Table 1 Modified lysines in the IAV polymerase

From: The ubiquitination landscape of the influenza A virus polymerase

Domain	K-ε-GG position	Rep. of 5	Exposed in monomer		Exposed in dimer		Rec. virus (titers [PFU/ml])		Ref.
Domain	K-ε-GG position	Rep. of 5	vRNA	cRNA	Symmetric	Asymmetric	Ala	Arg	Ref.
PB2
N-Terminal domain	32	2	−	−	−	−			⁸⁵
	41	4	−	−	+	−
	61	4	+	+	+	+
	80	4	+	+	+	+
	116	4	+	+	+	+
	121	4	−	−	−	Rep − \| Enc +
	126	4	+	+	+	+
	157	2	+	+	−	−	+ (2.0 × 10⁷)	+ (1.8 × 10⁷)
	187	4	+	+	+	−
	189	4	+	+	−	−
	197	2	+	+	+	+
Mid CapB Linker	312	2	+	−	−	−
	353	2	+	+	+	+			⁴⁴
	482	4	+	+	+	Rep+\| Enc −	LT (5.8 × 10⁴)	SP (1.7 × 10⁶)	³¹
627 Domain	586	2	+	+	+	+			⁸⁶
	617	2	−	+	−	−	−	−
	670	2	−	+	+	Rep − \| Enc +			⁸⁷
NLD	699	3	+	−	+	+	+ (1.0 × 10⁷)	−	³⁴
	718	2	+	+	+	−	SP (4.8 × 10⁶)	+	⁴⁶
	752	4	+	+	+	+			^31,46,47
PA
Endonuclease	22	4	+	+	+	+	+ (1.4 × 10⁸)	SP (1.4 × 10⁷)	⁵⁷
	29	5	+	+	+	+
	102	2	+	+	+	+			^48,50
	104	1	+	+	+	+			^48,50
	113^a	2	+	+	+	+			⁴⁸
	134	4	−	−	−	+	−	−	^48,49,88,89
	158	1	+	−	+	−			⁴⁹
Linker	213	3	+	+	+	+			⁴⁸
	245	3	+	+	+	+
	251	4	+	+	+	+
C-terminal domain	262	4	+	+	+	+
	281	2	−	−	−	−			^42,87,90,91
	309	2	+	+	−	Rep +\| Enc −
	339	2	+	+	+	Rep +\| Enc −			^9,48
	353	1	+	+	+	+			^11,12
	391	1	+	+	+	+
	536	4	+	+	+	Rep +\| Enc −	R/LT (7.3 × 10³)	R (3.6 × 10⁷)	⁵³
	605	4	+	+	+	Rep +\| Enc −	SP (2.2 × 10⁷)	+ (7.8 × 10⁶)
	609	2	+	+	+	+
	615	2	+	+	+	Rep +\| Enc −	−	+ (1.2 × 10⁷)	⁹²
	626	3	+	+	+	+			⁹³
	635	5	+	+	+	+	−	SP/LT (3.0 × 10⁴)	⁵¹
	643	3	−	−	−	−			^91,93
PB1
Core	121	3	−	−	−	−
Core	229	4	−	−	−	−	−	−	^{55,87,94,95,96}
ß-hairpin	347	2	−	−	−	−	−	−
	353	2	−	−	+	+			⁵⁵
	360	2	+	+	+	+	+ (3.6 × 10⁷)	LT (1.1 × 10⁵)
	379	2	+	+	+	+
Palm	471	1	−	−	−	−	−	SP (1.1 × 10⁷)	⁹⁷
Palm	480	3	+	+	+	+			^42,55,98
Thumb	578	2	+	+	+	+	+ (2.2 × 10⁷)	R (6.4 × 10³)
	586	1	+	+	+	+
	635	1	−	−	+	−	+ (9.4 × 10⁷)	+ (1.2 × 10⁸)
	669	2	−	−	−	+			⁹⁹
C-Ext	736	2	+	+	+	+
C-Ext	737	4	+	+	+	+

List of the positions of all K-ε-GG bound lysines in the PB2, PA, and PB1 subunits detected in 5 independent replicates (Rep.) at 5 h p. i. together with their location in functional domains. The number of replicates in which the site was detected is indicated (Rep. of 5). Surface exposure of the identified sites was assessed using 3D structural models of a WSN-adapted IAV polymerase monomer bound to either vRNA or cRNA as well as the 3D structures of the symmetric dimer of the H3N2 polymerase (PDB: 6QNW) and the ANP32A-stabilized asymmetric dimer of the influenza C virus polymerase. Surface exposure is indicated with (+), concealed location is indicated with (−). In the asymmetric dimer, surface exposure is also itemized regarding location in the replicating (Rep) or encapsidating (Enc) polymerase. Success or failure to generate recombinant viruses with the plasmids encoding the respective mutations after three independent rescue attempts is encoded by (+) = generation of virus, (−) = no virus after three attempts, (R) = generation of virus but reversion to wild type, (SP) = generation of virus but small plaque phenotype, (LT) = generation of virus but low rescue titer. Rescue titers are provided as [PFU/ml].
Studies that have previously described or functionally characterized the detected lysines are listed in the references (Ref.) column.
Following reanalysis of the MS data additional modified sites in PB2 (K113, K627) and PB1 (K278) were detected.
^aOne site in PA (K113) that was identified in previous analyses was excluded due to threshold adjustments.

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Table 1 Modified lysines in the IAV polymerase

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