Mapping the phosphoproteome of influenza A and B viruses by mass spectrometry

Abstract

Protein phosphorylation is a common post-translational modification in eukaryotic cells and has a wide range of functional effects. Here, we used mass spectrometry to search for phosphorylated residues in all the proteins of influenza A and B viruses--to the best of our knowledge, the first time such a comprehensive approach has been applied to a virus. We identified 36 novel phosphorylation sites, as well as confirming 3 previously-identified sites. N-terminal processing and ubiquitination of viral proteins was also detected. Phosphorylation was detected in the polymerase proteins (PB2, PB1 and PA), glycoproteins (HA and NA), nucleoprotein (NP), matrix protein (M1), ion channel (M2), non-structural protein (NS1) and nuclear export protein (NEP). Many of the phosphorylation sites detected were conserved between influenza virus genera, indicating the fundamental importance of phosphorylation for all influenza viruses. Their structural context indicates roles for phosphorylation in regulating viral entry and exit (HA and NA); nuclear localisation (PB2, M1, NP, NS1 and, through NP and NEP, of the viral RNA genome); and protein multimerisation (NS1 dimers, M2 tetramers and NP oligomers). Using reverse genetics we show that for NP of influenza A viruses phosphorylation sites in the N-terminal NLS are important for viral growth, whereas mutating sites in the C-terminus has little or no effect. Mutating phosphorylation sites in the oligomerisation domains of NP inhibits viral growth and in some cases transcription and replication of the viral RNA genome. However, constitutive phosphorylation of these sites is not optimal. Taken together, the conservation, structural context and functional significance of phosphorylation sites implies a key role for phosphorylation in influenza biology. By identifying phosphorylation sites throughout the proteomes of influenza A and B viruses we provide a framework for further study of phosphorylation events in the viral life cycle and suggest a range of potential antiviral targets.

Figure 1. Purification of viral proteins.

(A) Purification of WSN virus from the growth medium of infected MDBK cells. Samples of unpurified and purified material (0.01 ml from 120 ml and from 0.14 ml, respectively) were separated by 12% SDS-PAGE and stained with Coomassie Brilliant Blue. Key proteins are identified by electrophoretic mobility. (B) Negative-stain transmission electron micrographs of purified WSN virus. Two different magnifications are shown.

Figure 2. Location of phosphorylated residues in the matrix protein M1 and the ion channel M2.

(A) The M1 consensus sequences of influenza A and B viruses, aligned using ClustalW2. Letters are coloured green for experimentally-confirmed phosphorylation sites, blue for the nuclear localisation signal (NLS) of influenza A virus and orthologous basic residues in influenza B virus, and purple for a ubiquitination site. Where modifications could be assigned to more than one residue, all probable residues are coloured (see text for details). Confirmed phosphorylation sites, and their possible orthologues, are highlighted in green. (B) Location of phosphorylated residues in the N-terminal portion of influenza A virus M1 (PDB 1EA3 [47]). Hydrophobic residues in helices 1 and 4 are coloured yellow and basic residues of the NLS dark blue. (C) Sections of the M2 consensus sequences of influenza A, B and C viruses. Colours are as (A), with additional highlighting of structural features: alpha helices are orange (where experimentally determined; PDB 2L0J and PDB 2KJ1 [56]) or yellow (where predicted by JPred 3), and a beta sheet is purple (predicted by JPred 3).

Figure 3. Location of phosphorylated residues in the non-structural protein NS1 and the nuclear export protein NEP.

(A) Location of S48 in the dimeric NS1 RNA binding domain (PDB 2ZKO [60]). The subunits of the dimeric NS1 RNA binding domain are shown in light blue and pink, and RNA in gold. (B) Location of T197 and interacting residues in the dimeric NS1 effector domain (PDB 2GX9 [61]). (C) Consensus sequences of the N-terminal residues of influenza A and B virus NEP. Colours are as in Figure 2, with key hydrophobic residues of the nuclear export signal (NES) in red.

Figure 4. Location of phosphorylated residues in haemagglutinin.

(A) An influenza A virus H1-subtype HA monomer (PDB 1RVZ [75]) and an influenza B virus HA monomer (PDB 2RFU [81]). In the influenza A virus HA, T358 is indicated in the N terminus of HA2 and, after conformational rearrangement, in the fusion peptide (inset; PDB 2KXA [76]); the corresponding residue E377 is indicated in influenza B virus HA2. In influenza B virus HA S135 in indicated in the head domain (in the structure shown, position 136 is alanine), and S465 in the stem; the corresponding E446 residue is indicated in influenza A virus HA2. HA1 is shaded pink, HA2 light blue, hydrophobic residues of the fusion peptide yellow, glycosylations of the influenza B HA orange, the α(2,6)-sialic acid-containing pentasaccharide LSTc red and glutamic acids orthologous to phosphorylation sites purple. (B) The overall consensus sequence of the HA fusion peptide, compared to the consensus sequences of individual subtypes and lineages. Residue 15 of the fusion peptide, which corresponds to T358 of the H1 sequence, is shaded.

Figure 5. Location of phosphorylated residues in neuraminidase.

(A) The position of S160/S164/S166 in the head domain of an N1-subtype NA, viewed facing the virion surface (PDB 3BEQ [86]). Head domains of the NA tetramer are shown in light blue and pink. (B) The position of S164 in the NA active site, with catalytic residues in yellow and framework residues in pink.

Figure 6. Location of phosphorylated residues in the polymerase.

The position of S472 in the C-terminal tail of PB2 when (A) unbound (PDB 2GMO [91]) and (B) bound (PDB 2JDQ [91]) to importin α5. PB2 is shown in light blue and the importin in gold. In the bound form residues 742–747 are not resolved in the structure, and are indicated by a dotted line; the position of S742 has been estimated. (C) Portions of the consensus sequences of PB2, PB1 and PA from influenza A, B and C viruses. Colours are as in Figure 2; basic residues of the bipartite NLS of influenza A virus PB2, and orthologous residues in the influenza B and C virus sequences, are blue.

Figure 7. Location of phosphorylated residues in the nucleoprotein.

(A) Location of phosphorylated residues in NP of influenza A virus WSN (PDB 2IQH [105]) and influenza B virus (PDB 3TJ0 [108]). The structures do not include N-terminal residues, including S9 and Y10 of influenza A virus NP and S50 and T55-S58 of influenza B virus NP. The N-termini of the resolved structures and tail loops are indicated; in the orientation shown, the RNA-binding grooves are on the far side of the molecules. (B) The oligomerisation of NP of influenza A virus or influenza B virus via the tail loop (blue) and groove (pink), with phosphorylated residues highlighted.

Figure 8. Effect of mutating phosphorylated residues in the nucleoprotein.

(A) WSN viruses were generated containing the indicated mutations in NP. MDBK cells were infected at an MOI of 0.001, and virus harvested at the indicated time points. The mean and range of two experiments, or, for S402A and S402E, the mean and standard deviation of 4 experiments, is shown. For S402A and S402E differences from WT were tested by Student's unpaired 2-tailed t-tests at each time point; for both mutants the differences at 8 h are not significant, those at 24 h significant at p<0.05 and those at 30 h significant at p<0.005. (B) To assess the function of mutated NP proteins in transcription and replication, RNP reconstitutions were performed in 293 T cells and RNA species at 22 h post-transfection measured by primer extension and autoradiography. A representative image is shown, along with the mean and s.d. of 4 experiments, relative to WT. Differences from WT were tested using one-sample t-tests: * p<0.05, *** p<0.0005.