The quantitative profiling of biotinylation patterns method (Q-POP)—an experimental strategy for studying interactions between influenza virus polymerase subunits PB1 and PA. The PB1-TAP subunit was expressed in 293T cells cultured in ¹²C₆-lysine-containing medium, and the PB1-TAP-PA heterodimer was expressed in ¹³C₆-lysine-containing medium. They were separately partially purified, followed by in vitro biotinylation with NHS-biotin. They were then mixed at ∼1:1 ratio and concentrated by precipitation and separated on SDS-PAGE. The band containing both ¹²C₆-lysine- and ¹³C₆-lysine-labeled PB1 was excised followed by in-gel trypsin digestion. The biotinylated peptides were purified and finally analyzed by LC-MS/MS.

Characteristic fragment ions derived from immonium ions of biotinylated lysine. (A) MS/MS spectra of peptide 119VDKLTQR126 of PB1 with biotinylated K121 (MH⁺ = 1142.599 Da, SILAC light), (B) MS/MS spectrum of the corresponding peptide with an oxidized biotinylated K121 (MH⁺ = 1158.594 Da). b-ions are derived from the peptide N-termini, y-ions are derived from the C-termini.

Functional effects of mutations at positions 265 and 481 of PB1 upon PA binding and polymerase activity. (A) Alignment of amino acid sequences of PB1 subunits of influenza A, B, and C viruses near the K265 position. (B) Effects of the single, double, and triple mutant PB1 (see text) on 2P (PB1-PA) dimerization and 3P formation. 293T cells were transfected with plasmids expressing either wild-type PB1-TAP or the three mutant PB1-TAP, together with either a plasmid expressing PA for 2P heterodimerization (upper panel) or plasmids expressing PA and PB2 for 3P formation (lower panel). Protein size markers and positions of PB1-TAP, PA, PB2, and Hsp90 are shown. (C) In vitro ApG-primed transcription mediated by the wild type (WT), using samples from Figure 5(B) (lower panel), and the 3 indicated mutant PB1 polymerases. TP, transcription product. (D) Quantitation of four independent ApG-primed transcription reactions. Values are % of wild-type levels. Error bars = S.D; asterisks (*) indicate the values significantly different from 100% (P < 0.01, Student's t-test). (E) Primer extension assays of influenza vRNA, mRNA, and cRNA isolated from 293T cells expressing either wild-type RNPs or the indicated mutant RNPs (see Experimental Procedures). The positions of transcription products derived from NA reporter mRNA, cRNA, vRNA, and 5S RNA, used as a control, are indicated. (F) Quantitation of four independent primer extension assays. Values were expressed as % of the wild-type values set to 100%. Values significantly different from 100% (P < 0.01, Student's t-test) are indicated by an asterisk (*). (G) Alignment of amino acid sequences of PB1 subunits of influenza A, B, and C viruses near the K481 position. (H) Effect of the single mutant PB1 (K481A, see text) on 2P (PB1-PA) dimerization and 3P formation. A doublet of Hsp90α and β is visible. Additionally, the yields of the Hsp90 doublet appear to be high with respect to the PB1-TAP band, possibly because of partial degradation of PB1-TAP for unknown reasons. (I) In vitro ApG-primed transcription mediated by the wild-type (WT) and indicated mutant PB1 polymerases. (J) In vivo primer extension assay with wild-type and indicated mutant RNPs.

PB1 and PA polymerase subunits before and after biotinylation and mixing (A) partially purified PB1-TAP (L) and PB1-TAP-PA (H) before biotinylation, silver-stained, (B) mixture (L + H) of light PB1-TAP and heavy PB1-TAP-PA after biotinylation and concentration by precipitation, coomassie blue-stained. The positions of the PB1-TAP, PA, and Hsp90 bands are indicated on 7% SDS-PAGE.

Examples of SILAC pairs with (A) decreased accessibility, that is, peptide SICEKLEQSGLPVGGNEK modified at K265, (B) increased accessibility, that is, peptide VDKLTQGR modified at K121, and (C) similar accessibility, that is, peptide IKKEEFTEIMK modified at K737 to biotinylation in PB1-PA heterodimers compared with monomeric PB1.