Global remodeling of ADP-ribosylation by PARP1 suppresses influenza A virus infection

Domain: biology
Published: 2025-12-17
Editorial outcome: major_revision

Abstract
ADP-ribosylation is a highly dynamic and fully reversible post-translational modification performed by PARP enzymes that modulates protein function, abundance, localization, and turnover. Here we show that PARPs mount an antiviral response to influenza A virus infection causing a rapid and dramatic upregulation of global ADP-ribosylation that inhibits viral replication. Mass spectrometry analyzes define the global ADP-ribosylome during infection, creating an infection-specific profile with almost 4000 modification sites on ~1000 host proteins, as well as over 100 modification sites on viral proteins. Our data suggest that the global increase reflects a change in the form of ADP-ribosylation rather than modification of new targets. Functional assays demonstrate that modification of the viral replication machinery antagonizes its activity. We further show that the influenza A virus protein NS1 counteracts the anti-viral activity of PARPs and ADP-ribosylation, assigning a new activity to the primary viral antagonist of innate immunity. We identify PARP1 as the enzyme producing the majority of poly(ADP-ribose) present during infection. Influenza A virus replicates faster in cells lacking PARP1, linking PARP1 and ADP-ribosylation to the anti-viral phenotype. Together, these data establish ADP-ribosylation as an anti-viral innate immune-like response to viral infection antagonized by a previously unknown activity of NS1.

Introduction
Diverse cellular stresses, including infection, reprogram the post-translational modifications of proteins, rapidly altering existing cellular landscapes to promote survival of the host. These modifications dynamically modulate target protein function, abundance, turnover, and localization, playing important roles in immune activation during infection1–4. Multiple lines of evidence have recently implicated ADP-ribosylation, along with ADP-ribosyltransferases (commonly known as PARPs5), as an important part of this anti-pathogen response6–10. Many PARP genes are induced by interferon (IFN) signaling, suggesting a role in the response to pathogens11–15. Moreover, multiple PARP genes show signs of strong positive selection and other hallmarks of evolutionary conflicts at the virus:host interface16,17. Successful viruses, therefore, need to antagonize, evade, or co-opt PARP-mediated cellular countermeasures to ensure their fitness. Consequently, multiple positive-sense RNA viruses are known to encode enzymes that remove ADP-ribose (ADPr) from proteins6,8. PARPs and ADP-ribosylation are an under-appreciated arm of cellular anti-viral responses.

PARPs affect diverse biological functions, including DNA repair, gene regulation, and modulation of immune responses18. Of the 17 members of the human PARP family, up to 15 are catalytically active and transfer ADPr moieties from nicotinamide adenine dinucleotide onto target proteins or nucleic acids19,20. The extent and form of ADP-ribosylation can exert differential effects on the modified protein18,21,22. PARPs can attach a single ADPr to a target protein [mono(ADP-ribosyl)ation or MARylation] or create multimeric chains of ADPr [poly(ADP-ribosyl)ation or PARylation]. ADP-ribosylation is dynamic and reversible. PARPs add ADPr to targets while ADP-ribosylhydrolases reduce PAR chain lengths or completely remove ADPr23–25.

Influenza is a recurring global threat to animals, including humans. Viral replication is entirely dependent upon the infected cell, with influenza viruses usurping host factors to support replication while avoiding or evading anti-viral factors. Here, we show that influenza A virus infection causes a dramatic upregulation of PARylation that inhibits viral replication. We define the global ADP-ribosylome during influenza A virus infection, mapping thousands of modifications on viral and host proteins at single amino acid resolution. Infection induces a significant increase in PARylation to already modified proteins. Modifications on the viral replication machinery disable its activity, whereas the anti-viral activity of ADP-ribosylation is suppressed by the influenza A virus protein NS1. PARP1 is identified as the primary enzyme responsible for infection-induced PARylation and its anti-viral activity. Thus, ADP-ribosylation tempers viral replication as a key component of cellular anti-viral responses to influenza A virus, a process that is counteracted by the viral NS1 protein.

Results
Influenza A virus infection triggers PARylation with modification of viral proteins
We had previously performed a gene correlation analysis that linked expression of cellular genes to susceptibility to infection26. Among the putative anti-viral factors with the strongest negative correlation scores, we identified a PARP family member clustering with the interferon-induced transmembrane proteins (IFITMs) 1, 2, and 3, known inhibitors of several RNA viruses26,27. We therefore investigated whether influenza virus infections triggered an ADP-ribosylation response. Basal levels of ADP-ribosylation were detected in mock-infected lung cells, whereas infection with influenza A virus increased total ADP-ribosylation, as stained by a pan-ADPr reagent (Fig. 1A). The increase is typified by the appearance of discrete ADP-ribosylated proteins on blots as well as higher-molecular weight smears. As a positive control, lung cells were also treated with hydrogen peroxide (H2O2), a known inducer of ADP-ribosylation, resulting in robust detection of ADPr. Blotting was repeated using reagents specific for MARylation or PARylation, revealing that infection increases PARylation whereas MARylation was largely unchanged, if not slightly reduced.

ADP-ribosylation is highly dynamic and reversible through the activity of various enzymes such as poly(ADP-ribose) glycohydrolase (PARG)25. Therefore, we repeated infections in the presence of the PARG inhibitor PDD00017273. PDD treatment increased the PARylation signal from infected cells compared to untreated infected cells, whereas ADP-ribosylation was largely unchanged in all conditions for mock-infected cells (Fig. 1B). These data confirm that infection causes a marked induction of PARylation. PDD treatment was included in subsequent experiments to facilitate detection of PARylated proteins. Infection with influenza virus also upregulated ADP-ribosylation in cells derived from chickens, pigs, and bats (Fig. 1C), all of which are natural hosts of influenza A virus28. Next, we exposed cells to infectious virus, or the same dose of UV-inactivated particles. Only cells exposed to replication-competent virus showed an increase in ADP-ribosylation (Fig. 1D). Finally, we asked whether viral RNA alone or innate immune activation is sufficient to induce ADP-ribosylation. Genomic RNA was isolated from influenza virions and transfected into A549 cells. Cells were also treated with poly(I:C), a dsRNA mimetic that stimulates innate immune responses, and with interferon. We confirmed that viral RNA and poly(I:C) triggered immune sensing, indicating successful introduction of these nucleic acids (Supplementary Fig. 1A, B). Nonetheless, while infection again induced PARylation but not MARylation, the innate-immune stimulators did not (Fig. 1E and Supplementary Fig. 1C). Thus, global PARylation changes are a common response to replication of influenza A virus.

To specifically test whether infection-induced ADP-ribosylation results in modification of viral proteins, we immunopurified NP from infected cells (capturing NP, as well as NP assembled into RNPs with the viral polymerase). Blotting of NP purified from infected cells revealed robust ADP-ribosylation indicative of PARylation, which was absent in control conditions (Fig. 1F). In an orthogonal approach, we first enriched ADP-ribosylated proteins from cell lysates using the recombinant macrodomain protein Af1521, which has high affinity for ADPr, and then probed for NP. NP was selectively captured by Af1521, but not the ADPr-binding mutant Af1521G42E (Supplementary Fig. 1D). To further confirm the specificity of blotting and ensure detection of bona fide ADP-ribosylation events, we immuno-purified RNPs from infected cells and split samples into parallel blots. For the control membrane, western blots revealed ADP-ribosylation consistent with modifications to NP and the viral polymerase (Supplementary Fig. 1E, left). The other membrane was treated with hydroxylamine (HAM) (Supplementary Fig. 1E, right), which releases ADPr conjugates, primarily from modified aspartates, glutamates, and arginines29. HAM treatment markedly reduced detection of ADP-ribosylation, confirming that viral proteins are direct targets of PARP activity. HAM treatment did not completely eliminate the signal; fainter signal was still detected at the expected molecular weight of NP, suggesting that it may be modified at residues resistant to HAM treatment (e.g., histidines, cysteines, serines, or threonines). Together, these data show that influenza A virus infection induces a global upregulation of PARylation, including the modification of viral proteins.

Global identification of ADP-ribosylation sites during infection includes functionally important modification of viral proteins
Despite the broad impact of ADP-ribosylation on viral infections7, global analyzes of ADP-ribosylated proteins have largely been studied in the context of DNA damage and oxidative stress30,31. Therefore, we evaluated how influenza A virus infection alters the ADP-ribosylated proteome using enzymatic labeling of terminal ADPr coupled to mass spectrometry (ELTA-MS)31,32. ELTA selectively modifies ADP-ribosylated peptides with “clickable” N-azido-labeled tags, thereby enabling highly selective enrichment followed by MS for site identification. We defined the ADP-ribosylome in two biological replicates of A549 lung cells infected with the WSN strain of influenza A virus, mock-infected cells, or cells treated with H2O2 (positive control). Each sample was analyzed in technical triplicate. A peptide counted as ADP-ribosylated when it was detected in at least two technical replicates from the same biological replicate, one of which was by direct identification via tandem mass spectrometry (MS/MS). For increased stringency, we only considered modified peptides with site localization greater than 0.9. These results demonstrate high reproducibility across both technical and biological replicates, along with consistent identification of modification sites across ELTA-MS experiments at a range of amino acids, some of which were further validated through manual inspection (Supplementary Figs. 2 and 3 and Supplementary Data 1). For each condition, even with very strict cutoffs, we identified between ~850 and 1300 ADP-ribosylation sites on 500–700 proteins (Fig. 2A). When only considering those present in both biological replicates, we still identify ~800–1100 sites in ~440–530 proteins (Fig. 2B and Supplementary Fig. 2A). Interestingly, while infection produces a distinct ADP-ribosylation profile in A549 cells, there was a high degree of overlap among experimental conditions (i.e., mock, infected and H2O2-treated). Over half of the modified proteins, and almost three-quarters of all modified sites, were shared across all three conditions with a similar distribution of amino acid residues that were modified (Fig. 2B, C). This collection of shared proteins was highly enriched in gene ontology (GO) terms for protein translation (Supplementary Fig. 3B).

We also identified ADP-ribosylation sites on eight of the ten canonical influenza A virus proteins (Fig. 2D, E). The distribution of amino acids modified in viral proteins was similar to that of host proteins, with the exception that there were no modified cysteines detected in viral proteins (Fig. 2C). NP repeatedly had the highest number of modifications, consistent with our biochemical approaches that detected ADP-ribosylated NP (Fig. 1F and Supplementary Fig. 1D). It is unclear whether this is because NP is the most frequently modified viral protein, or because it is one of the most abundant proteins in infected cells. Together, these findings suggest the presence of a core ADP-ribosylome in A549 cells and show that influenza A virus induces a unique profile of ADP-ribosylated proteins and modification sites, including viral proteins.

Although blotting revealed significant increases in PARylation, ELTA-MS showed that the total number of modified proteins or sites does not change between mock and infected cells (Fig. 1A–C versus Fig. 2A, B). ELTA-MS uses a phosphodiesterase to cleave the pyrophosphate bond in ADPr to leave a single phosphoribosyl moiety that enables an unambiguous identification of ADP-ribosylation sites31. However, this cleavage eliminates the length information originally associated with the modified site (e.g., MAR versus PAR). To directly detect changes in polymer length, we used ELTA to label ADPr isolated from cells that were infected, mock-infected, or exposed to H2O2 as a positive control. Infection dramatically increased PAR chain length, whereas PARylation was below the level of detection on mock-infected samples (Fig. 2F). H2O2 treatment induced higher levels of PARylation consistent with our blotting. These results help resolve the apparent contradiction between our blotting and MS data and suggest that infection does not alter which sites are modified, but instead primarily changes the ADP-ribosylation form from MAR to PAR or increases the frequency of modification at a particular site.

A panel of 18 NP variants representing high-confidence modification sites was used to determine the functional effects of ADP-ribosylation (Supplementary Data 1). ADP-ribosylation sites were found in the head and body domains of NP, the tail loop that mediates NP oligomerization, and flanking the positively-charged RNA binding groove (Supplementary Fig. 4A, B). ADP-ribosylation sites were ablated by changing the modified residues to alanine, and NP function was assessed in a polymerase activity assay. Polymerase activity increased for several of the NP ADPr-site variants, with the most pronounced effects observed for variants D34A, S69A, and E294A (Fig. 2G). In some cases like NP T92A or E107A, the variants supported activity approaching that in control conditions with wild-type NP even though the amino acid substitutions appeared to affect the overall stability or expression of NP, perhaps masking a net increase in NP activity (Fig. 2G). These data suggest that ADP-ribosylation at certain sites on NP confers an anti-viral phenotype. Insights from deep mutational scanning of NP revealed selective pressure favoring substitution at residue 294 that cannot undergo ADP-ribosylation, reinforcing that ADP-ribosylation may attenuate NP function (Fig. 2G)33. Curiously, the E252A variant resulted in a complete loss of polymerase activity (Fig. 2G), in agreement with prior work33.

Influenza A virus NS1 suppresses PARylation
Our data indicated that ADP-ribosylation is part of the anti-viral response to influenza A virus infection. Influenza virus NS1 is the primary viral protein that antagonizes host anti-viral responses and was previously shown to reduce ADP-ribosylation of argonaute RISC catalytic component 2 (AGO2) complexes34,35. Therefore, we investigated the impact of NS1 on global ADP-ribosylation by infecting cells with two closely related strains of influenza A virus encoding NS1 proteins of different potency: WSN, which was used in experiments described above, and A/Puerto Rico/8/1934 (H1N1; PR8). Both strains induced ADP-ribosylation, although PARylation was lower in cells infected with PR8 compared to WSN, despite similar infection levels (Fig. 3A). PARylation was transiently induced regardless of the strain, as indicated by the ability of PARG inhibitor to preserve the signal. PARP1, in addition to functioning as an ADP-ribosyltransferase, is a target for caspases and its cleavage is frequently used as a marker for the activation of apoptosis36,37. Influenza virus infection triggers apoptosis, which plays dual pro- and anti-viral roles during infection, and the regulation of apoptosis by influenza A virus is important for successful replication38–40. PARP1 cleavage was more pronounced in cells infected with WSN compared to PR8 (Fig. 3A). Curiously, WSN-infected cells had higher levels of PARylation despite also exhibiting higher levels of PARP1 cleavage.

Next, we tested a direct role of NS1 in regulating PARylation by infecting cells with a virus that lacks the NS1 gene (PR8ΔNS1). PARylation was dramatically increased in cells infected with PR8ΔNS1 compared to PR8, even though PR8ΔNS1 virus is highly attenuated as revealed by lower levels of NP (Fig. 3B). As before, ELTA labeling revealed that infection caused a dramatic increase in ADPr polymer length and the amount of ADPr polymers was even more pronounced in cells infected with ΔNS1 virus (Fig. 3C). NS1 expression also reduced viral protein PARylation. Influenza A virus RNPs were assembled by expression in cells in the presence or absence of NS1. RNPs were immunopurified with antibodies against NP and probed for PARylation, revealing much less PARylation of viral proteins when NS1 was present (Fig. 3D). These data show that NS1 antagonizes global PARylation during infection while also reducing the PARylation intensity associated with viral proteins.

To further investigate the kinetics of PARylation and the associated role of NS1, we performed single-cycle infections in A549 cells with PR8 or PR8ΔNS1. For PR8, a slight increase in PARylation was first detectable at 8 hpi, i.e., in a later stage of infection (Fig. 4A). PARylation levels then continued to increase over the time course. In contrast, PARylation was apparent as early as 4 hpi in cells inoculated with PR8ΔNS1. As before, PARylation continued to increase over the time course, but at much higher levels. The increase in PARylation with PR8ΔNS1 was associated with higher levels of PARP1 cleavage (Fig. 4A). This is consistent with the ability of NS1 to downregulate apoptosis41. NP is also a target for caspase cleavage, resulting in the removal of ~16 amino acids from the N-terminus42. Blotting revealed earlier NP cleavage in PR8ΔNS1-infected cells compared to PR8-infected cells, again linking increased PARylation with apoptotic activity (Fig. 4A).

We capitalized on the robust ADP-ribosylation during PR8ΔNS1 infection to test functions of NS1 that are important for controlling PARP activity. We induced PARylation by inoculating cells with PR8ΔNS1 and then attempted to rescue the ΔNS1 phenotype by providing NS1 in trans by co-infection with virus encoding different NS1 proteins. Paralleling prior results, PR8 NS1 completely suppressed PARylation while WSN NS1 reduced, but did not eliminate it (Fig. 4B). We confirmed that this difference between PR8 and WSN was due solely to NS1 by swapping this gene between each virus. WSN encoding NS1PR8 (WSN NS1PR8) was now able to potently suppress PARylation, whereas PR8 encoding NS1WSN (PR8 NS1WSN) caused less suppression (Fig. 4B). One of the major mechanisms by which NS1 antagonizes host responses is through binding double-stranded RNA to prevent its sensing by innate immune activators43,44. We therefore generated virus encoding a variant that no longer binds double-stranded RNA (NS1 R38A/K41A)43. NS1 R38A/K41A lost its ability to suppress PARylation, with results indistinguishable from infection by PR8ΔNS1 alone (Fig. 4B). These data reveal that NS1 counteracts the anti-viral activity of PARPs and ADP-ribosylation, possibly through its ability to bind dsRNA.

NS1 suppresses PARylation but does not reduce the number of ADP-ribosylation sites
As NS1 suppresses global PARylation, we performed ELTA-MS to compare infections in the presence or absence of NS1. In one set of experiments, we compared infections with WSN, PR8, and PR8ΔNS1 (ELTA-MS #2 in Fig. 5A, C, E). In the other set, we also included WSNΔNS1 (ELTA-MS #3 in Fig. 5B, D, F). Because viruses encoding NS1 deletions are attenuated, infection conditions were optimized for each virus to ensure equivalent expression levels of viral proteins. Each condition identified between ~1050 and 2400 modified sites on ~380–660 proteins (Fig. 5A–D and Supplementary Data 2 and 3). ADP-ribosylation sites were identified on up to eight different viral proteins, where again NP was the viral protein with the greatest number of ADP-ribosylation sites (Fig. 5E, F).

Across all datasets, we detected ADP-ribosylation of proteins involved in diverse cellular functions, including consistent enrichment of proteins involved in protein binding, RNA binding, cadherin binding, and cell adhesion molecule binding (Supplementary Data 4). Infections induced a unique profile of ADP-ribosylation, while significant overlap among conditions reinforced the idea of a core ADP-ribosylome in A549 cells. Most of the modifications were shared across independent experiments, highlighting the reproducibility of ELTA-MS (Supplementary Fig. 5A). There was a high degree of overlap among modified proteins and sites for cells infected with WSN or PR8 with no notable change in the distribution of residues that were modified (Supplementary Fig. 5B, C). When considering all modified proteins identified for each condition in ELTA-MS #1-3, we again see a core ADP-ribosylome with most modified proteins are shared across conditions (Supplementary Fig. 5D). Remarkably, despite large differences in the intensity of PARylation revealed by blotting, cells infected with WT and ΔNS1 mutant virus had comparable numbers of ADP-ribosylation sites and modified proteins, with the majority of sites and proteins found in both conditions (Fig. 5C, D). This result was consistent among WSN and PR8 strains.

PARP1 directs anti-viral PARylation
Our data show that infection primarily changes PARylation. PARylation is performed by a limited subset of PARPs – PARP1, PARP2, PARP5a (TNKS) and PARP5b (TNKS2)19. ELTA-MS identified ADP-ribosylation at two canonical auto-modification sites in PARP1, E488 and E491, indicating that at least PARP1 was active in our infected cells (Supplementary Fig. 2 and Supplementary Data 2 and 3)45. To specifically test which PARPs are involved in the response to influenza virus infection, we measured PARylation in infected cells treated with a series of inhibitors possessing different specificities: PJ34 is a broad-specificity PARP inhibitor; Olaparib and Rucaparib target PARP1 and PARP2 with some off-target activity against other PARPs; AG14361 selectively inhibits PARP1; and, XAV939 primarily targets PARP5a/TNKS and PARP5b/TNKS218,46. ADP-ribosylation was unaffected by XAV939 in cells infected with either WSN or PR8, eliminating PARP5a/TNKS and PARP5b/TNKS2 as candidates (Fig. 6A). By contrast, PARylation was inhibited by Olaparib, Rucaparib, and the PARP1-specific AG14361, implicating PARP1 as the dominant enzyme mediating PARylation. Interestingly, caspase cleavage of NP still occurred under all conditions, indicating that infection-induced apoptosis does not require PARylation (Fig. 6A). Next, we purified viral RNPs from infected cells and probed them for MARylation or PARylation. Treating infected cells with Olaparib did not affect MARylation of the purified RNPs, but dramatically reduced the degree of PARylation (Fig. 6B). We generated two independent PARP1 knockout A549 cell lines to directly test its activity during infection (Fig. 6C). We infected these cells with PR8ΔNS1 to create a highly sensitized setting to observe changes in ADP-ribosylation. Infection-induced PARylation was almost completely absent in both PARP1 knockout cell lines (Fig. 6D). We repeated these experiments using WT PR8 and WSN. Again, the global increase was absent in PARP1 knockouts (Fig. 6E). Together, these data show that PARP1 mediates the majority of PARylation on viral proteins as well as a global increase in the cell, whereas viral proteins are MARylated by an unknown PARP.

PARP1 both initiates ADP-ribosylation via MARylation and extends chains via PARylation47. We investigated these activities by complementing our knockout cells with PARP1, PARP1 E988A (inactive catalytic site), or PARP1 E988K (only able to MARylate)47,48. Complementation with WT PARP1 restored PARylation in cells infected with WSN or PR8 as revealed by blotting for PAR and pan-ADPr (Fig. 7A and Supplementary Fig. 6A). This result confirms that the knockout cell phenotype was due to the loss of PARP1, rather than off-target effects. Expression of the catalytically inactive PARP1 E988A did not restore PARylation, nor did expression of the MARylating PARP1 E988K change MARylation patterns, but some of this may be due to its lower enzymatic activity. Complementation with WT PARP1 resulted in an apparent minor increase in MARylation compared to the parental cells, overlapping with the high-molecular smearing in the PAR blot. Whether this represents actual MARylation by PARP1, or results from the low levels of cross-reactivity towards PAR reported for the MAR detection reagent, needs to be determined49. Curiously, complementation with a PARP1 variant D214A, which cannot undergo caspase cleavage, failed to restore PARylation in infected cells (Fig. S6B). These data indicate that PARP1 drives global PARylation during infection, but that other PARPs likely contribute the majority of MARylation.

Finally, we tested the effect of PARP1 on viral replication by infecting PARP1 knockout cells. Multi-cycle replication assays were initiated in A549 and PARP1 knockout cells. Virus replicated to ~fivefold higher titers in cells lacking PARP1 during the early stages of infection (Fig. 7B). Viral titers ultimately reached the same plateau in both cell lines. In a single-cycle replication assay, PARP1 knockout cells again produced fivefold more virus than parental A549 cells (Fig. 7C). Thus, PARP1 orchestrates a global increase in PARylation during infection, establishing an anti-viral activity that slows the replication of influenza A virus.

Methods
Cell lines
Authenticated stocks of the following cell lines were purchased from the American Type Culture Collection: human embryonic kidney 293T cells (CRL-3216), human lung A549 cells (CCL-185), Madin-Darby canine kidney cells (MDCK; CCL-34), chicken UMNSAH/DF-1 cells (CRL-3586), pig kidney PK(15) cells (CCL-33), and Brazilian free-tailed bat lung Tb1Lu cells (CCL-88). The A549 innate immune reporter cell line encodes an integrated IFN-stimulated response element (ISRE) promoter expressing nanoluciferase-2A-GFP fusion protein. All cell lines were grown at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum. Media for the A549 ISRE reporter cells was supplemented with 5 μg/ml blasticidin. Cells were regularly tested and verified as free of Mycoplasma contamination using MycoAlert (Lonza LT07-318).

A549 PARP1 knockout cells were generated by co-transfecting the Cas9-expressing plasmid pMJ920 (Addgene 42234) and two different sgRNA-expressing plasmids targeting CCACCTCAACGTCAGGGTGC and TGGGTTCTCTGAGCTTCGGT creating a ~35bp deletion. Cells were transfected with TransIT-X2 (Mirus MIR6005) in 6-well plates using the recommended protocols. 24 h after transfection, clonal cell lines were generated by seeding on average 0.5 cells per well in a 96-well plate, expanding clones, and confirming the loss of PARP1 by blotting.

Antibodies and ADPr detection reagents
Immunoblotting was performed with mouse α-tubulin DM1A (Sigma T9026), goat α-RNP (BEI Resources NR-3133), α-β actin (Proteintech 66009-1-Ig), and α-PARP1 (Cell Signaling 9542S). Immunoprecipitations were performed with mouse α-FLAG M2 (Sigma F1804) or α-NP H16-L10 (Bio X Cell BE0159) that was then captured on protein A agarose beads. ADP-ribosylation was detected by blotting with reagents specific for MAR (Bio-Rad AbD43647 and EMD Millipore MABE1076), PAN (EMD Millipore MABE1016) and PAR (EMD Millipore MABE1031)49,73. ADP-ribosylated proteins were enriched with GST-Af1521 that was expressed and purified from bacteria as described30. Where indicated, membranes were incubated overnight at room temperature with 1 M hydroxylamine (HAM; NH2OH) diluted in Tris-buffered saline and 0.5% Tween 20 before blotting for PAR.

Stable expression of PARP1
A549 PARP1 knockout cells were complemented using the Sleeping Beauty transposon system74. Briefly, A549 PARP1 knockout cells were co-transfected with 0.2 μg of pSB100 and 1.8 μg of pSBbiBP-PARP1-V5 or PARP1 mutant plasmids. Cells were transfected with TransIT-X2 (Mirus MIR6005) in 6-well plate using the recommended protocols. 24 h after transfection, cells were selected with to 1 μg/ml puromycin for 7 d. Expression of PARP1 or mutants was confirmed by blotting with anti-PARP1 or anti-V5-tag antibody.

Viruses and infections
Influenza A virus A/WSN/33 (H1N1; WSN), WSN encoding FLAG-tagged PB2 polymerase subunit (WSN-PB2-FLAG), and A/Puerto Rico/8/1934 (H1N1; PR8) were generated using the influenza A virus reverse genetics system75,76. PR8 that lacks NS1 (PR8ΔNS1) or encodes the RNA-binding mutant (NS1 K38A/R41A), as well as WSN that does not encode NS1 (WSNΔNS1), were propagated and titered on MDCK cells overexpressing NS1-GFP.

293T cells were infected in virus growth medium (VGM; DMEM supplemented with penicillin/streptomycin, 25 mM HEPES, 0.3% BSA). A549 cells were infected in VGM with 0.5 μg/ml TPCK-trypsin or Opti-MEM I medium with 2% FBS and 0.5 μg/ml TPCK-trypsin. For the detection of global ADP-ribosylation, A549 cells were infected at MOI 0.1, UMNSAH/DF-1 cells were infected in OptiMEM + 0.2% heat-inactivated FBS at MOI 1, and PK(15) and Tb1Lu cells were infected in OptiMEM + 2% FBS at MOI 1.

Multicycle replication infections were performed by inoculating cells with PR8 at MOI 0.01 in VGM with 1.0 μg/ml TPCK-trypsin and supernatants were collected from 8 to 72 hpi. Single cycle infections were performed by inoculating cells with PR8 at MOI 5 in VGM with 1 μg/ml TPCK-trypsin and progeny virus was collected at 8 hpi. Viruses were titered by plaque assay on MDCK cells.

Detection of ADP-ribosylation
Cells were mocked-treated or infected with the indicated viruses. In some experiments, the PARG inhibitor PDD00017273 (MedChemExpress HY-108360) was added to a final concentration of 3 μM for 24 h before lysis. As a positive control, uninfected cells were exposed to 1 mM H2O2 for 10 min immediately before lysis. Cells were lysed in cold MOPS-RIPA buffer (16 mM MOPS pH 7.5, 150 mM NaCl, 1% deoxycholic acid, 0.2% SDS, 2% NP-40 with protease inhibitors, 1 μM ADP-HPD [Sigma 118415], and 40 μM PJ-34 [Millipore Sigma 528150]) or Tris-RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% deoxycholic acid, 0.1% SDS, 1% NP-40 with protease inhibitors and 1 μM ADP-HPD). The generic PARP inhibitor PJ-34 was included to prevent spurious ADP-ribosylation that can occur in lysates50. ADP-ribosylation was detected by blotting with purified MAR, PAN or PAR detection reagents49. ADP-ribosylation was measured in the presence of the PARP inhibitors Olaparib (5 μM; MedChemExpress HY-10162), rucaparib (5 μM; MedChemExpress HY-10617A), AG14361 (10 μM; Sigma SML3081), XAV939 (5 μM; TOCRIS 3748), or PJ34 (4 μM; MedChemExpress HY-13688A). Stocks were prepared by dissolving inhibitors in DMSO. Cells were treated 1 h after infection.

Innate immune activation and ADP-ribosylation
To test the ability of influenza virus genomes to induce ADP-ribosylation, viral RNA was purified from virions using Trizol (Invitrogen) and transfected into A549 ISRE reporter cells using TransIT-X2 (Mirus, MIR 6005) according to the manufacturer’s instructions. As controls, cells were either mock treated, transfected without nucleic acid, transfected with high-molecular weight poly(I:C) (Invivogen tlrl-pic), incubated with 250 U/mL IFN-β, treated with 1 mM hydrogen peroxide (H₂O₂) for 10 min, or infected with WSNΔNS1 at an MOI of 1. Cells were analyzed at 24 h post transfection or infection. ADP-ribosylation was detected by blotting as described above. ISRE activation was measured by imaging GFP fluorescence using EVOS FL Auto Imaging System (Thermo Fisher) under consistent exposure settings or by using the Nano-Glo Luciferase Reporter Assay System (Promega, N1120) according to the manufacturer’s instructions.

RNA sequencing
RNA-sequencing was previously reported77. Briefly, A549 cells were mock-infected or inoculated with WSN at an MOI of 0.02 in VGM with 0.25 μg/ml TPCK-trypsin. Infections were allowed to proceed for 24 h before RNA was harvested. In a separate condition, cells were mock-infected for 16 h followed by 8 h of interferon β (IFN-β) treatment (250 μ/ml). Each condition was repeated in biological triplicate. RNA was extracted from cells using TRIzol (Invitrogen 15596026) per manufacturer’s specifications. RNA samples were submitted to Novogene and prepared for mRNA sequencing using NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs E7530). Reads were trimmed and mapped to a concatenated hg19-WSN genome using Spliced Transcripts Alignment to a Reference (STAR)78. Differential expression was evaluated with DESeq279.

Polymerase activity assay
293T cells were transfected with plasmids encoding WSN PA, PB1, PB2, and NP, a viral RNA-like firefly luciferase reporter, and a Renilla luciferase internal control reporter using TransIT-2020 (Mirus MIR5400). 18 sites in NP above or near our 0.9 localization cutoff were selected for mutagenesis. Plasmids encoding mutant versions of viral proteins were created by PCR-mediated mutagenesis and verified by sequencing. Firefly and Renilla luciferase activities were assayed ~24 h post transfection. Firefly luciferase was normalized to Renilla luciferase within each sample.

ELTA labelling of ADPr
ADPr isolation and labeling was performed following prior approaches31. For WT virus samples, A549 cells were infected with WSN at an MOI of 2 for 24 h with PDD added to 3 μM 10 h before harvest. A mock-infected control was performed in parallel. For NS1 mutant virus samples, A549 cells were infected with WSNΔNS1 at an MOI of 2 for 10 h in the presence of 3 μM PDD. For H2O2-treated samples, cells were treated with 3 μM PDD for 10 h and then 1 mM H2O2 for 10 m. Incubations were terminated by washing cells with cold PBS and lysing them in 20% (w/v) cold trichloroacetic acid for 15 m. Proteins were precipitated, washed in 70% ethanol, dried, and resuspend in 0.5 M KOH with shaking at 37°C for 90 m to release ADPr from proteins. Sample were neutralized by the addition of MOPS to 0.8 M. Nucleic acids and proteins were digested by the addition of 25 μg DNase I (Roche 10104159001), 25 μg RNase A (Thermo EN0531) and 100 μg proteinase K (part of Roche 05080576001) with overnight incubation at 37°C with shaking. ADPr was isolated from each samples using the High Pure miRNA Isolation Kit (Roche 05080576001) following the manufacturer’s instruction eluting samples with 100 μl distilled water. ADPr samples were labeled in 10 μl reactions with 0.5 μg of OAS1, 5 μCi α-32P-dATP (Revity BLU512H250UC), and 50 μg/ml low molecular weight poly(I:C) (Invivogen tlrl-picw) in 20 mM Tris pH 7.5, 20 mM magnesium acetate, and 2.5 mM DTT at 37°C for 2 h. 6% of total isolated ADPr was used in each labeling reaction from infected and mock-treated samples. Given the extremely high degree of ADP-ribosylation caused by H2O2 treatment, only 1–1.5 % of the sample was used for labeling to allow for comparison on the same gel. Samples were denatured at 70°C for 10 min 25% urea, 12.5 mM NaCl, and 1 mM EDTA before separation on a 7 M urea 15% acrylamide gel using 0.5x TBE. Gels were analyzed by phosphorimaging.

Sample preparation for proteomic analyses
Three distinct ELTA-MS experiments were performed. In each experiment, each condition was performed in biological duplicate and samples were analyzed in technical triplicate. A549 cells were infected with WSN or PR8 at an MOI of 1 for 8 h and then treated with 1 mM H2O2 for 10 m or mock treated. In experiments with ΔNS1 viruses, infections with WSNΔNS1 or PR8ΔNS1 were allowed to proceed for 16 h such that viral protein levels were roughly equivalent with that in WT-infected cells. PDD was added to the cell culture prior to the end of the incubation. Cells were then placed on ice and quickly washed with cold PBS, which was then fully removed. Cells were lysed with freshly made guanidine-hydrochloride (GdnHCl) buffer heated to 99°C (6 M GdnHCl, 100 mM HEPES pH 8.0, 5 mM tris(2-carboxyethyl)phosphine [TCEP], 10 mM 2-chloroacetamide). Cells were scraped from the dish and transferred to an Eppendorf tube where they were incubated for an additional 10 min at 99°C. Samples from the same biological replicate were pooled, flash frozen in liquid nitrogen, and stored at –80°C until processing. Samples were thawed, and total protein concentration of lysates was determined by Bradford assay. Protein digestion reactions were prepared by diluting lysates containing 10 mg total protein sixfold in 100 mM Tris-HCl pH 8.0 and adding 100 µg Trypsin (Thermo Fisher 90058) and 100 µg LysC (Wako 129-02543). Samples were incubated overnight at 37°C while shaking at 200 rpm. Next, 1 M triethylammonium acetic acid (TEAA) pH 7.5 was added to peptide solutions to a final concentration of 100 mM before clarifying peptide solutions by centrifugation for 30 min at 4°C. 1 mg SepPak t-C18 Classic cartridges (Waters) were conditioned with 80% acetonitrile (ACN) and equilibrated with 100 mM TEAA pH 7.5 before loading peptide solutions. Peptides were washed with 100 mM TEAA pH 7.5 and eluted with 40% ACN. Samples were dried down to completion by vacuum centrifugation.

Enrichment of ADP-ribosylated peptides
ADP-ribosylated peptides were resuspended in MilliQ H2O and enriched using the ELTA-MS proteomics workflow31,32. First, peptides were incubated with 100 µM N6-(6-Azido)hexyl-dATP (Jena Bioscience CLK-NU-002), 20 µg/mL low molecular weight poly(I:C) (Invivogen tlrl-picw), and 20 µg/mL human 2´-5´-oligoadenylate synthase 1 (OAS1; prepared in-house as in ref. 31) in buffer containing 100 mM Tris-HCl pH 7.5, 20 mM magnesium acetate, and 2.5 mM dithiothreitol (DTT) for 1 h at 37°C while shaking at 800 rpm. 100 µL of 50% dibenzocyclooctyne (DBCO)-agarose (Click Chemistry Tools 1034) equilibrated in 1× PBS was added to each OAS1 reaction before rotating end-over-end at room temperature for 1 h. The samples were centrifuged to pellet the agarose resin and the supernatant was discarded. The agarose resin was washed three times with 5 M NaCl, three times with 20% ACN, and three times with 1× PBS. After washing the resin twice in phosphodiesterase buffer containing 100 mM HEPES pH 8.0 and 15 mM MgCl2, the samples were transferred to new Eppendorf tubes. Human nudix 16 (NudT16) was prepared as described80, 5 μg was added to each sample, and the volume was brought up to 200 µL with phosphodiesterase buffer. Samples were incubated for 2 h at 37°C while shaking at 1400 rpm. The samples were centrifuged to pellet the agarose resin and 150 µL of the eluate was transferred to new Eppendorf tubes. 10% trifluoroacetic acid (TFA) was added to samples to a final concentration of 1% TFA, and the samples were loaded onto homemade Stage-Tips that were conditioned with 80% ACN/0.1% TFA and equilibrated with 5% ACN/0.1% TFA. Peptide were washed with 0.1% TFA, eluted with 40% ACN/0.1% TFA, and dried down to completion by vacuum centrifugation.

LC-MS/MS analysis of enriched peptides
All enriched peptide samples were analyzed on a Thermo Orbitrap Fusion Lumos mass spectrometer coupled to an EASY-nLC 1200 system (Thermo) with a column type and stationary phase. LC Buffer A was 0.1% formic acid (FA), LC Buffer B was 0.1% FA/95% ACN, and the column oven was maintained at 40°C. Peptides were separated with a 95-min gradient from 8 to 27% Buffer B, followed by an increase to 50% Buffer B over 5 min, another increase to 90% Buffer B over 5 min, and finally a 10-min washing block. MS data were collected with a spray voltage of 2.4 kV, capillary temperature of 200°C, and RF lens of 30%. MS1 scans were performed at a resolution of 120 k, a scan range of 300 to 1800 m/z, a maximum injection time of 50 ms, and an automatic gain control (AGC) target of 1,000,000 charges. Precursor ions were isolated with a width of 1.6 m/z and an AGC target of 50,000 charges. Higher-energy collisional dissociation (HCD) fragmentation was performed with a normalized collision energy of 25% and a resolution of 30 k.

MS data analysis
Thermo .RAW files were converted to .mzML files using MSConvert from the ProteoWizard library81. Database searching was performed in FragPipe (v20.0) with MSFragger (v3.8), IonQuant (v1.9.8), and Philosopher (v5.0.0)82–84. The UniProt FASTA file for PR8 proteins PA, PA-X, PB1, PB1-F2, PB2, NP, HA, NA, M1, M2, NS1, and NEP/NS2 were downloaded from influenza A virus (strain A/Puerto Rico/8/1934 H1N1; Taxon ID 211044). The UniProt FASTA file for WSN proteins PA, PB1, PB2, NP, HA, NA, M1, M2, and NS1 were downloaded from influenza A virus (A/WSN/1933 H1N1; Taxon ID 382835) and WSN proteins PA-X and NEP/NS2 from influenza A virus (strain A/Wilson-Smith/1933 H1N1; Taxon ID 381518). The UniProt FASTA file for the human proteome was downloaded from UniProt Proteome ID UP000005640. The four FASTA files were manually concatenated in Python, and then decoys and contaminant proteins were added in FragPipe. A closed search in nonlabile mode was performed using the default FragPipe parameters with exceptions specified below. Fully enzymatic digestion used Trypsin and LysC rules. Variable modifications included 212.0086 (phospho-ribosylation) on DESKTYRCH (max 2 per peptide), 15.9949 (oxidation) on M (max 3 per peptide), 42.0106 (acetylation) on protein N-terminus (max 1), and –0.98401 (amidation) on protein C-terminus (max 1). The fixed modification was indicated as 57.02146 (carbamidomethylation) on C. The activation type filter was HCD and fragment ion series included b and y. Peptide-spectrum match (PSM) validation was performed with Percolator, PTM site localization was performed with PTMProphet, and protein inference was performed via ProteinProphet85–87. “Match between runs” (MBR) was turned on and the MBR ion false discovery rate (FDR) was set to 1%. “Normalize intensity across runs” was turned on and “peptide-protein uniqueness” was set to unique and razor peptides.

ELTA-MS uses high-energy collisional dissociation (HCD) for ADPr site localization, which can lead to mislocalization in some instances, especially on aspartate and glutamate residues88. We therefore also manually confirmed localization of representative spectra from host and viral proteins (Supplementary Fig. 2). Nonetheless, we cannot fully exclude the potential loss of Asp-ADPr and Glu-ADPr adducts under our experimental conditions that could lead to rare underreporting of modifications at these residues.

MS data filtering and visualization
All downstream analyzes of the DESKTYRCH_212.0086.tsv and combined_ion.tsv output files of the FragPipe search and plotting of the processed data were performed with homemade scripts in the statistical environment R (v. 4.3.2). Incorrectly assigned phospho-ribose sites on C were removed from the DESKTYRCH_212.0086 dataframe by comparing it to the carbamidomethylation sites on C in the combined_ion dataframe. Site indices [proteinID_aminoacid_residue] were calculated for the combined_ion data frame. Peptides with one phospho-ribose were labeled as “pep_one-mod” and peptides with two phospho-riboses were labeled as “pep_multi-mod” and then two separate index rows were made for each multiply-modified peptide. The MS data (RT, m/z, charge, PSMs, etc.) were copied to each separate index row associated with the multiply-modified peptide. To ensure reproducibility across technical replicates and filter for modified peptide identifications with direct MS evidence, we developed a formula to calculate a score for each site index. MS/MS identifications were assigned an arbitrary value of 3 and MBR identifications were assigned an arbitrary value of 1. For each biological replicate, we scored all cases in which the site was identified in at least two technical replicates. The score equaled the sum of MS/MS identifications multiplied by 3 plus the sum of MBR identifications multiplied by 1. Modified peptide identifications were filtered for scores greater than or equal to 4, indicating at least one technical replicate containing direct MS/MS evidence of the modified peptide and another replicate with either matching or direct evidence. For plotting, modified peptide identifications were condensed into unique site indices. For PSMs of site indices derived from multiple modified peptide sequences, the PSMs for each modified peptide associated with that site index were summed. For PSMs of modified proteins, the PSMs for every modified peptide/site index associated with that protein were summed, excluding the ones labeled “pep_multi-mod” to avoid double counting.

For the clustering heatmaps, unique site indices were filtered for localization probabilities greater than or equal to 0.9. Then, identifications with scores greater than or equal to 4 were assigned a value of one (present) and identifications that were unmatched or had a score of less than 4 were assigned a value of zero (absent). Clustering heatmaps were generated using the pheatmap function in R (v. 4.3.2) and the Jaccard (“binary”) clustering distance for categorical data. Heatmaps showing the number of sites, max score, or number of PSMs were generated using ggplot2 in R.

For Venn diagrams, modified site indices were filtered for localization probabilities greater than or equal to 0.9, whereas modified proteins were filtered less stringently for localization probabilities greater than or equal to 0.6 in order to increase the number of modified peptide identifications, since ADPr is a labile modification during HCD fragmentation and thus can be difficult to localize. Venn diagrams were generated using InteractiVenn89.

Statistics and reproducibility
All experiments were repeated with at least three independent biological replicates, with at least three technical replicates for each experiment. Representative replicates are shown as the mean ± standard deviation. Pairwise comparisons were made using a two-tailed Student’s t-test. Multiple comparisons were performed with an ANOVA followed by an ad hoc Dunnett’s multiple comparisons test. GO enrichment was performed in gProfiler (2.0) and ShinyGO (0.8)90,91.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Review on manuscript # NCOMMS-24-64945 submitted to nature communications\nIn this study “Global remodeling of ADP-ribosylation by PARP1 suppresses influenza A virus infection” the authors, encouraged by results from a previous study (gene correlation analysis), started with investigating the effect of different PARP enzymes on influenza virus replication. They all seem to interfere with viral replication and most PARPs also with viral polymerase activity. PARP8, identified by their gene correlation analysis, seems to reduce Influenza virus replication and gene expression, shown for several virus strains.\nFurther, they demonstrate an increase in PARylation signal upon infection of A459 cells with Influenza virus. To gain insight into this global change in PARylation signal, they aimed at identifying modified proteins and mapping modification sites using ELTA-MS. Host as well as viral proteins were identified with the viral NP protein carrying the most ADP-ribosylated sites among the viral proteins, which fits their westernblot analyses of the NP being modified.\nIn addition, the authors show robust evidence that the viral NS1 protein can modulate PARylation levels by an unknown mechanism. PARP1 has been identified to be the main protein contributing to changes in PARylation upon influenza virus infection and seems to have antiviral activity as overexpression interferes with viral replication, whereas the knockout allows for higher replication rates.\nIn the following minor comments on the text and Figures and major concerns are outlined.\nMinor comments on the text:\nLines 30 and 60\nThe ADP-ribosylation community agreed on a nomenclature for ADP-ribosyltransferases according to which “PARP” should be used as a name on its own right, rather than an abbreviation for poly-ADP-ribose, which also some of the authors agreed on. Polymerase does not reflect the enzymatic activity of the transferases, which transfer as ADP-ribose units to their substrates. That should be adapted.\nLines 72, 73\nLine 76\nPARPs do not attach multimeric chains to a target protein but ADPr by ADPr in an iterative process, which then results in PAR chains.\nLine 81\nRather state host factors then “Co-factors”, which doesn’t seem the right term in this context.\nLine 102\nWhy not naming PARP8 here as the PARP enzyme, they identified in their gene correlation analysis?\nLine 104\nSpecify PARPs (namely PARP8, 9, 10, 12, and 14) that are expressed upon infection or IFNß treatment. Figure S1C should be referred to as well here.\nLine 106:\nAs a more recent reference describing ADP-ribosyltransferases at the host virus interface, Luescher et al., 2022 “Intracellular ADP-ribosyltransferases at the host-virus interface” could be included as reference as well.\nLine 138:\nPDD treatment was included in subsequent experiments to facilitate the detection of ADP-ribosylated proteins. This should be changed to PARylated proteins, as PARG is inhibited which can only cleave glycosidic linkages and thus degrade PAR chains.\nLine 162:\n...induced expression of multiple PARPs and causes global up-regulation of ADP-ribosylation...\n5 out of 16 tested PARPs showing high expression upon infection are several but not multiple. Overstatements should be avoided.\nBecause MARylation is hardly if at all increased upon infection, the authors should talk about “global up-regulation of PARylation, instead of ADP-ribosylation.\nLine 191:\n“... with the exception that there were no modified cysteinyls detected in viral proteins (Figure 2C).” Figure 2C also does not indicate modified cysteines among the human proteins. Why do the authors point on this?\nLine 200:\n“...although blotting revealed massive increases in PARylation”. This seems overstated, when looking at Figures 2B, C, D and E. A massive increase is detectable upon H2O2 treatment.\nLine 201:\n“... does not change does not change”...\nLines 255, 256:\n„For PR8, increased PARylation was detected at 8 hpi, i.e. in a later stage of infection (Figure 4A). PARylation levels then continued to increase over the time course. In contrast, PARylation was apparent as early as 4 hpi in cells inoculated with PR8ΔNS1.”\nLooking at Figure 4A an increase in PARylation for PR8 can be seen 10 hpi, whereas an increase in PARylation was apparent as early as 6hpi in cells inoculated with PR8∆NS1.\nLines 288, 289:\nAs before, we identified ~ 2,600-3,000 modified sites in ~380-660 proteins (Figure 5A-B, Figure S5, Table S2-S3).\nWhere do these numbers come from?\nLine 321\n“PJ34 generically inhibits PARPs” compared to Wahlberg et al., 2012, Nature Biotechnology this statement is wrong, especially with regard to the IFN-sensitive PARPs which are also upregulated in this study (Supplementary Figure S1C), which are hardly inhibited by this compound.\nLines 368-372\nThis seems very speculative based on the data generated in this study.\nMinor comments on Figures:\nAmong all figures and panels representing westernblot analyses molecular weight marks should be added. This always makes it easier to comprehend the data. Further the figure legends as well as the labeling of the blots are quite minimalistic. Cells that have been used for infection should be indicated, the MOI of infection should be indicated and especially the hours post infection (hpi), when the data was obtained should be indicated. That would make it way easier to understand the figures without going through the text and material and method section. Further, in several panels controls are missing, such as a confirmation of infection or a loading control. This seems especially important when stating that there is in increase in overall ADP-ribosylation signal, which is hard to comprehend, when a loading control is missing. Also most input blots are missing these, as I assume that Input reflects the analysis of the whole cell lysate, before IP. Blots showing IPs should be labeled accordingly (IP: anti-FLAG for example). I recommend adapting these issues accordingly.\nAdditional issues to each figure or panel are outlined below.\nSupplementary Figure 1A\nThis Figure does like Figure 1D in reference 30 (same authors) a lot and should be indicated by including the respective reference.\nSupplementary Figure 1B/C\nAs stated in the figure legend, these graphs have been created from three replicates. I recommend visualizing individual data points as has been done for other graphs in this study as well.\nFigure 1A, Supplementary Figure S2D:\nA proof, that the individual PARPs are indeed expressed and to what extend they are overexpressed is missing here.\nFigure 1B:\nIt is hard to see the increase of MAR signal, the authors claim to see. MAR-signal appears rather reduced, as single bands appear fainter upon infection with IAV. Statements in the text should be adapted accordingly.\nFigure 1C/3A:\nAccording to the supplementary data, cutout of the PAR blot and cutout for NP and Actin signals seem to come from different experiments when looking at the overall loading pattern. Can the authors please comment on this?\nSupplementary Figure 2A-C\nFor these experiments the authors generated stable A549 cells, via lentiviral transduction, constitutively expressing PARP8. A proof is missing.\nFigure 3D\nJust out of curiosity, could the highly modified NP protein also be pulled out by AD1521 in Figure 3D?\nMajor concerns on Figures and Data:\nFigure 2A-D, Supplementary Figure S4, Table S1\nHere the authors performed ELTA-MS to analyze the global change in PARylation upon IAV infection.\nFirst, only two biological replicates have been performed which doesn’t allow identifying statistically significantly enriched ADP-ribosylated peptides.\nTable S1 supporting this figure is hard to understand, as neither the different conditions (H2O2-treatment, mock or IAV infection) can be identified within this table, nor the biological replicates.\nThe analysis of the technical triplicates is presented in Supplementary Figure S4. Here, Venn diagrams indicate ADP-ribosylated proteins and identified sites, meant to show high reproducibility between the technical replicates.\nOn the right-hand site the data from technical replicates is somehow summarized to reflect similarity of the biological replicates. How are numbers calculated for this? The numbers of identified proteins within the biological replicates do not fit at all to anything indicated with the technical replicates. In the text it is stated, that “a peptide counted as ADP-ribosylated when it was detected at least in two technical replicates” (lines 175-176), but even taken this into account does not sum up to the numbers indicated for the biological replicates. Based on these hardly understandable numbers, and poor Table, Figures 2B and 2C were created as a summary of the MS analysis and GO term analysis has been performed (Supplementary Figure S4B). Then there is another Venn diagram presented in Supplementary Figure S4C, which the authors do not refer to within the text. This figure indicates an overlap between H2O2 treated HeLa and A549 cells and Mock-infected A549 cells. I don’t see what this Figure supports within this study. Taken together, this makes it challenging to fully trust the results of the MS analysis.\nFigure 2E/F, Supplementary Figure S3B\nHere, the identified ADP-ribosylated sites within the viral proteins, especially the heavily modified NP are indicated. Based on this, they individually substituted the respective NP sites to Alanines. These mutants were then used to investigate polymerase activity and three were identified allowing higher activity.\nThe authors conclude, “that mutating these residues to prevent ADP-ribosylation enhanced polymerase activity, indicating that ADP-ribosylation at these positions disrupts function” (lines 391-393). This cannot be stated like this, because ADP-ribosylation of these sites has formally not been proven in an independent experiment. This could have been done by enrichment of the mutant and detection of ADP-ribosylation or by exploiting AF1521 for enrichment, that in turn would allow enriching the wildtype NP but not the respective mutant. This also could have been done in their polymerase assay with co-expression of e.g. PARP1, which should leave NP unmodified and allow for high activity.\nFigure 5, Supplementary Figure S5, Supplementary Table S2, S3, S4\nHere the authors performed ELTA-MS to confirm the effect of NS1 on global ADP-ribosylation events using two different Influenza strains either wildtype or ∆NS1. Again, two biological replicates have been analyzed in technical triplicates. Neither the different settings analyzed in each experiment, nor the biological replicates are indicated with the respective supplementary table. Both tables are hard to understand.\nHere, biological replicates are compared to each other and among the two experiments.\nThe authors state, that infections induced a unique profile of ADP-ribosylation, while significant overlap among conditions reinforced the idea of a core ADP-ribosylome in A549 cells (lines 294, 295). Supplementary Figure S6A shows these overlaps, either for PR8 of ELTA-MS#2 and #3 or WSN of all three ELTA-MS approaches. In total they now have 4 biological replicates of PR8 infected cells analyzed by ELTA-MS and 6 biological replicates of WSN infected cells. If identified proteins and sites are as conserved as claimed, why are Venn diagrams in Supplementary Figure S6B then separated based on the individual data sets? Which if these was taken to generate Supplementary Table S4, the GO term analysis? Combining all these data sets would allow for principal component analysis to visualize the clustering of the individual biological replicates and indeed allow to test significance of the individual hits. None of this has been done.\nTo claim, that a unique profile of ADP-ribosylation is induced upon infection a comparison to mock treated cells is missing to visualize proteins and sites that are uniquely enriched upon infection. Further, I wonder, why the analysis of the ∆NS mutants was not considered. As NS has been shown to somehow counteract ADP-ribosylation events, similar protein and sites should have been identified here but more enriched over control but this seems not the case (lines 306-308).\nThere are substantial things missing to come to their conclusion and the presentation of the MS analysis is not convincing.\nFigure 6A\nCut outs indicated in the supplementary showing NP and ß-actin do not fit the blots shown here.\nTaken together, this study shows a robust increase in PARylation signal upon IAV infection, confirmed by several immunblot analyses. Further, data indicating that this can be manipulated be NS1 of IAV in an unknown fashion is solid. Additionally, PARP1 has been identified as the PARP enzyme responsible for this increase in PARylation. Also, PARP1 has been identified being restrictive for IAV. Whether this indeed is dependent on its catalytic activity has not been formally investigated. Also, whether PARP1-mediated ADP-ribosylation of the NP protein is the main mechanism to interfere with IAV replication has not been proven. However, modification of NP during infection has been demonstrated.\nIn addition, the authors put a lot of effort and work in gaining insights into this global change of ADP-ribosylation upon IAV infection. However, this part of the study is challenging to comprehend and trust as outlined above. In the end it also seems that the MS-analyses do not reflect the initial results seen by Westernblot analyses, so conclusions are hard to be drawn.\nFor the discussion, the authors did neither consider reports on IAV-induced DNA damage events nor NS1 which also has been linked to DNA damage, suppressing DNA-repair mechanisms and manipulating DRR pathways. As PARP1 is best studied with regard to its function in DNA damage it seems obvious to discuss their results in light of this as well.\nIn the end an author contribution statement is missing!\nWith this said and considering all the concerns outlined above, I am unable to recommend this study for publishing in nature communications at this time.", "reviewer_2": "SUMMARY:\nThis paper provides an interesting study on the antiviral response to influenza by PARPs and is noteworthy for providing the first ADPr proteomics study during infection. The important conclusion is that PARP1 as the main enzyme responsible for infection-induced PAR. This original work is of significance to the ADPr/PARP field as well as to researchers interested in studying antiviral responses. It is generally well written and elegantly uses a variety of different approaches. I have technical concerns that need to be addressed to validate the conclusions related to the proteomics analyses.\nMajor points:\n1) Fig 2. The authors observe that infection does not significantly alter the number of modified sites in contradiction to their western blotting results and conclude that infection changes the ADPr for MAR to PAR. However, the same sites could be identified in different conditions but they might have different ratios. A quantitative proteomics analyses similar to the analyses performed by the Nielsen lab in their ADPr-related paper, would have been better, although for this 3-4 biological replicates should have been included per condition instead of the 2 the authors have in their current experimental setup.\n2) Fig 2, 5. Prior to the proteomics analysis, cells were lysed by incubation at 99 C followed by overnight tryptic digestion at pH 8. In light of recent studies on the lability of Asp-/Glu-ADPr (Weixler Life Science Alliance 2023; Tashirto J Am Chem Soc 2023; Longarini Nat Comms 2024), these conditions have most likely led to a significant loss of Asp-/Glu-ADPr prior to analyses by mass spectrometry. They authors might reply that their identification of a number of Asp-/Glu-ADPr sites is evidence that their approach works for this type of ADPr as well. However, the reported Asp-/Glu-ADPr sites might been identified due to mislocalisation by HCD (see comment below), similarly to the study by Martello Nature Comms 2016, whose Asp-/Glu-ADPr sites were not identified again in studies of the same lab once ETD was used. The authors should at least discuss the probable loss of Asp-/Glu-ADPr in their proteomics analyses.\n3) The authors use HCD for proteomics analyses of ADPr sites, but this fragmentation technique can lead to mislocalisation of some forms of ADPr, especially abundant Ser-ADPr. While they use a stringent cut-off of 0.9, the complete loss of phospho-ribose is probably interpreted automatically by the algorithms used for localization score as the erroneous evidence of lack of ADPr on a particular residue leading to a high score despite the actual uncertainty in localization. In HCD spectra the presence of phospho-ribose is actual evidence that that fragment ion contains phospho-ribose and this information can definitely be used for localization. In contrast, the absence of the modification can mean actual absence of the modification or compete loss of the modification due to its lability and standard software, made for stable PTMs, cannot distinguish between these two possibilities. Problematically it uses this lack of evidence as evidence of its absence providing a potentially misleading high localization score. None of this would have been an issue if the authors had used ETD, but perhaps this fragmentation technique is available on the Fusion used for these analyses? Another possibility would have been to manually validate the HCD spectra and provide those that allow confident localization, but this might not be feasible considering the high number of sites identified? Still the authors could provide a few annotated MSMS spectra showing confident localization for the most important sites identified, such as on NP. At least the authors should discuss this and provide all their raw files deposited in a public repository.\nMinor points:\nFig. 1B: A recently derived MAR antibody (clone AbD43647) has been shown to work well for the detection of MAR induced by interferon treatment of A549 cells (Kar et al. EMBO J 2024). While the vast majority of band in western blots performed by the authors don’t change when comparing A549 cells infected with WSN and mock-infected cells, these bands don’t appear to be recognized by AbD43647 in blots performed by Kar et al. EMBO J 2024 and at the same time several specific bands recognized by AbD43647 are induced by interferon. In addition, did the author consider the sensitivity of Asp-/Glu-ADPr to high temperature (Weixler Life Science Alliance 2023; Tashirto J Am Chem Soc 2023; Longarini Nat Comms 2024)? In particular, if the authors had performed the conventional boiling step prior to SDS-PAGE, much of Asp-/Glu-ADPr would have been lost favoring detection of more stable forms of ADPr, such as on serine. Therefore, AbD43647 together with cell lysis at room temperature could give cleaner and more informative results.\nPage 3, lines 63 - 64: ‘gene loss or duplication m— processes’ A few words seem to be missing.\nLines 201 – 202: ‘number of modified proteins or sites does not change does not change in mock and infected cells’: ‘does not change’ is repeated twice", "reviewer_3": "The manuscripts aims to describe and define the role of host mediated ADP-ribosylation on the antiviral immune response against Influenza A virus infection. The focus on ADP-ribosylation is largely based on pervious results as well as earlier reports that demonstrate a role of PARPs in the antiviral immune response as well as direct antiviral effects.\nThe manuscripts includes a large number of experiments. While the majority of the experiments and results presented are of high quality, well performed and interesting, the manuscripts addresses too many different aspects and thereby looses its focus and clarity. It partially reads like an assembly of old and new data that are related but just not made to fit together. Sometimes less really is more!\nAlready the characterization of the infection induced switch in mono and poly-ribosylation combined with the potential antagonistic function of NS1, including the dependency on its RNA binding activity, is so complex that it is would be sufficient for a manuscript.\nWhile all the open ends are addressed and well discussed in the final part, it remains unclear why so many aspects were included in the first place (what about PARP8 after all?). I strongly suggest to reduce the amount of data and lines of investigation to strengthen the focus on the remaining data and to improve the overall quality of the manuscript.\nE.g. the function and antiviral effect of ribosylation sites in viral proteins is an important aspect but it is here only investigated in the context of NP. The results are interesting but leave an incomplete picture on the functional repertoire of ADP-ribosylation on other viral proteins. The importance of these data for the overall message of this manuscript remains unclear and may be better placed in a separate manuscript.\nIn addition to the general comments there are several specific aspects to the figures and text that need to be addressed and improved.\nSpecific comments\nWhat is the relevance of the data in the first paragraph of the results? They address PARP related aspects, but don’t quite fit to the following layout of the manuscript. Why the focus on PARP8 here? How does this benefit the following data?\nFigure 1 A: Expression levels for the PARP overexpression are missing\nFigure 1B: Since many proteins are stained on the blot, the inclusion of size markers would be very helpful for orientation between panels B and C. It is very difficult to judge whether the similarity between the ADP-ribosylation patterns without size markers. Please adapt accordingly.\nFigure 1D: Why is there a decrease in PARylation in the UV treated virus infected cells? Can PARylation be induced by transfection of viral RNA?\nFigure 1 F: Is NP also modified when it is overexpressed alone, or is the induction through infection required?\nFigure 2E: Why are there orange marks on the upper and the lower side? Are these reflecting the results of the replicates? Please specify in the figure.\nLine 201: “does not change” is duplicated in the sentence and should be deleted.\nLines 235-239: The context of PARP1 cleavage is not sufficiently addressed here. Why was this assessed and what is the conclusion. How does this relate to the different viruses. The potential connection to NS1 is not sufficiently explained. Please add more explanation.\nFigure 3D: PB1 has only one ADP-ribosylation site so it may not be the best protein to be used in this assay as it easily underlies technical issues. An additional blot with NP that has multiple modified sites also show the same/similar effect of signal reduction would strengthen this result. How do these data fit to the results of the mass spec analysis with viruses lacking NS1 (Fig. 5)? These Data show no effect on PB1 ribosylation. Together these data leave a rather unclear role of NS1 on ribosylation.\nFigure 4: The results with NS1 are intriguing. The anti-PARylation activity was associated with the NS1 RNA binding activity. Does this mean, viral RNA sensing is the trigger for the changes in ADP-ribosylation? And can it be stimulated by transfection of viral RNA? These are important experiments to foster the claim of a novel function of NS1. It may rather be an indirect downstream effect of NS1 mediated inhibition of the RIG-I pathway. This needs crarification.\nFigure 5A: mock r2 is named strangely (mock r12) is that correct? Otherwise it should be corrected. There is huge difference in the ribosylation sites in the mock samples (r1, r2). How can this be explained?\nFigure 5C: Would you not expect to have more ribosylation sites on viral proteins under conditions lacking NS1? Results in Fig.3D and Fig. 4 suggested this outcome? Please explain. Maybe the data in Fig 3 and 4 should be omitted as they are kind of misleading. It is difficult to judge whether this is because of technical issues or the chosen analysis using only the anti PAR-antibody but not the anti-MAR-antibody.\nAlthough the authors suggest that instead of inhibiting/affecting ribosylation sites, NS1 could affect the transition from mono to poly-ribosylation. However there is no strong data for this hypothesis as direct comparisons between both ribosylation forms is missing. Unless there is an additional assay with clear results that support this hypothesis it should be strongly tuned down throughout the manuscript and the abstract. Results in Fig. 5C also suggest that there are other NS1-independent reasons for the observed differences the ribosylation patterns of viral proteins.\nFigure 6: The use of inhibitors in combination with KO cells is strongly appreciated as both approaches heavily affect cells and easily lead to artifacts and misinterpretation.\nWhat is the proof that the inhibitors work specifically at the used concentrations in the used cells? Positive controls and cytotoxicity assay should be provided.\nWhat is the effect of the inhibitors on viral replication? At least one PARP1 inhibitor should be investigated to complement the replication data in PARP1 KO cells.\nDiscussion:\n1. The first paragraph reads like a different paper due to the simplified and overstated summary of the presented results. Please refine this paragraph by including a more balanced data summary of the strengths, weaknesses and open questions.\n2. The data in supplementary figure one demonstrate that several PARPs are induced by viral infection, which is interesting. How is this finding related to the reported changes in the ADP-ribosylation? Later data include a major role of PARP1 in the switch to poly-ribosylation but it is not upregulated by IAV infection."}, "author_response": "We thank the reviewers for their constructive criticism that have significantly improved the\nmanuscript. We appreciate their assessment that “the majority of the experiments and results\npresented are of high quality, well performed and interesting” and that they report “original work\nof significance to the ADPr/PARP field as well as to researchers interested in studying antiviral\nresponses.”\nThe manuscript has been significantly improved by addressing the reviews that can be\nconsolidated into three major areas. As these changes address critiques that span across all\nthree reviews, we briefly summarize them here:\n• Additional experiments needed to support conclusions: A significant amount of new\ndata is included that directly tests PAR chain length (Fig 2F and 3C), assesses the\nability of viral RNA to trigger PARylation (Fig 1E and S1A-C), confirms ADPr site\nlocalization by manually inspecting spectra (Fig S2), repeats our results with UV-\ninactivated virus (Fig 1D), and obtains similar data with the new MAR-specific\nantibody (Fig 1A).\n• A concern on MS approaches: We provide additional detail, validation and\nappropriate caveats regarding technical issues in the ELTA-MS that might affect our\ninterpretation. Of note, we ensure our manuscript reports highly reproducible data by\nperforming 6 biological replicates of WSN, mock, and H O conditions, 4 biological\n2 2\nreplicates of PR8 and PR8∆NS1, and 2 biological replicates of WSN∆NS1. Each\nbiological replicate is then analyzed in 3 technical replicates and subject to strict\ncutoff thresholds as described in the Methods. Manual validation of representative\nspectra in Fig S2 shows replication across independent ELTA-MS experiments,\nreplication across conditions within a replicate, and modification of Ser, Asp, and Glu\nresidues.\n• A need for a clearer and more streamlined manuscript: The manuscript has been re-\norganized and focused with the removal of text and data that are no longer needed\nto support the main conclusions.\nWe have addressed each criticism in full to create a much stronger manuscript that enables\nbetter understanding for the reviewers/readers. Individual changes are detailed in the point-by-\npoint rebuttal below.\nReviewer #1 (Remarks to the Author):\nReview on manuscript # NCOMMS-24-64945 submitted to nature communications\nIn this study “Global remodeling of ADP-ribosylation by PARP1 suppresses influenza A virus\ninfection” the authors, encouraged by results from a previous study (gene correlation analysis),\nstarted with investigating the effect of different PARP enzymes on influenza virus replication.\nThey all seem to interfere with viral replication and most PARPs also with viral polymerase\nactivity. PARP8, identified by their gene correlation analysis, seems to reduce Influenza virus\nreplication and gene expression, shown for several virus strains.\nFurther, they demonstrate an increase in PARylation signal upon infection of A459 cells with\nInfluenza virus. To gain insight into this global change in PARylation signal, they aimed at\nidentifying modified proteins and mapping modification sites using ELTA-MS. Host as well as\nviral proteins were identified with the viral NP protein carrying the most ADP-ribosylated sites\namong the viral proteins, which fits their westernblot analyses of the NP being modified.\nIn addition, the authors show robust evidence that the viral NS1 protein can modulate\nPARylation levels by an unknown mechanism. PARP1 has been identified as the main\nprotein contributing to changes in PARylation upon influenza virus infection and seems to have\nantiviral activity as overexpression interferes with viral replication, whereas the knockout allows\nfor higher replication rates.\nIn the following minor comments on the text and Figures and major concerns are outlined.\nMinor comments on the text: We appreciate the reviewer’s close reading of our manuscript and\nhave made all the necessary text changes.\nLines 30 and 60\nThe ADP-ribosylation community agreed on a nomenclature for ADP-ribosyltransferases\naccording to which “PARP” should be used as a name on its own right, rather than an\nabbreviation for poly-ADP-ribose, which also some of the authors agreed on. Polymerase does\nnot reflect the enzymatic activity of the transferases, which transfer as ADP-ribose units to their\nsubstrates. That should be adapted.\nWe thank you for this suggestion and now use PARP as a name as opposed to an acronym.\nLines 72, 73\nascribed catalytic activity in complex with DTX3L based on more recent literature.\nThe text has been changed on line 70 to reflect that up to 16 PARPs are active.\nLine 76\nPARPs do not attach multimeric chains to a target protein but ADPr by ADPr in an iterative\nprocess, which then results in PAR chains.\nThe text on lines 73-75 has been changed to say PARPs attach single ADPr or create\nmultimeric chains.\nLine 81\nRather state host factors then “Co-factors”, which doesn’t seem the right term in this context.\nThis change has been made on line 79.\nLine 102\nWhy not naming PARP8 here as the PARP enzyme, they identified in their gene correlation\nanalysis?\nFollowing suggestions from Reviewers 1 and 3, we have refocused the manuscript and simply\nrefer to our prior publication identifying PARPs as regulators of viral replication.\nLine 104\nSpecify PARPs (namely PARP8, 9, 10, 12, and 14) that are expressed upon infection or IFNß\ntreatment. Figure S1C should be referred to as well here.\nFollowing suggestions from all Reviewers to focus the manuscript, this section has been\nremoved.\nLine 106:\nAs a more recent reference describing ADP-ribosyltransferases at the host virus interface,\nLuescher et al., 2022 “Intracellular ADP-ribosyltransferases at the host-virus interface” could be\nincluded as reference as well.\nThis citation is now included on line 60 when describing PARPs as exerting anti-pathogen\nresponses.\nLine 138: PDD treatment was included in subsequent experiments to facilitate the detection of\nADP-ribosylated proteins. This should be changed to PARylated proteins, as PARG is inhibited\nwhich can only cleave glycosidic linkages and thus degrade PAR chains.\nThis phrasing is now included on line 112.\nLine 162:\n...induced expression of multiple PARPs and causes global up-regulation of ADP-ribosylation...\n5 out of 16 tested PARPs showing high expression upon infection are several but not multiple.\nOverstatements should be avoided.\nFollowing suggestions from all Reviewers to focus the manuscript, this section has been\nremoved.\nBecause MARylation is hardly if at all increased upon infection, the authors should talk about\n“global up-regulation of PARylation, instead of ADP-ribosylation.\nHaving confirmed the changes are due to PARylation, we now take care to be specific and refer\nto PARylation where appropriate. Changes have been made throughout the manuscript.\nLine 191:\n“... with the exception that there were no modified cysteinyls detected in viral proteins (Figure\n2C).” Figure 2C also does not indicate modified cysteines among the human proteins. Why do\nthe authors point on this?\nWe apologize that it was hard to see that modified cysteines were indeed present in human\nproteins and our original figure. We have re-ordered the stack plots and labelling in Fig 2C and\nS5C to make it clearer that modified Cys were detected in human proteins, but none in viral\nproteins.\nLine 200:\n“...although blotting revealed massive increases in PARylation”. This seems overstated, when\nlooking at Figures 2B, C, D and E. A massive increase is detectable upon H2O2 treatment.\nThis sentence has been toned down on line 182.\nLine 201:\n“... does not change does not change”...\nCorrected.\nLines 255, 256:\n„For PR8, increased PARylation was detected at 8 hpi, i.e. in a later stage of infection (Figure\n4A). PARylation levels then continued to increase over the time course. In contrast, PARylation\nwas apparent as early as 4 hpi in cells inoculated with PR8ΔNS1.”\nLooking at Figure 4A an increase in\nPARylation for PR8 can be seen 10 hpi,\nwhereas an increase in PARylation was\napparent as early as 6hpi in cells inoculated\nwith PR8∆NS1.\nWe politely disagree. Figure 4A shows the\nbeginnings of an increase in PAR smearing\naround 100 kDa for PR8∆NS1 at 4 hpi, and\na similar faint increase at 8 hpi for WT PR8.\nThe snapshot below shows a zoomed\nversion of the relevant portion of Fig 4A,\nwith the emerging PARylation signal boxed\nFrom Fig 4A. Lysates were prepared from A549 cells were infected\nin red.\nwith PR8 or PR8∆NS1 and blotted to detect PARylation. The initial\nincrease in PARylation for each infection is boxed in red.\nLines 288, 289:\nAs before, we identified ~ 2,600-3,000 modified sites in ~380-660 proteins (Figure 5A-B, Figure\nS5, Table S2-S3).\nWhere do these numbers come from?\nThe numbers are derived from Table S2-3, making sure to only count unique sites and proteins\nthat pass our filtering thresholds. Note that for completeness, Tables S1-S3 report all detected\nsite with a localizations ≥0.6, and not just the filtered data (e.g. it retains peptides with replicate\nscores <4). This also includes some sites that were detected in multiple peptides. Thus, totals\ncannot be derived from a simple summation of entries in the table. The text has been updated to\nreflect our most recent counts. To aid the reader, we now include a new table with a complete\nlist of any protein or site that passed threshold in any of our experiments. This is the new Table\nS5.\nLine 321\n“PJ34 generically inhibits PARPs” compared to Wahlberg et al., 2012, Nature Biotechnology this\nstatement is wrong, especially with regard to the IFN-sensitive PARPs which are also\nupregulated in this study (Supplementary Figure S1C), which are hardly inhibited by this\ncompound.\nWe apologize for the lack of precision. The text on lines 303-304 now refers to PJ34 as a broad-\nspectrum PARP inhibitor.\nLines 368-372\nThis seems very speculative based on the data generated in this study.\nWe agree that this was a speculative conclusion based on the original data. We directly tested\nwhether infection changes ADPr chain length. New data in Fig 2F and 3C show a marked\nincrease in PAR chain length in infected cells (data are copied below in response to Reviewer 2;\nsee also lines 187-191 and 233-235). These data help resolve the apparent contradiction\nbetween our blotting and MS results and suggest that infection does not alter which sites are\nmodified, but instead primarily changes the ADP-ribosylation form from MAR to PAR or\nincreases the frequency of modification at a particular site. These data now provide strong\nsupport the conclusion in our Discussion.\nMinor comments on Figures:\nAmong all figures and panels representing westernblot analyses molecular weight marks should\nbe added. This always makes it easier to comprehend the data.\nThank you for the suggestion. We have included molecular weight markers in all figures where\nappropriate.\nFurther the figure legends as well as the labeling of the blots are quite minimalistic. Cells that\nhave been used for infection should be indicated, the MOI of infection should be indicated and\nespecially the hours post infection (hpi), when the data was obtained should be indicated. That\nwould make it way easier to understand the figures without going through the text and material\nand method section.\nWe agree with the desire to have more detail in the legends, but are constrained by Nature\nCommunication editorial guides that limit word counts in the legends. We added more\ninformation where appropriate and possible and have ensured all experimental details are\npresent in either the results and methods.\nFurther, in several panels controls are missing, such as a confirmation of infection or a loading\ncontrol. This seems especially important when stating that there is in increase in overall ADP-\nribosylation signal, which is hard to comprehend, when a loading control is missing. Also most\ninput blots are missing these, as I assume that Input reflects the analysis of the whole cell\nlysate, before IP. Blots showing IPs should be labeled accordingly (IP: anti-FLAG for example). I\nrecommend adapting these issues accordingly.\nWe apologize that the presence of the controls in our original submission was not clear. Each\nfigure has loading controls and infection controls, where appropriate. New blots generated for\nthis rebuttal also include loading and infection controls (see Fig 1D, 1E). In some cases, a single\nloading control is shown for an experiment that might have multiple blots (e.g. Fig 1A). All of the\noriginal figures involving immunoprecipitated samples labeled these conditions with “IP,” and\nthat remains true in the resubmission (see Fig 1F, 3D, 6B).\nAdditional issues to each figure or panel are outlined below.\nSupplementary Figure 1A\nThis Figure does like Figure 1D in reference 30 (same authors) a lot and should be indicated by\nincluding the respective reference.\nIn response to suggestions from Reviewer 3, these data have been removed from the\nmanuscript and we now simply refer to the prior work in the first line of the Results section.\nSupplementary Figure 1B/C\nAs stated in the figure legend, these graphs have been created from three replicates. I\nrecommend visualizing individual data points as has been done for other graphs in this study as\nwell.\nIn response to suggestions from Reviewer 3, these data have been removed from the\nmanuscript.\nFigure 1A, Supplementary Figure S2D:\nA proof, that the individual PARPs are indeed expressed and to what extend they are\noverexpressed is missing here.\nIn response to suggestions from Reviewer 3, these data have been removed from the\nmanuscript.\nFigure 1B:\nIt is hard to see the increase of MAR signal, the authors claim to see. MAR-signal appears\nrather reduced, as single bands appear fainter upon infection with IAV. Statements in the text\nshould be adapted accordingly.\nWe agree with the Reviewer and appreciate them pointing this out. Following suggestions from\nReviewer 2, we performed additional experiments using a new anti-MAR antibody. These data\nshow no significant change in MARylation (Fig 1A, right). The new data is copied below in\nresponse to Reviewer 2. Our text now reflects the conclusion reached by Reviewer 1 with Line\n103-105 now saying “Blotting was repeated using reagents specific for MARylation or\nPARylation, revealing that infection increases PARylation whereas MARylation was largely\nunchanged, if not slightly reduced.”\nFigure 1C/3A:\nAccording to the supplementary data, cutout of the PAR blot and cutout for NP and Actin signals\nseem to come from different experiments when looking at the overall loading pattern. Can the\nauthors please comment on this?\nWe are sorry for any confusion. We confirmed that all cropped blots in the figures were correct\nand are appropriately paired with their originals in Fig S8.\nSupplementary Figure 2A-C\nFor these experiments the authors generated stable A549 cells, via lentiviral transduction,\nconstitutively expressing PARP8. A proof is missing.\nIn response to suggestions from Rev 3 to streamline the manuscript, these data have been\nremoved from the manuscript.\nFigure 3D\nJust out of curiosity, could the highly modified NP protein also be pulled out by AD1521 in\nFigure 3D?\nThis is an interesting idea and those data were present in the\noriginal submission and now in Fig S1D. These experiments\nshow that Af1521 captures NP from infected cells. Those data\nare copied here. Additional lines of evidence indicate NP is\nADP-ribosylated. Blotting immunopurified NP with anti-PAR\nantibodies repeatedly detected ADP-ribosylation (Fig 1F, 3D,\nS1E) and this was reduced when ADPr was removed by\nhydroxylamine treatment (Fig S1E). Finally, ELTA-MS directly\ndemonstrates multiple modifications on NP (Fig 2 and 5).\nFrom S1D: Human lung A549 cells were\ninfected with WSN. ADP-ribosylated\nproteins were affinity purified with\nrecombinant Af1521 or the binding mutant\nG42E. Influenza A virus NP was detected\nin the input and purified fractions by\nblotting with anti-RNP antibody.\nMajor concerns on Figures and Data:\nFigure 2A-D, Supplementary Figure S4, Table S1\nHere the authors performed ELTA-MS to analyze the global change in PARylation upon IAV\ninfection.\nFirst, only two biological replicates have been performed which doesn’t allow identifying\nstatistically significantly enriched ADP-ribosylated peptides.\nWe perform 3 large-scale experiments resulting in a total of 6 biological replicates of WSN,\nmock, and H O conditions, 4 biological replicates of PR8 and PR8∆NS1, and 2 biological\n2 2\nreplicates of WSN∆NS1. Each biological replicate is analyzed in 3 technical replicates. We then\nemploy strict cutoffs to identify modified proteins. We further manually inspected representative\nspectra to ensure accurate site localization (Fig S2). These procedures ensure reproducible\nresults, as we detail in Fig S3 and S5. To capture the totality of our data, we now include Table\nS5 that consolidates all of these data to provide a composite list of proteins and sites that\npassed our threshold. We agree with the reviewer that ELTA-MS is better suited for site\nidentification, as opposed strict quantitation. We include this limitation in our Discussion\nbeginning on Line 393.\nTable S1 supporting this figure is hard to understand, as neither the different conditions (H2O2-\ntreatment, mock or IAV infection) can be identified within this table, nor the biological replicates.\nWe apologize that the table legends for the supplemental tables were not obvious. There were\nincluded at the end of the original manuscript. To aid the reader, Table S1-S5 were modified\nand now contain the legend on the first tab of the spreadsheet. All of the data requested by\nReviewer 1 are present in the table as described in the associated legends.\nThe analysis of the technical triplicates is presented in Supplementary Figure S4. Here, Venn\ndiagrams indicate ADP-ribosylated proteins and identified sites, meant to show high\nreproducibility between the technical replicates.\nOn the right-hand site the data from technical replicates is somehow summarized to reflect\nsimilarity of the biological replicates. How are numbers calculated for this? The numbers of\nidentified proteins within the biological replicates do not fit at all to anything indicated with the\ntechnical replicates. In the text it is stated, that “a peptide counted as ADP-ribosylated when it\nwas detected at least in two technical replicates” (lines 175-176), but even taken this into\naccount does not sum up to the numbers indicated for the biological replicates. Based on these\nhardly understandable numbers, and poor Table, Figures 2B and 2C were created as a\nsummary of the MS analysis and GO term analysis has been performed (Supplementary Figure\nS4B).\nWe apologize for the lack of clarity. As described in the original submission, and now in the\nresubmission on lines 154-157 and with details in the Methods on line 701 “MS data filtering and\nvisualization,” we used rigorous cutoffs to only report highly reproducible hits in our MS data\n(See line 711-719). This required that data from technical replicates pass threshold cutoffs to be\nincluded as hits in their associated biological replicate. Thus, we would not expect a direct\nrelationship between the numbers presented in the technical replicates (Fig S3A, left) and the\nfiltered biological replicates (Fig S3A, right). We have amended the associated Figure S3A\nlegend on line 1203 to make this clear – “Note that technical replicates include all data meeting\nthe minimum localization score, whereas biological replicates are further filtered based on\nreplicate scores to ensure accurate and reproducible identification as detailed in the Methods.”\nThen there is another Venn diagram presented in Supplementary Figure S4C, which the authors\ndo not refer to within the text. This figure indicates an overlap between H2O2 treated HeLa and\nA549 cells and Mock-infected A549 cells. I don’t see what this Figure supports within this study.\nAs suggested by the reviewer, these data were removed.\nFigure 2E/F, Supplementary Figure S3B\nHere, the identified ADP-ribosylated sites within the viral proteins, especially the heavily\nmodified NP are indicated. Based on this, they individually substituted the respective NP sites to\nAlanines. These mutants were then used to investigate polymerase activity and three were\nidentified allowing higher activity. The authors conclude, “that mutating these residues to\nprevent ADP-ribosylation enhanced polymerase activity, indicating that ADP-ribosylation at\nthese positions disrupts function” (lines 391-393). This cannot be stated like this, because ADP-\nribosylation of these sites has formally not been proven in an independent experiment.\nWe politely disagree. Independent biological replicates of ELTA-MS #1 detected direct\nmodification at these sites (Fig 2E, Table S1), many of which were identified again in the\nseparate ELTA-MS #2 and ELTA-MS #3 experiments with two different strains of influenza virus\n(Fig 5, Tables S2-3, Fig S7 and S8). This is further supported by orthogonal approaches like\naffinity purification based on ADP-ribosylation followed by blotting, all of which show\nmodification of NP. Together this provides strong support for our claims that these site in NP are\nmodified by ADP-ribosylation.\nThis could have been done by enrichment of the mutant and detection of ADP-ribosylation or by\nexploiting AF1521 for enrichment, that in turn would allow enriching the wildtype NP but not the\nrespective mutant. This also could have been done in their polymerase assay with co-\nexpression of e.g. PARP1, which should leave NP unmodified and allow for high activity.\nWe thank the reviewer for their interest and suggestion. We too considered this approach, but it\nassumes that each NP molecule is only modified at a single site and that this would allow for\nsubstantial changes in modification when that site is mutated. Given that at least 18 different\nsites are modified, it is highly unlikely that mutating any one site will change the total ADP-\nribosylation on NP that would allow us to differentially purify WT or mutant proteins.\nFigure 5, Supplementary Figure S5, Supplementary Table S2, S3, S4\nHere the authors performed ELTA-MS to confirm the effect of NS1 on global ADP-ribosylation\nevents using two different Influenza strains either wildtype or ∆NS1. Again, two biological\nreplicates have been analyzed in technical triplicates. Neither the different settings analyzed in\neach experiment, nor the biological replicates are indicated with the respective supplementary\ntable. Both tables are hard to understand.\nWe apologize that the conditions were not apparent. Table legends in the original submission\nand the revised version indicate that the table used column headings to clearly identify the\nexperimental condition (“Condition”) and the type of replicate for each entry (e.g. technical\nreplicates identified as “Tec1,” “Tec2,” and “Tec3”). As indicated above, to aid the reader Table\nS1-S5 were modified and now contain the legend on the first tab of the spreadsheet.\nHere, biological replicates are compared to each other and among the two experiments.\nThe authors state, that infections induced a unique profile of ADP-ribosylation, while significant\noverlap among conditions reinforced the idea of a core ADP-ribosylome in A549 cells (lines 294,\n295). Supplementary Figure S6A shows these overlaps, either for PR8 of ELTA-MS#2 and #3 or\nWSN of all three ELTA-MS approaches. In total they now have 4 biological replicates of PR8\ninfected cells analyzed by ELTA-MS and 6 biological replicates of WSN infected cells. If\nidentified proteins and sites are as conserved as claimed, why are Venn diagrams in\nSupplementary Figure S6B then separated based on the individual data sets?\nThank you for raising this point and allowing us to further highlight reproducibility of our data\n(now presented in Fig S5). We performed new comparisons of data from all of our runs in Fig\nS5D to show ~600 proteins are in a shared core ADP-ribosylome. This is described in Line 288-\nruns (ELTA-MS #2 and #3 for PR8 and ELTA-MS #1, #2 and #3 for WSN). This highlights the\nreproducibility across runs. Fig S5B then shows a high degree of overlap during WSN and PR8\ninfections within a run, supporting our claim of a core ADP-ribosylome.\nWhich if these was taken to generate Supplementary Table S4, the GO term analysis?\nGO analysis was performed on all combined data for a given experimental condition. This can\nbe found in the table legend and the methods.\nCombining all these data sets would allow for principal component analysis to visualize the\nclustering of the individual biological replicates and indeed allow to test significance of the\nindividual hits. None of this has been done.\nWe agree with the reviewer that it would be useful to combine the data and now provide\ncomposite data from all ELTA-MS experiments in Table S5. The relatedness of each biological\nreplicate is already represented in the hierarchical clustering at the top of each heat map.\nTo claim, that a unique profile of ADP-ribosylation is induced upon infection a comparison to\nmock treated cells is missing to visualize proteins and sites that are uniquely enriched upon\ninfection. Further, I wonder, why the analysis of the ∆NS mutants was not considered. As NS\nhas been shown to somehow counteract ADP-ribosylation events, similar protein and sites\nshould have been identified here but more enriched over control but this seems not the case\n(lines 306-308).\nThese data were presented in our original submission and now in Fig 2A and 5A-B, which show\nthe profiles for each condition allowing for easy visualization of overlap. Fig 2B also provides\nVenn diagrams comparing mock and infected cells. Specific comparisons for ∆NS1 have been\nmade in Fig 5 C-D. While NS1 appears to suppress PARylation as shown by blotting and\nmeasurement of PAR chain length (Fig 3B, 3C, 4A and 4B), it does not dramatically alter the\nproteins or sites that are modified as identified by ELTA-MS. Combined, our data suggest that\nNS1 functions primarily to reduce how often any particular site is PARylated, although a\ndefinitive answer requires further experimentation. We offer cautious speculation on this in the\nDiscussion beginning on line 442.\nFigure 6A\nCut outs indicated in the supplementary showing NP and ß-actin do not fit the blots shown here.\nWe are sorry for any confusion. We confirmed that all cropped blots in the figures were correct\nand are appropriately paired with their originals in Fig S8.\nTaken together, this study shows a robust increase in PARylation signal upon IAV infection,\nconfirmed by several immunoblot analyses. Further, data indicating that this can be manipulated\nbe NS1 of IAV in an unknown fashion is solid. Additionally, PARP1 has been identified as the\nPARP enzyme responsible for this increase in PARylation. Also, PARP1 has been identified\nbeing restrictive for IAV. Whether this indeed is dependent on its catalytic activity has not been\nformally investigated. Also, whether PARP1-mediated ADP-ribosylation of the NP protein is the\nmain mechanism to interfere with IAV replication has not been proven. However, modification of\nNP during infection has been demonstrated.\nWe thank the reviewer for their summary of the numerous strengths in our manuscript.\nIn addition, the authors put a lot of effort and work in gaining insights into this global change of\nADP-ribosylation upon IAV infection. However, this part of the study is challenging to\ncomprehend and trust as outlined above. In the end it also seems that the MS-analyses do not\nreflect the initial results seen by Westernblot analyses, so conclusions are hard to be drawn.\nWe agree that there was an apparent contradiction between the blotting and MS data in our\noriginal submission. As described above, we addressed this by measuring ADPr chain length\nduring infection. New data in Fig 2F and 3C show infection dramatically increased PAR chain\nlength, whereas PARylation was below the level of detection on mock-infected samples. These\nresults help resolve the apparent contradiction between our blotting and MS data and suggest\nthat infection does not alter which sites are modified, but instead primarily changes the ADP-\nribosylation form from MAR to PAR or increases the frequency of modification at a particular\nsite. This is consistent with our data in Fig 6 that show the PARylating activity of PARP1 is\nresponsible for most of the changes we detect during infection. We describe these findings on\nlines 187-194 and 233-235 and discuss their implications on lines 377-379 and 395-396.\nFor the discussion, the authors did neither consider reports on IAV-induced DNA damage\nevents nor NS1 which also has been linked to DNA damage, suppressing DNA-repair\nmechanisms and manipulating DRR pathways. As PARP1 is best studied with regard to its\nfunction in DNA damage it seems obvious to discuss their results in light of this as well.\nWhile these are interesting ideas, our discussion covers the main findings in the paper and\navoids highly speculative claims on the role of NS1 regulating DNA damage responses.\nIn the end an author contribution statement is missing!\nAn author contribution statement has been added for resubmission on line 1012.\nReviewer #2 (Remarks to the Author):\nSUMMARY:\nThis paper provides an interesting study on the antiviral response to influenza by PARPs and is\nnoteworthy for providing the first ADPr proteomics study during infection. The important\nconclusion is that PARP1 as the main enzyme responsible for infection-induced PAR. This\noriginal work is of significance to the ADPr/PARP field as well as to researchers interested in\nstudying antiviral responses. It is generally well written and elegantly uses a variety of different\napproaches.\nWe thank the reviewer for their positive assessment of the work and its quality and noting its\nbroad impact.\nI have technical concerns that need to be addressed to validate the conclusions related to the\nproteomics analyses.\nWe appreciate the constructive feedback and address each of these below.\nMajor points:\n1) Fig 2. The authors observe that infection does not significantly alter the number of modified\nsites in contradiction to their western blotting results and conclude that infection changes the\nADPr for MAR to PAR. However, the same sites could be\nidentified in different conditions but they might have different\nratios. A quantitative proteomics analyses similar to the\nanalyses performed by the Nielsen lab in their ADPr-related\npaper, would have been better, although for this 3-4\nbiological replicates should have been included per\ncondition instead of the 2 the authors have in their current\nexperimental setup.\nWe agree with the reviewer. ELTA-MS is designed to\nidentify ADP-ribosylation sites, not determine what percent\nof any given site is modified. We follow the reviewer’s\nsuggestion to be cautious with our interpretation and now\ninclude text to outline the limitation of ELTA-MS (beginning\non line 390) and account for the possibility that our western\nblot might reflect higher usage at an ADPr site or changes in\nchain length (e.g. line 187-190, 377-379). New data in Fig\n2F and 3C (copied here for convenience) show infection\ndramatically increased PAR chain length, whereas From Figure 2F and 3C: ELTA labeling of\nPARylation was below the level of detection on mock- total ADPr isolated from A549 cells infected,\ninfected samples. These results help resolve the apparent mock infected, or treated with H2O2. ADPr\nwas labeled with 32P-dATP by OAS1,\ncontradiction between our blotting and MS data and suggest\nseparated by 15% urea-PAGE, and detected\nthat infection does not alter which sites are modified, but\nby phosphorimaging. * = non-specific band\ninstead primarily changes the ADP-ribosylation form from from 32P-dATP stock.\nMAR to PAR or increases the frequency of modification at a particular site. This is consistent\nwith our data in Fig 6 that show the PARylating activity of PARP1 is responsible for most of the\nchanges we detect during infection. We describe these findings on lines 187-194 and 233-235\nand discuss their implications on lines 377-379 and the paragraph beginning on line 386.\nWhile Fig 2 is based on 2 biological replicates, our manuscript includes a total of 3 large-scale\nexperiments resulting in a total of 6 biological replicates of WSN, mock, and H O conditions, 4\n2 2\nbiological replicates of PR8 and PR8∆NS1, and 2 biological replicates of WSN∆NS1. Each\nbiological replicate is analyzed in 3 technical replicates. To help readers capture the totality of\nour data, we now include Table S5 that consolidates all of these data to provide a composite list\nof proteins and sites that passed our threshold.\nWe appreciate the interest in quantitative comparison of ADPr occupancy between conditions.\nHowever, quantitative approaches like tandem mass tagging are not currently compatible with\nthe ELTA-MS workflow, making these questions outside the scope of the current manuscript.\n2) Fig 2, 5. Prior to the proteomics analysis, cells were lysed by incubation at 99 C followed by\novernight tryptic digestion at pH 8. In light of recent studies on the lability of Asp-/Glu-ADPr\n(Weixler Life Science Alliance 2023; Tashirto J Am Chem Soc 2023; Longarini Nat Comms\n2024), these conditions have most likely led to a significant loss of Asp-/Glu-ADPr prior to\nanalyses by mass spectrometry. They authors might reply that their identification of a number of\nAsp-/Glu-ADPr sites is evidence that their approach works for this type of ADPr as well.\nHowever, the reported Asp-/Glu-ADPr sites might been identified due to mislocalisation by HCD\n(see comment below), similarly to the study by Martello Nature Comms 2016, whose Asp-/Glu-\nADPr sites were not identified again in studies of the same lab once ETD was used. The\nauthors should at least discuss the probable loss of Asp-/Glu-ADPr in their proteomics analyses.\nWe appreciate the reviewer’s insightful comment and acknowledge the potential loss of Asp-\n/Glu-ADPr under our experimental conditions. We would like to note that ELTA-MS has been\ndemonstrated to identify all classes of ADP-ribosylation at different amino acids1,2. It is well\nestablished that all existing proteomics approaches may lead to some degree of modification\nloss, particularly for Asp-/Glu-ADPr. Even enrichment techniques such as boronate affinity\npurification, which specifically target Asp-/Glu-ADPr, operate at alkaline pH and can result in\nmodification instability.\nRegarding HCD-based site localization, we have applied stringent criteria to ensure accurate\nsite assignment. This includes a site probability threshold of >0.9, which is more stringent than\nsome ADPr-site identification papers3 and matches localization threshold used by other4–6. and\na technical replicate score of ≥4, which required detection in at least two technical replicates\nfrom the same biological replicate with at least one direct identification via tandem mass\nspectrometry (MS/MS). This is detailed in the methods beginning on line 694 and in the spectra\nwe now include in Fig S2.\nDespite these precautions, we recognize the possibility of mislocalization or signal loss as noted\nin previous studies. We include this caveat in the Discussion in the paragraph beginning on line\n390 and the Methods on line 694-699. This is further addressed in the next comment where we\nmanually validate representative spectra.\n3) The authors use HCD for proteomics analyses of ADPr sites, but this fragmentation\ntechnique can lead to mislocalisation of some forms of ADPr, especially abundant Ser-ADPr.\nWhile they use a stringent cut-off of 0.9, the complete loss of phospho-ribose is probably\ninterpreted automatically by the algorithms used for localization score as the erroneous\nevidence of lack of ADPr on a particular residue leading to a high score despite the actual\nuncertainty in localization. In HCD spectra the presence of phospho-ribose is actual evidence\nthat that fragment ion contains phospho-ribose and this information can definitely be used for\nlocalization. In contrast, the absence of the modification can mean actual absence of the\nmodification or compete loss of the modification due to its lability and standard software, made\nfor stable PTMs, cannot distinguish between these two possibilities. Problematically it uses this\nlack of evidence as evidence of its absence providing a potentially misleading high localization\nscore. None of this would have been an issue if the authors had used ETD, but perhaps this\nfragmentation technique is available on the Fusion used for these analyses? Another possibility\nwould have been to manually validate the HCD spectra and provide those that allow confident\nlocalization, but this might not be feasible considering the high number of sites identified? Still\nthe authors could provide a few annotated MSMS spectra showing confident localization for the\nmost important sites identified, such as on NP. At least the authors should discuss this and\nprovide all their raw files deposited in a public repository.\nWe thank the reviewer for their detailed feedback and acknowledge the potential for\nmislocalization when using HCD for ADP-ribosylation site assignment. As the reviewer points\nout, the loss of phospho-ribose can be misinterpreted by standard localization algorithms, which\nare primarily designed for stable PTMs. However, we note that such mislocalization would likely\nresult in a reduced number of confidently assigned sites rather than an inflation of incorrect\nassignments.\nGiven the large number of identified sites, manual validation of all spectra is not feasible.\nHowever, in response to the reviewer’s suggestion, we now provide annotated MS/MS spectra\nfor key sites, including those on NP, to support our localization assignments (Fig S2 and copied\nhere for convenience.) We include 6 different spectra, similar to publications in Nature Comms\nand Cell Reports4,5.\nFrom Fig S2. Manual\nconfirmation of site\nlocalization for\nrepresentative spectra\nRepresentative spectra were manually annotated to confirm proper ADPr site localization for\nmodified sites in viral and host proteins. Spectra were chosen to show replication across\nindependent ELTA-MS experiments (e.g. ELTA MS #1 and #2 for NP), replication across\nconditions within a replicate (e.g. ELTA MS #3 WSN vs PR8 for NS1), and modification of Ser,\nAsp, and Glu residues.\nAdditionally, our raw data have already been deposited in a public repository, allowing for\nfurther independent evaluation. The dataset can be accessed on the MassIVE database under\naccession code MSV000095351 with a ProteomeXchange ID PXD053989.\nMinor points:\nFig. 1B: A recently derived MAR antibody (clone AbD43647) has been shown\nto work well for the detection of MAR induced by interferon treatment of A549\ncells (Kar et al. EMBO J 2024). While the vast majority of band in western\nblots performed by the authors don’t change when comparing A549 cells\ninfected with WSN and mock-infected cells, these bands don’t appear to be\nrecognized by AbD43647 in blots performed by Kar et al. EMBO J 2024 and\nat the same time several specific bands recognized by AbD43647 are\ninduced by interferon.\nThis is an excellent suggestion. We acquired AbD43647 and used it to\nperform the requested experiments. Those data are in the revised Fig 1A and\ncopied here for convenience. Moreover, this addresses an experimental\nconcern of Reviewer 1 above. Curiously, our lysates from FLUAV-infected\ncells do not contain the obvious IFN-induced bands like those in Kare, et al.\npossibly because only very few infected cells trigger interferon responses\nunder our infection conditions7–9.\nFrom Fig 1A. A549\nlysates probed for\nIn addition, did the author consider the sensitivity of Asp-/Glu-ADPr to high\nMAR using AbD43647\ntemperature (Weixler Life Science Alliance 2023; Tashirto J Am Chem Soc\n2023; Longarini Nat Comms 2024)? In particular, if the authors had performed the conventional\nboiling step prior to SDS-PAGE, much of Asp-/Glu-ADPr would have been lost favoring\ndetection of more stable forms of ADPr, such as on serine. Therefore, AbD43647 together with\ncell lysis at room temperature could give cleaner and more informative results.\nWe are aware of the impact of high temperatures on sample integrity and have tried different\nconditions, like denaturing samples 70C, and did not detect obvious differences in blotting.\nNonetheless, we include this when we discuss limitations of our approach in the Discussion\n(beginning on line 390).\nPage 3, lines 63 - 64: ‘gene loss or duplication m— processes’ A few words seem to be missing.\nThis has been corrected.\nLines 201 – 202: ‘number of modified proteins or sites does not change does not change in\nmock and infected cells’: ‘does not change’ is repeated twice\nThis has been corrected.\nReviewer #3 (Remarks to the Author):\nThe manuscripts aims to describe and define the role of host mediated ADP-ribosylation on the\nantiviral immune response against Influenza A virus infection. The focus on ADP-ribosylation is\nlargely based on pervious results as well as earlier reports that demonstrate a role of PARPs in\nthe antiviral immune response as well as direct antiviral effects.\nThe manuscripts includes a large number of experiments. While the majority of the experiments\nand results presented are of high quality, well performed and interesting, the manuscripts\naddresses too many different aspects and thereby looses its focus and clarity. It partially reads\nlike an assembly of old and new data that are related but just not made to fit together.\nSometimes less really is more!\nAlready the characterization of the infection induced switch in mono and poly-ribosylation\ncombined with the potential antagonistic function of NS1, including the dependency on its RNA\nbinding activity, is so complex that it is would be sufficient for a manuscript.\nWhile all the open ends are addressed and well discussed in the final part, it remains unclear\nwhy so many aspects were included in the first place (what about PARP8 after all?). I strongly\nsuggest to reduce the amount of data and lines of investigation to strengthen the focus on the\nremaining data and to improve the overall quality of the manuscript.\nE.g. the function and antiviral effect of ribosylation sites in viral proteins is an important aspect\nbut it is here only investigated in the context of NP. The results are interesting but leave an\nincomplete picture on the functional repertoire of ADP-ribosylation on other viral proteins. The\nimportance of these data for the overall message of this manuscript remains unclear and may\nbe better placed in a separate manuscript.\nIn addition to the general comments there are several specific aspects to the figures and text\nthat need to be addressed and improved.\nWe thank the reviewer for their assessment that the “results presented are of high quality, well\nperformed and interesting.” We also appreciate their constructive feedback on how to focus the\nmanuscript to make the most impactful claims.\nWe embraced the Reviewer’s suggestion that “less is more” and have streamlined and\nreorganized the manuscript. Portions of Fig 1, all of Fig S1 and S2, Fig 3C-D, Fig 5C, Fig S7B,\nand their associated text have been removed. This allowed us to focus more on the\nidentification of ADP-ribosylation sites and their effects on infection and strengthen those data\nwithout lessening the impact of the overall conclusions. This has produced a more concise and\naccessible manuscript.\nSpecific comments\nWhat is the relevance of the data in the first paragraph of the results? They address PARP\nrelated aspects, but don’t quite fit to the following layout of the manuscript. Why the focus on\nPARP8 here? How does this benefit the following data?\nWe agree with the reviewer that these data are not needed to motivate the work and they have\nbeen removed from the resubmission.\nFigure 1 A: Expression levels for the PARP overexpression are missing\nThis panel is no longer in the manuscript.\nFigure 1B: Since many proteins are stained on\nthe blot, the inclusion of size markers would be\nvery helpful for orientation between panels B and\nC. It is very difficult to judge whether the similarity\nbetween the ADP-ribosylation patterns without\nsize markers. Please adapt accordingly.\nThank you for the suggestion. We have included\nmolecular weight markers in all figures where\nappropriate.\nFigure 1D: Why is there a decrease in PARylation\nin the UV treated virus infected cells? Can\nPARylation be induced by transfection of viral\nRNA?\nWe have performed another replicate of our\nexperiment where cells are treated with UV- From Fig 1: (D) A549 cells were inoculated with infectious or\ninactivated virus and included new loading UV-inactivated virus, or mock-treated. PARylation was\ncontrols (Fig 1D and copied here for detected in whole cell lysates by blotting.\nconvenience). These data show that PARylation (E) A549 cells were transfected with the indicated amounts\nrequires active replication. We also performed of viral genomic RNA (vRNA), poly(I:C), or transfected\nnew experiments to determine if viral RNA alone without nucleic acid (“empty”). Cells were also subjected to\nis sufficient for induction of PARylation (new Fig interferon b (IFN-b)-, mock-, or H2O2-treatment as controls.\n1E copied here, new Supp Fig 1A-C, and Total ADP-ribosylation, viral NP and PA, and b-actin were\nassociated text on lines 116-123). Genomic RNA was purified from influenza virus and\ntransfected back into A549 cells. While H O and infection induced PARylation, exposing cells\n2 2\nto genomic RNA, the artificial agonist poly I:C, or IFN-b had no effect of PAN, PAR, or\nMARylation (Fig 1E, S1C). These experiments used an ISRE reporter cell line to detect innate\nimmune activation by the viral RNA, serving as a positive control for transfection and RNA\nsensing. Viral genomes activated innate immune sensing, on par with the artificial agonist poly\nI:C (Fig S1A-B). The absence of ADP-ribosylation was not because genomes were not sensed.\nThus, genomic RNA is not sufficient to trigger anti-viral PARylation. This aligns with our data\nwith UV-inactivated virus, which also deposits genomic RNA but does not activate PARylation.\nFigure 1 F: Is NP also modified when it is overexpressed alone, or is the induction through\ninfection required?\nPilot experiments indicate that NP is not modified when it is expressed alone.\nFigure 2E: Why are there orange marks on the upper and the lower side? Are these reflecting\nthe results of the replicates? Please specify in the figure.\nWe apologize for the lack of clarity. These represent findings from the two independent\nbiological replicates, as detailed in the figure legend.\nLine 201: “does not change” is duplicated in the sentence and should be deleted.\nThis error has been corrected.\nLines 235-239: The context of PARP1 cleavage is not sufficiently addressed here. Why was this\nassessed and what is the conclusion. How does this relate to the different viruses. The potential\nconnection to NS1 is not sufficiently explained. Please add more explanation.\nAs requested, we provide some more context. Line 222-228 now say:\nPARP1, in addition to functioning as an ADP-ribosyltransferase, is a target for caspases\nand its cleavage is frequently used as a marker for the activation of apoptosis. Influenza\nvirus infection triggers apoptosis, which plays dual pro- and anti-viral roles during\ninfection, and the regulation of apoptosis by influenza A virus is important for successful\nreplication. PARP1 cleavage was more pronounced in cells infected with WSN\ncompared to PR8 (Figure 3A). Curiously, WSN-infected cells had higher levels of\nPARylation despite also exhibiting higher levels of PARP1 cleavage.\nWe also indicate that NS1 down regulates apoptosis, consistent with us detecting higher\ncaspase activation during infection with ∆NS1 viruses. Starting on line 247:\nThe increase in PARylation with PR8∆NS1 was associated with higher levels of PARP1\ncleavage (Figure 4A). This is consistent with the ability of NS1 to down regulate\napoptosis. NP is also a target for caspase cleavage, resulting in the removal of ~16\namino acids from the N-terminus. Blotting revealed earlier NP cleavage in PR8∆NS1-\ninfected cells compared to PR8-infected cells, again linking increased PARylation with\napoptotic activity (Figure 4A).\nFigure 3D: PB1 has only one ADP-ribosylation site so it may not be the best protein to be used\nin this assay as it easily underlies technical issues. An additional blot with NP that has multiple\nmodified sites also show the same/similar effect of signal reduction would strengthen this result.\nHow do these data fit to the results of the mass spec analysis with viruses lacking NS1 (Fig. 5)?\nThese Data show no effect on PB1 ribosylation. Together these data leave a rather unclear role\nof NS1 on ribosylation.\nChanges to focus the manuscript resulted in the removal of the data in the original Fig 3D.\nRegarding the perceived discrepancy between the original Figure 3C-D and our ELTA-MS data\nin Figure 5, we note that ELTA-MS identifies ADP-ribosylation sites but does not provide\nquantitative information on site occupancy or characterize ADP-ribose chain length. As a result,\nthe substantial changes in ADP-ribosylation observed by blotting may not be directly reflected in\nELTA-MS data. As detailed in response to a similar point was raised by Reviewer 2, we are\ncautious with our interpretation and now include text to outline the limitation of ELTA-MS\n(beginning on line 390) and account for the possibility that our western blot might reflect higher\nusage at an ADPr site or changes in chain length (e.g. line 190-193, 350-352). New data in Fig\n2F and 3C (copied above) show infection dramatically increased PAR chain length, whereas\nPARylation was below the level of detection on mock-infected samples. These results help\nresolve the apparent contradiction between our blotting and MS data and suggest that infection\ndoes not alter which sites are modified, but instead primarily changes the ADP-ribosylation form\nfrom MAR to PAR or increases the frequency of modification at a particular site. This is\nconsistent with our data in Fig 6 that show the PARylating activity of PARP1 is responsible for\nmost of the changes we detect during infection. We describe these findings on lines 187-194\nand 233-235 and discuss their implications on lines 377-379 and the paragraph beginning on\nline 386.\nFigure 4: The results with NS1 are intriguing. The anti-PARylation activity was associated with\nthe NS1 RNA binding activity. Does this mean, viral RNA sensing is the trigger for the changes\nin ADP-ribosylation? And can it be stimulated by transfection of viral RNA? These are important\nexperiments to foster the claim of a novel function of NS1. It may rather be an indirect\ndownstream effect of NS1 mediated inhibition of the RIG-I pathway. This needs crarification.\nThis was an excellent suggestion and we performed these experiments as noted above in the\nresponse to comments on Fig 1D (new Fig 1E and S1A-C). In brief, transfecting cells with\ngenomic RNA does not trigger PARylation. Neither does treating cells with IFN-b or transfecting\nthem with poly I:C. In all cases, we confirmed these treatments activated innate immunity.\nThese leads us to conclude in the Discussion on line 452: “As the RNA binding activity of NS1\nhas pleiotropic effects, it is not clear if dsRNA binding directly antagonizes ADP-ribosylation.\nRather, the fact that treating cells with the dsRNA mimetic poly(I:C) does not induce ADP-\nribosylation (Figure 1E and S1C) argues that NS1:dsRNA interactions likely play an indirect\nrole.”\nFigure 5A: mock r2 is named strangely (mock r12) is that correct? Otherwise it should be\ncorrected. There is huge difference in the ribosylation sites in the mock samples (r1, r2). How\ncan this be explained?\nOur apologies for the mislabeling. It has been corrected. Variation between mock samples likely\nreflects a combination of inherent biological variability and technical limitations of ELTA-MS.\nGiven our stringent criteria for identifying ADP-ribosylation sites, our approach is inherently\nconservative, which may further contribute to the extent of overlap between conditions.\nFigure 5C: Would you not expect to have more ribosylation sites on viral proteins under\nconditions lacking NS1? Results in Fig.3D and Fig. 4 suggested this outcome? Please explain.\nMaybe the data in Fig 3 and 4 should be omitted as they are kind of misleading. It is difficult to\njudge whether this is because of technical issues or the chosen analysis using only the anti\nPAR-antibody but not the anti-MAR-antibody.\nThese are the first data sets reporting the APD-ribosylome during any infection. Our primary\ngoal was to detect as many sites as possible. ∆NS1 viruses exhibit delayed replication kinetics.\nTo control for this, as described in the Methods, we allowed ∆NS1 infections to proceed for 16\nhr before harvesting lysates. This corresponded roughly to 8 hr post-infection with WT virus,\nwhich is when we harvested lysates for those infections. We agree that a potential expectation\nwould be more sites on viral proteins during infection with a ∆NS1 virus. The apparent\ndifference in ADP-ribosylation may reflect differences in viral protein levels due to different times\nof harvest as much as they represent changes in ADP-ribosylation. In addition, we use different\nassays to measure ADP-ribosylation on viral proteins (affinity capture and blotting in original Fig\n3C-D) and the whole proteome (ELTA-MS Fig 5). As the reviewer alludes to, perhaps it is not an\nappropriate comparison. Following their recommendation, we have removed Fig 3C-D and 5C.\nTo fully resolve this point, future studies on temporal profiling of ADP-ribosylation will be\nneeded.\nAlthough the authors suggest that instead of inhibiting/affecting ribosylation sites, NS1 could\naffect the transition from mono to poly-ribosylation. However there is no strong data for this\nhypothesis as direct comparisons between both ribosylation forms is missing. Unless there is an\nadditional assay with clear results that support this hypothesis it should be strongly\ntuned down throughout the manuscript and the abstract. Results in Fig. 5C also suggest that there are other\nNS1-independent reasons for the observed differences the ribosylation patterns of viral proteins.\nFollowing the reviewers suggestion, we measured the transition from MAR to PAR during\ninfection with WT or ∆NS1 viruses (Fig 3C, also copied above in response to Reviewer 2).\nInfection caused a dramatic increase in ADPr polymer length and the amount of ADPr polymers\nwas even more pronounced in cells infected with ∆NS1 virus. This suggest that NS1 is\nregulating how frequently any particular site is modified. This is described in the Results sub-\nsection “NS1 suppresses PARylation but does not reduce the number of ADP-ribosylation sites”\nbeginning line 270 and then in the Discussion on line 441.\nFigure 6: The use of inhibitors in combination with KO cells is strongly appreciated as both\napproaches heavily affect cells and easily lead to artifacts and misinterpretation.\nWhat is the proof that the inhibitors work specifically at the used concentrations in the used\ncells? Positive controls and cytotoxicity assay should be provided.\nInhibitors are used following manufacturers’ conditions for inhibition of the targeted PARP (with\nconcentrations described in the Methods). Re-validating all of these inhibitors for each target\nPARP is beyond the scope of this paper. Uncertainty is ameliorated in complementary\nexperiments using a clean genetic knockout of PARP1 reproduce phenotypes associated with\nPARP1 inhibitors. cf Fig 6A to Fig 6D-E, Fig 7A, Fig S6A-B.\nWhat is the effect of the inhibitors on viral replication? At least one PARP1 inhibitor should be\ninvestigated to complement the replication data in PARP1 KO cells.\nAs the reviewer points out above, while chemical inhibitors are useful tools, their use can be\nconfounded by off-target specificity and toxicity. As such, we preferred to test viral replication\nwith a clean genetic approach that selectively queries the role of PARP1 and avoids concerns\nabout specificity and toxicity. Fig 7B-C demonstrates that a knockout of PARP1 increases viral\ntiters at early times during infection. This is reversed when the cells are complemented with\nwild-type PARP1, ensuring that the effects are due to PARP1. Together, these data support the\nconclusion that PARylation and PARP1 exert an anti-viral effect.\nDiscussion:\n1. The first paragraph reads like a different paper due to the simplified and overstated summary\nof the presented results. Please refine this paragraph by including a more balanced data\nsummary of the strengths, weaknesses and open questions.\nWe have changed this paragraph to better reflect the content in the current version.\n2. The data in supplementary figure one demonstrate that several PARPs are induced by viral\ninfection, which is interesting. How is this finding related to the reported changes in the ADP-\nribosylation? Later data include a major role of PARP1 in the switch to poly-ribosylation but it is\nnot upregulated by IAV infection. To follow the reviewers’ suggestion to streamline the\nmanuscript, these data have been removed from the resubmission\n1. Ando, Y. et al. ELTA: Enzymatic Labeling of Terminal ADP-Ribose. Molecular Cell 73, 845-\n856.e5 (2019).\n2. McPherson, R. L., Ong, S.-E. & Leung, A. K. L. Ion-Pairing with Triethylammonium Acetate\nImproves Solid-Phase Extraction of ADP-Ribosylated Peptides. Journal of proteome\nresearch 19, 984–990 (2020).\n3. Martello, R. et al. Proteome-wide identification of the endogenous ADP-ribosylome of\nmammalian cells and tissue. Nature Communications 7, 1–13 (2016).\n4. Hendriks, I. A. et al. The regulatory landscape of the human HPF1- and ARH3-dependent\nADP-ribosylome. Nat Commun 12, 5893 (2021).\n5. Larsen, S. C., Hendriks, I. A., Lyon, D., Jensen, L. J. & Nielsen, M. L. Systems-wide Analysis\nof Serine ADP-Ribosylation Reveals Widespread Occurrence and Site-Specific Overlap with\nPhosphorylation. Cell Reports 24, 2493-2505.e4 (2018).\n6. Bonfiglio, J. J. et al. Serine ADP-Ribosylation Depends on HPF1. Molecular Cell 65, 932-\n940.e6 (2017).\n7. Russell, A. B., Trapnell, C. & Bloom, J. D. Extreme heterogeneity of influenza virus infection\nin single cells. Elife 7, e32303 (2018).\n8. Sun, J. et al. Single cell heterogeneity in influenza A virus gene expression shapes the innate\nantiviral response to infection. PLOS Pathogens 16, e1008671 (2020).\n9. Killip, M. J., Jackson, D., Pérez-Cidoncha, M., Fodor, E. & Randall, R. E. Single-cell studies\nof IFN-β promoter activation by wild-type and NS1-defective influenza A viruses. J Gen Virol\n98, 357–363 (2017)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have substantially improved the manuscript in response to previous comments and criticism. The new version is clearer and better structured with a more streamlined presentation of data and removal data present in the earlier version of the manuscript. The newly generated data support the authors‘ initial findings and further strengthen their conclusions. I appreciate the considerable effort the authors have made to address all previously raised concerns.\nIn detail, the authors have implemented all suggested text revision. Regarding the Westernblot analyses, the missing controls have been included in all relevant experiments. The supplementary figures and image croppings are now consistent with the main figure data.\nThe data obtained through ELTA-MS are presented in a more accessible manner in this version, as the authors have provided a clearer description of their data analysis and revised the underlying tables, making them easier to understand. Also new comparisons of the data from all runs underlines the robustnes of their analysis and solidity of their data.\nAn author contribution statement has been added.\nIn my oppinion, the authors have addressed all previous concerns in a satisfactory manner, and the revised version represents a clear, well supported and technically sound contribition to the field. With this in mind, I recommend this manuscript for publication in Nature Communications.", "reviewer_2": "The authors have satisfactorily addressed my concerns.", "reviewer_3": "The authors have done a tremendous job to comprehensively and convincingly address all reviewer comments. I am fully satisfied with the revised version of the article and have no further comments."}, "author_response": ""}]

Decision letter