Phosphorylation at the Homotypic Interface Regulates Nucleoprotein Oligomerization and Assembly of the Influenza Virus Replication Machinery

<div><p>Negative-sense RNA viruses assemble large ribonucleoprotein (RNP) complexes that direct replication and transcription of the viral genome. Influenza virus RNPs contain the polymerase, genomic RNA and multiple copies of nucleoprotein (NP). During RNP assembly, monomeric NP oligomerizes along the length of the genomic RNA. Regulated assembly of the RNP is essential for virus replication, but how NP is maintained as a monomer that subsequently oligomerizes to form RNPs is poorly understood. Here we elucidate a mechanism whereby NP phosphorylation regulates oligomerization. We identified new evolutionarily conserved phosphorylation sites on NP and demonstrated that phosphorylation of NP decreased formation of higher-order complexes. Two phosphorylation sites were located on opposite sides of the NP:NP interface. In both influenza A and B virus, mutating or mimicking phosphorylation at these residues blocked homotypic interactions and drove NP towards a monomeric form. Highlighting the central role of this process during infection, these mutations impaired RNP formation, polymerase activity and virus replication. Thus, dynamic phosphorylation of NP regulates RNP assembly and modulates progression through the viral life cycle.</p></div

Mechanistic model for the regulated oligomerization of NP and RNP assembly.

<p>A portion of NP is phosphorylated by an unknown host kinase at S165 and S407 (represented as red dots), preventing tail loop:binding groove interactions and preventing self-association. Conversely, at the appropriate stage of the viral life cycle, dephosphorylation of NP by an unknown cellular phosphatase or synthesis of new NP molecules that do not get phosphorylated permits efficient self-association, genome replication, and RNP assembly.</p

Phosphorylation of the tail loop and binding groove maintains monomeric NP.

<p>Binary tail loop-binding groove interactions were directly probed using an NP tail loop deletion mutant (NPΔTL) and a tail loop fused to eGFP (eGFP-TL). (A) Binding groove mutants were investigated by co-expressing WT or mutant NPΔTL with eGFP-TL in 293T cells. Lysates were immunoprecipitated with anti-GFP antibody and co-precipitating NP was visualized by blotting with anti-RNP antibody. Expression levels of interacting partners were analyzed by blotting total proteins with anti-RNP or anti-GFP antibodies. (B) The impact of tail loop mutants was probed by co-expressing WT or mutant eGFP-TL with NPΔTL. Complexes were immunoprecipitated and detected as above. (C) Schematic depiction of how phosphorylation might block entry of the tail loop into the binding groove and inhibiting NP oligomerization.</p

NP phospho-mutants disrupt RNP formation.

<p>(A and B) Viral RNPs were reconstituted in 293T cells by expressing WT or mutant NP-V5, a vRNA template, and polymerase containing either PB2-HA (A), or PA-FLAG (B). Assembled RNPs were immunoprecipitated using anti-HA or anti-FLAG antibodies and co-precipitated NP was visualized by blotting with anti-RNP antibody (upper panels, IP). Expression of PB2, PA and WT or mutant NP was confirmed by blotting whole cell lysate (lower panels, Input). (C and D) NP mutations do not disrupt binding to PB2 or PB1. Lysates were prepared from 293T cells expressing PB2-HA (C) or PB1-HA (D) and WT or mutant NP-V5, treated with RNaseA, and immunoprecipitated with anti-V5 antibody. Co-precipitating PB2 or PB1 was detected by blotting with anti-HA antibody (upper panels, NP IP). Equivalent expression of WT and mutant NP were confirmed by blotting whole cell extracts with anti-V5 antibody (lower panels, input).</p

Identification of serine residues and a phosphorylation site in NP that are critical for polymerase activity.

<p>(A) The structure of NP (PDB 2IQH) reveals the core head and body regions as well as a tail loop that directs oligomerization. Conserved, solvent-exposed serine residues were identified in the NP protomer and selected for mutagenesis. (B) High-throughput polymerase activity assays were performed in human 293T cells expressing the viral polymerase, WT or mutant NP, and reporter constructs representing negative- (vNA-Luc) or positive-sense (cNA-Luc) RNA templates. (C, and D) Purified NP was prepared for mass spectrometry, tryptically digested, and enriched for phosphopeptides. Targeted MS identified the phosphopeptide 401-ASSGQISIQPTFSVQR-416 with phosphorylations localized to S407 and to S413 on NP.</p

Mechanistic model for the regulated oligomerization of NP and RNP assembly.

<p>A portion of NP is phosphorylated by an unknown host kinase at S165 and S407 (represented as red dots), preventing tail loop:binding groove interactions and preventing self-association. Conversely, at the appropriate stage of the viral life cycle, dephosphorylation of NP by an unknown cellular phosphatase or synthesis of new NP molecules that do not get phosphorylated permits efficient self-association, genome replication, and RNP assembly.</p

NP phosphorylation sites are important for RNP activity and virus replication.

<p>(A) Polymerase activity assay were performed with a vNA-luc reporter in the presence of excess WT or mutant NP. Equivalent expression of the wild type and mutant proteins were confirmed by western blotting cell lysates. Polymerase activity was normalized to that of WT (n = 3 +/- SD). (B) Primer-extension analysis was performed on RNA extracted from cells expressing the viral polymerase, vNA-luc, and WT or mutant NP. Polymerase products were identified by their predicted molecular weight. * = non-specific band. (C) Multicycle replication kinetics were examined in MDCK cells infected at an MOI of 0.01 with recombinant viruses encoding WT or mutant NP. Viral titers at the indicated time points were determined by plaque assay (n = 3 independent infections +/- SD).</p

Critical serine residues control NP oligomerization in cells.

<p>Lysates were prepared from 293T cells expressing WT, mutant or phosphomimetic NP and fractionated by size exclusion chromatography. Fractions were analyzed by western blot with anti-NP antibodies. WT NP and the oligomerization defective NP R416A were used as internal standards to determine the position of the oligomeric and monomeric populations, respectively. Elution peaks for calibration standards are shown.</p

Segmentation of Precursor Mass Range Using “Tiling” Approach Increases Peptide Identifications for MS¹‑Based Label-Free Quantification

Label-free quantification is a powerful tool for the measurement of protein abundances by mass spectrometric methods. To maximize quantifiable identifications, MS¹-based methods must balance the collection of survey scans and fragmentation spectra while maintaining reproducible extracted ion chromatograms (XIC). Here we present a method which increases the depth of proteome coverage over replicate data-dependent experiments without the requirement of additional instrument time or sample prefractionation. Sampling depth is increased by restricting precursor selection to a fraction of the full MS¹ mass range for each replicate; collectively, the <i>m</i>/<i>z</i> segments of all replicates encompass the full MS¹ range. Although selection windows are narrowed, full MS¹ spectra are obtained throughout the method, enabling the collection of full mass range MS¹ chromatograms such that label-free quantitation can be performed for any peptide in any experiment. We term this approach “binning” or “tiling” depending on the type of <i>m</i>/<i>z</i> window utilized. By combining the data obtained from each segment, we find that this approach increases the number of quantifiable yeast peptides and proteins by 31% and 52%, respectively, when compared to normal data-dependent experiments performed in replicate

Absence of Vitamin K-Dependent γ-Carboxylation in Human Periostin Extracted from Fibrotic Lung or Secreted from a Cell Line Engineered to Optimize γ-Carboxylation

<div><p>Periostin (PN, gene name POSTN) is an extracellular matrix protein that is up-regulated in bronchial epithelial cells and lung fibroblasts by TH-2 cytokines. Its paralog, TGF-β-induced protein (βig-h3, gene name TGFBI), is also expressed in the lung and up-regulated in bronchial myofibroblasts by TGF-β. PN and βig-h3 contain fasciclin 1 modules that harbor putative recognition sequences for γ-glutamyl carboxylase and are annotated in UniProt as undergoing vitamin K-dependent γ-carboxylation of multiple glutamic acid residues. γ-carboxylation profoundly alters activities of other proteins subject to the modification, e.g., blood coagulation factors, and would be expected to alter the structure and function of PN and βig-h3. To analyze for the presence of γ-carboxylation, proteins extracted from fibrotic lung were reacted with monoclonal antibodies specific for PN, βig-h3, or modification with γ-carboxyglutamic acid (Gla). In Western blots of 1-dimensional gels, bands stained with anti-PN or -βig-h3 did not match those stained with anti-Gla. In 2-dimensional gels, anti-PN-positive spots had pIs of 7.0 to >8, as expected for the unmodified protein, and there was no overlap between anti-PN-positive and anti-Gla-positive spots. Recombinant PN and blood coagulation factor VII were produced in HEK293 cells that had been transfected with vitamin K 2, 3-epoxide reductase C1 to optimize γ-carboxylation. Recombinant PN secreted from these cells did not react with anti-Gla antibody and had pIs similar to that found in extracts of fibrotic lung whereas secreted factor VII reacted strongly with anti-Gla antibody. Over 67% coverage of recombinant PN was achieved by mass spectrometry, including peptides with 19 of the 24 glutamates considered targets of γ-carboxylation, but analysis revealed no modification. Over 86% sequence coverage and three modified glutamic acid residues were identified in recombinant fVII. These data indicate that PN and βig-h3 are not subject to vitamin K-dependent γ-carboxylation.</p></div