferase activity was determined at 24 h posttransfection and normalized to that of the control, WT NP. NP levels (black columns) indicate basal RdRp activity in the absence of exogenous CNOT4; NP plus CN levels (gray columns) indicate RdRp activity in CNOT4-overexpressing cells. Red asterisks indicate the KR mutants that had statistically significantly lower RdRP activities than those of the wild-type NP; they are further highlighted with green boxes. Black asterisks indicate the KR mutants that had statistically significantly higher RdRP activities than the wild-type NP in the presence of overexpressed CNOT4. The black # sign indicates the mutants whose RdRP activities were enhanced by CNOT4 to a statistically significant level (means ± SD, n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (versus WT NP). #, P < 0.05; ###, P < 0.001 (versus KR-NP), based on a one-way ANOVA with Tukey’s multiple-comparison test. (B) Locations of the critical lysine residues on the NP molecule. The three-dimensional structure of the influenza A virus NP was modified from that described by Chenavas et al. (44). K184, K227, and K273 are located in the RNA-binding groove of NP (with blue indicating a positive charge); K91 and K351 are located on the NP surface.

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