E3 ligase Mindbomb 1 facilitates nuclear delivery of adenovirus genomes

Stephanie L. Sarbanesa, Vincent A. Blomenb, Eric Lamc, Søren Heisseld, Joseph M. Lunaa, Thijn R. Brummelkampb,e,f, Erik Falck-Pedersenc, H.-Heinrich Hoffmanna,1, and Charles M. Ricea,1

aLaboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065; bOncode Institute, Division of Biochemistry, The Netherlands Institute, 1066CX Amsterdam, The Netherlands; cMolecular Biology Graduate Program, Department of Microbiology and Immunology, Hearst Research Foundation, Weill Medical College of Cornell University, New York, NY 10021; dProteomics Resource Center, The Rockefeller University, New York, NY 10065; eCeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, A-1090, Vienna, Austria; and fCancer Genomics Center, 1066CX Amsterdam, The Netherlands

Edited by Thomas Shenk, Princeton University, Princeton, NJ, and approved November 4, 2020 (received for review July 30, 2020) The journey from plasma membrane to nuclear pore is a critical . By hijacking MT tracks, the virus scales most of the step in the lifecycle of DNA viruses, many of which must success- distance to its nuclear destination (9). fully deposit their genomes into the nucleus for replication. Viral Even upon arrival in the vicinity of the nucleus, a series of capsids navigate this vast distance through the coordinated hijacking coordinated events are still required to successfully deliver the of a number of cellular host factors, many of which remain viral genome into the nucleus for early transcription and repli- unknown. We performed a -trap screen in haploid cells to iden- cation using host cell machinery. The viral capsid must transfer tify host factors for adenovirus (AdV), a DNA virus that can cause from the MTOC to the nuclear pore where it docks directly onto severe respiratory illness in immune-compromised individuals. This the nuclear pore complex (NPC) via Nup214 (10, 11). Here, work identified Mindbomb 1 (MIB1), an E3 involved unable to traverse the pore, the capsid must be physically dis- in neurodevelopment, as critical for AdV infectivity. In the absence rupted. The liberated genomic viral DNA (vDNA) and associ- of MIB1, we observed that viral capsids successfully traffic to the ated are imported into the nucleus while the now-empty proximity of the nucleus but ultimately fail to deposit their genomes capsid shell is relocated toward the periphery. The involvement within. The capacity of MIB1 to promote AdV infection was depen- of numerous cellular proteins including CRM1, histone H1, se-

dent on its ubiquitination activity, suggesting that MIB1 may medi- lect nucleoporins, import factors, and MT motors highlight the MICROBIOLOGY ate proteasomal degradation of one or more negative regulators of complexity of these processes (12–16). However, a comprehen- AdV infection. Employing complementary proteomic approaches to sive view of the factors governing capsid disassembly and genome characterize proteins proximal to MIB1 upon AdV infection and dif- release at this interface is still lacking. ferentially ubiquitinated in the presence or absence of MIB1, we In this work, we used a gene-trap screening approach in observed an intersection between MIB1 and ribonucleoproteins haploid cells with AdV5 infection and identify the E3 ubiquitin (RNPs) largely unexplored in mammalian cells. This work uncovers ligase Mindbomb 1 (MIB1) as a host factor required by the virus. yet another way that viruses utilize host cell machinery for their We demonstrate that MIB1 functions early in the AdV lifecycle own replication, highlighting a potential target for therapeutic in- to promote delivery of the viral genome to the nucleus. Specif- terventions that counter AdV infection. ically, in the absence of MIB1 ubiquitination, capsids successfully arrive at the nucleus but the release of vDNA into the nucleus is adenovirus | viral entry | host factor | Mindbomb 1 | E3 ubiquitin ligase Significance uman adenoviruses (hAdVs) are DNA viruses that can cause severe respiratory illness predominantly affecting children H Adenoviruses (AdVs) are DNA viruses that can cause severe and immune-compromised individuals. Aside from respiratory respiratory illness in . A better understanding of the illness, AdVs can also cause meningitis, conjunctivitis, and gas- complex ways AdVs utilize cellular processes in service of their trointestinal disease with recent outbreaks in hospitals and col- replication is critical to the development of new therapies to leges resulting in fatalities (1, 2). The contagiousness of certain counter viral infection and disease. Using a genome-wide AdV serotypes poses a serious public health concern. However, screen, we identified a cellular Mindbomb 1 (MIB1) there are currently neither vaccines nor specific antiviral treat- required for AdV infection and show that it functions in the ments. Adenoviruses can also be reengineered as vectors to treat a AdV entry process to mediate release of the viral genome into variety of other diseases (3, 4). A better understanding of the the nucleus. We further show that MIB1, as an E3 ubiquitin li- factors mediating AdV genome delivery to the nucleus has im- gase, mediates AdV capsid disassembly and genome release plications for the development of antiviral therapeutics as well as through ubiquitination of a target protein(s), demonstrating in application of AdV vectors in vaccine development and the importance of this rapid posttranslational modification to oncolytic cancer therapies. virus infection. AdV entry encompasses all steps from attachment of the virus via its receptor at the plasma membrane to the successful de- Author contributions: S.L.S., H.-H.H., and C.M.R. designed research; S.L.S., V.A.B., E.L., S.H., position of the viral genome into the nucleus of the host cell. For and H.-H.H. performed research; J.M.L., T.R.B., and E.F.-P. contributed new reagents/ adenovirus serotype 5 (AdV5), binding of the fiber protein to the analytic tools; S.L.S., V.A.B., E.L., S.H., and H.-H.H. analyzed data; S.L.S. wrote the paper; coxsackievirus and adenovirus receptor (CAR) triggers endocy- and E.F.-P. contributed expertise. tosis of the virus particle (5). As this endosome matures and The authors declare no competing interest. acidifies, the capsid becomes partially degraded, shedding its This article is a PNAS Direct Submission. fibers and exposing the membrane-lytic pVI protein. Puncture of Published under the PNAS license. the endosome by pVI releases the compromised yet intact capsid 1To whom correspondence may be addressed. Email: [email protected] or into the cytoplasm where it can couple to motors [email protected]. (6–8). (MTs) are dynamic cytoskeletal components This article contains supporting information online at https://www.pnas.org/lookup/suppl/ that nucleate predominantly from the microtubule organizing doi:10.1073/pnas.2015794118/-/DCSupplemental. center (MTOC), often synonymous with the nuclear-proximal Published December 28, 2020.

PNAS 2021 Vol. 118 No. 1 e2015794118 https://doi.org/10.1073/pnas.2015794118 | 1of12 Downloaded by guest on September 28, 2021 blocked. Using complementary proteomic approaches, we iden- Appendix,Fig.S1B). Together these results corroborated our tified cellular proteins typically associated with ribonucleopro- findings from the screen supporting MIB1 as a host factor tein (RNP) granules enriched for ubiquitination and in close for AdV5. proximity to MIB1 during AdV DNA release while observing a MIB1 emerged from the screen as the sole additional host depletion of cytoskeletal components. These findings pave the factor for AdV5 infection besides known viral receptors. Given way for future mechanistic work to identify the specific MIB1 MIB1’s well-characterized roles in regulating surface level ex- ubiquitination target(s) mediating AdV genome delivery. pression of Notch and Wnt receptors by endocytosis (in the latter case coupled to degradation), we were curious whether the ef- Results fects of MIB1 were mediated by indirect modulation of CAR MIB1 Is a Host Factor for AdV Infection. To identify host expression. Detection of CAR in both whole cell lysates and at factors required for AdV5 infection, we performed a genetic loss- the cell surface, assessed by Western blot and flow cytometry, of-function screen in near-haploid (Hap1) cells, which can be ef- respectively, however, revealed comparable levels between WT, ficiently mutagenized via insertional mutagenesis. This approach MIB1 KO, and WT MIB1-reconstituted Hap1 cell lines (Fig. 1C has been successfully employed in the identification of receptors and SI Appendix, Fig. S2 A and B). CAR independence of the and host factors for several viruses (17, 18). Retroviral delivery of observed AdV5 inhibition was further confirmed by infection a gene-trap cassette introducing a strong splice acceptor site and with alternate AdV serotypes that utilize distinct entry receptors. polyadenylation signal was used to disrupt and generate a While AdV2 similarly utilizes CAR, AdV7 and AdV35 utilize saturated library of loss-of-function mutants (19). Although the desmoglein-2 (DSG-2) and CD46, respectively (24). Infections − orientation in which the gene-trap cassette integrates into genes is with AdV5rep -GFP modified to express the fiber proteins of stochastic, the cassette was designed to disrupt a gene upon in- either AdV7 (AdV5-F7) or AdV35 (AdV5-F35), while readily tegration in the same transcriptional orientation. Therefore, genes able to infect CAR KO cells, were highly impaired in GFP ex- that confer resistance to AdV5 infection upon inactivation should pression in MIB1 KO cells (25, 26) (Fig. 1 G and H). MIB1 KO be enriched for disruptive sense orientation gene-trap insertions. cells infected with WT AdV2, AdV7, and AdV35 displayed po- We infected this library with replication-competent GFP-expressing tent inhibition of vDNA replication across all viral serotypes adenovirus 5 (AdV5) and isolated surviving cells (Fig. 1A). (Fig. 1I). Based on these data, we concluded that MIB1 plays a Three genes emerged as significantly enriched for disruptive role in AdV infection downstream of receptor engagement. gene-trap insertions in the infected population, indicating that Lastly, infection of MIB1 KO Hap1 cells with a broad panel of their disruption protected cells from AdV infection and/or cyto- both RNA and DNA viruses showed no clear dependence on toxicity and marking them as potential host factors necessary for MIB1 for infection. Therefore, among all viruses tested in Hap1 AdV5 infection (Fig. 1B). Two of these genes were the well-known cells, MIB1 appears to be an AdV-specific host factor (SI Ap- receptors of AdV5: CXADR encoding CAR and integrin ITGB5, pendix, Fig. S3). validating the effectiveness of the technique in identifying specific determinants of AdV infection (5, 20, 21). Adenovirus Infection Requires the Ubiquitination Activity of MIB1. E3 The third gene identified by the screen was Mindbomb E3 ubiquitin ligases are the effectors of ubiquitination, a post- ubiquitin ligase 1 (MIB1) (Fig. 1B). MIB1 is a large E3 ubiquitin translational modification (PTM) characterized by the attach- ligase with well-described roles in the Notch and Wnt signaling ment of ubiquitin to a target protein. Modification can result in pathways during neural development (22, 23). However, there rapid degradation of the protein by the or alter its were no reported roles for MIB1 in the context of DNA virus localization or binding partners. MIB1 is a member of the RING infection. To validate MIB1 as a necessary host factor for AdV family of E3 ligases. At the N terminus of MIB1 are two sub- infection, we generated Hap1 cells deficient for MIB1 using strate-binding domains termed MZM (Mib-Herc2/ZZ Zinc CRISPR-Cas9 editing and two independent single-guide RNAs Finger/Mib-Herc2) and REP (consisting of two Mib-repeat do- (sgRNAs) targeting exons 1 and 2 of MIB1, respectively (Fig. 1C mains) that are cumulatively designated the “MIB” domain and and SI Appendix,Fig.S1A). To rule out the possibility of CRISPR which serve as the primary determinants of target specificity. off-target effects, we then reconstituted selected sequence- and These domains then connect via a series of ankyrin repeats to a C Western blot-confirmed MIB1 knockout (KO) clones with wild- terminus comprised of three RING finger domains (Fig. 2A). type (WT) MIB1. In addition, we generated Hap1 cells deficient These RING domains, the defining feature of these ligases, act for CAR using CRISPR-Cas9 editing to serve as positive controls by binding E2-conjugating enzymes and facilitating the direct for AdV5 infection inhibition. This panel of cells was then infected transfer of ubiquitin from E2 onto specific target proteins. Re- with two different recombinant AdV5-GFP reporter viruses. constitution of Hap1 MIB1 KO clone 2-1 with domain mutants Replication-competent AdV5 (AdV5rep+), lacking only E4orf3, is of MIB1 (lacking combinations of the MIB1 domain, the RING capable of viral replication and spread following the initial round domain, etc.) demonstrated that only full-length MIB1 was ca- of infection at 24 to 30 hours postinfection (hpi). In contrast, the pable of rescuing AdV infection (SI Appendix, Fig. S4 A and B). − replication-incompetent AdV5 (AdV5rep ) cannot progress to To more specifically determine whether MIB1’s effect on AdV later stages of infection (late mRNA transcription, genome rep- was dependent upon its ubiquitination activity, we reconstituted lication, and virion progeny production) due to the absence of MIB1 KO clone 2-1 with a set of previously characterized point viral proteins E1A and E1B. While Hap1 WT cells supported virus mutants. These included mutations to RING domains 1 (C834S/ infection (GFP expression), all MIB1 KO cells were refractory to H836N) and 2 (C890S), three distinct mutations to RING do- − GFP expression upon infection with both AdV5rep - and AdV5- main 3 that abrogate ubiquitination activity (C977S, C985S, and rep+-GFP viruses (almost to the degree of inhibition observed for M998R), and a patient variant, V943F, which maps to the coiled- CAR KO cells) (Fig. 1 D–F). Reconstitution with WT MIB1 was coil domain between RING domains 2 and 3. This variant has sufficient to restore infection (Fig. 1 D–F). Inhibition of both been shown to eliminate ubiquitination of Notch substrates but is − AdV5rep as well as AdV5rep+ in MIB1 KO cells served as our also posited to disrupt MIB1 homodimerization (22, 27, 28). first indication that MIB1 must act at a step in the viral lifecycle Consistent with previous literature reporting lack of function for preceding late stages of infection. Additional CRISPR KOs of RING domains 1 and 2, only those MIB1 constructs with mu- − − MIB1 generated in A549 cells and primary STAT1 / human fi- tations in RING domain 3 failed to restore AdV infection when broblasts, as well as partial KO in HeLa cells, also resulted in added back to MIB1 KO cells (22, 29) (Fig. 2 B and C). Therefore, dramatic inhibition of AdV5 infection, further validating the im- the ubiquitination activity of MIB1 is required for AdV infection portance of MIB1 to infection in diverse cellular contexts (SI in Hap1 cells.

2of12 | PNAS Sarbanes et al. https://doi.org/10.1073/pnas.2015794118 E3 ubiquitin ligase Mindbomb 1 facilitates nuclear delivery of adenovirus genomes Downloaded by guest on September 28, 2021 1.00 ACB CXADR MIB1 KO MIB1 KO + MIB1

ITGB5 MIB1 1-2 2-1 2-2 1-1 1-2 2-1 2-2 1-1 CAR KO 1 CAR KO 2 WT kDa 0.75 α-MIB1 100 Hap1 cells mutagenized by lentivirus delivery 0.50 50 of gene-trap 8 (complexity ~1x10 ) α-CAR 37 sense/total insertions 0.25

α-β-actin 0.00 Log (No. of insertions) 37

AdV5-GFP infection 0 2 4 6 (~1,000 p/cell) log [number of insertions]

rep- rep+ rep+ AdV5 24 hpi AdV5 24 hpi AdV5 72 hpi D 30 EF40 60 ] 30 20 40

20 Selection by AdV5-GFP -positive cells [% (~2 weeks) -positive cells [%] 10 20

GFP-positive cells [%] GFP-positive 10 GFP GFP

0 0 0 WT WT WT ex 1 ex 2 ex 1 ex 2 ex 1 ex 2 ex 2 ex 1 ex 1 ex 2 ex 1 ex 2

MIB1 KO MIB1 KO MIB1 KO MIB1 KO MIB1 KO MIB1 KO MICROBIOLOGY CAR KO CAR KO + MIB1 + MIB1 + MIB1 CAR KO

AdV5-F7 - 24hpi AdV5-F35 - 24hpi AdV2 AdV7 AdV35 GHI30 40 1.5 receptor: CAR receptor: DSG-2 receptor: CD46 ] ]

30 collect as pool and 20 1.0 deep sequence insertion sites 20

- count independent insertions/gene 10 viral DNA 0.5 -positive cells [% - calculate per gene the significance 10 GFP of enrichment in AdV-infected dataset cells [% GFP-positive compared to an uninfected control 0 0 [norm to WT for each virus] 0.0 1-1 2-1 2-1 1-1 1-1 2-1 WT WT WT WT WT ex 1 ex 1 ex 1 ex 2 ex 1 ex 2 ex 2 ex 2 MIB1 KOMIB1 KO MIB1 KO MIB1 KO MIB1 KO MIB1 KO MIB1 KO CAR KO CAR KO CAR KO CAR KO + MIB1 + MIB1 CAR KO

Fig. 1. Haploid genetic screen identifies E3 ubiquitin ligase MIB1 as a host factor necessary for AdV infection independent of AdV receptor. (A) Outline of haploid genetic screen for AdV5 infection. (B) Fishtail plot showing enrichment of genes in haploid screen upon AdV5 selection identified by deep sequencing and plotted as number of distinct gene-trap insertions (x axis) and the ratio of sense over total insertions (y axis) per gene. Genes significantly enriched for sense orientation integrations (FDR < 0.05) labeled by gene name (red). (C) Western blot of MIB1 CRISPR KO clones in Hap1 cells generated by guide RNAs targeted to either exon 1 (1-1, 1-2) or exon 2 (2-1, 2-2), KO clones reconstituted with MIB1, and two CAR KO clones probed for MIB1, CAR (two isoforms), or β-actin expression. (D–F) Infection of Hap1 WT cells, MIB1 KO clones, WT MIB1-reconstituted KO clones, and CAR KO clones with (D) AdV5rep−-GFP (MOI = 2,500 p/cell; 24 hpi). (E and F) AdV5rep+-GFP [(E) MOI = 75 p/cell; 24 hpi and (F) MOI = 50 p/cell; 72 hpi] and (G and H) Infection of Hap1 WT cells, a CAR KO − clone, MIB1 KO clones, and WT MIB1-reconstituted KO clones with recombinant AdV5rep -GFP expressing the fiber protein of (G) AdV7 or (H) AdV35 (MOI = 1 × 104 p/cell; 24 hpi). (I) Hap1 WT cells, a CAR KO clone, and individual MIB1 KO clones (1-1, 2-1), infected with WT AdV serotypes using distinct entry receptors: AdV2 (CAR), AdV7 (DSG-2), and AdV35 (CD46) at MOI = 1 × 103 p/cell (vDNA was harvested at 72 hpi and measured by qPCR). All experiments were performed in triplicate and, if not indicated otherwise, harvested cells were analyzed by flow cytometry and plotted as the percentage of GFP-positive cells averaged across the two selected MIB1 KO clones for guide RNAs targeting either exon 1 (ex 1) or exon 2 (ex 2).

Of note, levels of MIB1 appear to be tightly regulated as re- window in the viral lifecycle. This early phase encompasses a constitution of WT and MIB1 KO cells with WT MIB1 de- series of entry steps where the virus traverses the cytosol from creased cell proliferation in the majority of clones as well as in the plasma membrane to the nuclear pore, releases its genome, the WT cells (SI Appendix, Fig. S4C). MIB1 KO clone 2-1 and its and begins early transcription. To examine the specific step at WT MIB1-reconstituted counterpart were chosen for functional which MIB1 acts, we utilized a recombinant AdV5 (JR34) and mechanistic follow-up due to comparable cell growth ki- modified to express a FLAG-tagged pVII protein (30). The pVII netics and efficiency of AdV rescue upon reconstitution. protein is a component of the incoming capsid and arranged in tight complex to the vDNA. Within an intact capsid, the FLAG- MIB1 and the Proteasome Are Required for the Delivery of AdV5 DNA tagged pVII is obscured from binding. However, upon into the Nucleus upon Capsid Arrival at the Nuclear Envelope. The release of the vDNA from the capsid into the nucleus, FLAG- − MIB1 dependence of AdV5rep which does not progress to late- tagged pVII (still associated with this vDNA) can be detected by stage infection allowed us to narrow MIB1’s role to the early anti-FLAG and visualized by immunofluorescence

Sarbanes et al. PNAS | 3of12 E3 ubiquitin ligase Mindbomb 1 facilitates nuclear delivery of adenovirus genomes https://doi.org/10.1073/pnas.2015794118 Downloaded by guest on September 28, 2021 A C890S C977S M998R below the level observed in untreated WT MIB1-reconstituted C834S/H836N V943F C985S cells (SI Appendix, Fig. S5 B and C). Loss of vDNA deposition in MIB1 KO and MG132-treated N C WT MIB1-reconstituted cells suggested there might be a defect MZM REP RING1 RING2 RING3 along the trajectory of viral capsids from plasma membrane to nucleus. To resolve first whether MIB1 and the proteasome act MIB ankyrin repeats RING upstream or downstream of centrosome arrival, we examined the spatial distribution of JR34 virus particles in MIB1 KO and MG132-treated WT MIB1-reconstituted cells following treat- B 80 reconstituted MIB1 KO ment with leptomycin B (LMB). LMB, an inhibitor of CRM1- mediated export, has been shown to boost virion association with MT motors leading to dramatic aggregation and arrest of virions 60 directly at the MTOC (11). Indeed, in WT MIB1-reconstituted KO cells treated with LMB prior to and throughout JR34 in- fection we observed robust capsid accumulation at the MTOC 40 upon staining for AdV5 hexon and pericentrin (a defining structural component of the MTOC) (Fig. 3D). Treatment with -positive cells [%]

P LMB also blocked vDNA release into the nucleus as quantified

GF 20 by FLAG-pVII staining (SI Appendix, Fig. S5A). Aggregation of capsids at the MTOC was similarly observed for both MIB1 KO cells and MG132-treated WT MIB1-reconstituted cells only upon treatment with LMB, indicating that MIB1 and the pro- 0 teasome act downstream of arrival at and departure from the centrosome (Fig. 3 D and E). Consistent with this, in untreated V943F C890S C977S C985S M998R C834S/ H836N MIB1 KO and MG132-treated WT MIB1-reconstituted cells, MIB1 KO WT MIB1 Hap1 WT AdV capsids could be readily observed in the vicinity and along MIB1 point mutants the surface of the nucleus (Fig. 3D). Notably, in MIB1 KO and MG132-treated WT MIB1-reconstituted cells this close associ- kDa C MIB1 ation with the nucleus persisted even at 4 hpi, a timepoint when 100 WT MIB1-reconstituted cells show cytoplasmic aggregates of hexon stain indicative of DNA deposition and retrafficking of the β-actin emptied capsids away from the nuclear pore (12) (SI Appendix, 37 Fig. S5B). We therefore concluded that both MIB1 and the proteasome (either independently or cooperatively) mediate Fig. 2. The ubiquitination activity of MIB1 is required for AdV infection. (A) some critical step upon arrival at the nuclear envelope required Schematic of the MIB1 protein with domains and point mutations anno- tated. (B) AdV5rep+-GFP infection (MOI = 200 p/cell; 24 hpi) of Hap1 WT cells, for capsid disassembly and release of vDNA into the nucleus. MIB1 KO clone 2-1 and KO 2-1 reconstituted with panel of point mutants (annotated in A). Experiments were performed in triplicate and harvested MIB1 Localizes to Centriolar Satellites in Hap1 Cells. Our microscopy cells were analyzed by flow cytometry and plotted as the percentage of GFP- studies with the JR34 virus had placed the block on infection in positive cells. (C) Western blot analysis of MIB1 expression levels in Hap1 WT, MIB1 KO cells after arrival at the nuclear envelope but prior to MIB1 KO clone 2-1, and KO 2-1 reconstituted with MIB1 constructs in B. successful vDNA delivery to the nucleus. We were interested in defining the corresponding spatial/cellular context of MIB1 during early AdV infection. To do so we employed APEX2 proximity (IF) microscopy (Fig. 3A). Detection of pVII has been widely labeling to biochemically tag proteins proximal (∼10-nm radius) to employed as a proxy for genome release (31, 32). MIB1 KO MIB1 (33). We reasoned that proteins captured in the immediate clone 2-1 and clone 2-1 reconstituted with WT MIB1 were in- vicinity of MIB1 would not only provide insight into MIB1’slo- fected with JR34 and stained for both capsid (anti-hexon) and calization but might also encompass MIB1 ubiquitination targets. released pVII-vDNA complex (anti-FLAG). Strikingly, while MIB1 KO clone 2-1 cells were stably reconstituted with WT MIB1 MIB1 KO and WT MIB1-reconstituted cells displayed a similar or the RING3 ubiquitination-defective mutant C985S MIB1 tag- quantity of viral particles in the cytoplasm, the subsequent re- ged with FLAG-APEX2 and assayed for their capacity to restore lease of vDNA (quantified as FLAG-pVII foci) into the nucleus AdV infection. Infection was reduced in KO cells reconstituted by 1.25 hpi was severely compromised in KO relative to recon- with APEX2-tagged MIB1 relative to untagged MIB1, possibly stituted cells (Fig. 3 B and C). due to low levels of functional APEX2-tagged protein (SI Ap- Given that MIB1’s ubiquitination activity is required for in- pendix,Fig.S6A). To boost WT MIB1 expression/activity, we in- fection and that one well-characterized consequence of ubiq- troduced the V943F mutation into these constructs which we had uitination is to target the modified protein to the proteasome for found in our MIB1 mutagenesis studies to enhance AdV infection degradation, we were curious whether the proteasome might also over WT MIB1 (Fig. 2B). Indeed, V943F mutants restored the play a role in AdV5 genome delivery. Brief pretreatment with capacity of APEX2-tagged WT MIB1 to rescue virus infection (SI the proteasome inhibitor MG132 before and throughout JR34 Appendix,Fig.S6A). All subsequent APEX2 experiments were infection significantly reduced (five- to sixfold) the appearance conducted in the background of the V943F mutation with APEX2 of FLAG-pVII foci in WT MIB1-reconstituted cells by 1.25 hpi fused to the C terminus of MIB1. In parallel, we generated MIB1 (Fig. 3 B and C). Thus, blocking proteasomal degradation in WT KO cells expressing APEX2 fused to the fluorescent protein MIB1-reconstituted cells phenocopied MIB1 KO at this stage of mKate. As a ubiquitously localized cytoplasmic protein, the mKate infection, suggesting in both cases that aberrant stabilization construct served as a generic baseline control over which enrich- of some target protein may impede virus infection. Inhibition of ment for specific proximal partners of MIB1 could be identified. vDNA release by MG132, however, reflects a delay rather than We first established the expression and labeling specificity of the sustained block imposed by lack of MIB1 as FLAG-pVII our three different constructs by IF in uninfected cells. MIB1 KO signal increased over time in MG132-treated cells, although still cells reconstituted with WT MIB1-APEX2, C985S MIB1-APEX2,

4of12 | PNAS Sarbanes et al. https://doi.org/10.1073/pnas.2015794118 E3 ubiquitin ligase Mindbomb 1 facilitates nuclear delivery of adenovirus genomes Downloaded by guest on September 28, 2021 A B +MG132 MIB1 KO + WT MIB1 MIB1 KO MIB1 KO + WT MIB1 MIB1 KO FLAG-pVII hexon hexon FLAG-pVII

NPC +DRAQ5 merge

C E 1.3 1.3 3000 untrt

1.0 1.0 LMB MICROBIOLOGY 2000

0.5 0.5 1000 (over MTOC region) (over MTOC mean FITC (hexon) intensity hexon foci/nuclear area FLAG-pVII foci/ hexon foci (norm. to KO +WT MIB untrt) (norm. to KO +WT MIB untrt) 0.0 0.0 0 +WT MIB1KO +WT MIB1 KO +WT MIB1KO +WT MIB1 KO +WT MIB +WT MIB1 MIB1 KO +MG132 +MG132 +MG132

D untreated +LMB +MG132 +LMB +MG132 MIB1 KO +WT MIB1 KO

hexon pericentrin DRAQ5

Fig. 3. MIB1 and the proteasome are required for AdV DNA delivery upon capsid arrival at the nuclear envelope. (A) Schematic of recombinant AdV5 (JR34) expressing FLAG-tagged pVII protein which is inaccessible to antibody binding within capsid but becomes accessible upon DNA release at the NPC. (B) Representative images of MIB1 KO clone 2-1 and its WT MIB1-reconstituted counterpart infected with JR34 (MOI = 2,000 p/cell; 1.25 hpi) in the presence or absence of MG132 (30 μM) stained for capsid (anti-hexon) and pVII (anti-FLAG) and merged with DRAQ5 nuclear stain. (Scale bar: 10 μm, maximum projected.) (C) Quantification of hexon foci and FLAG foci seen in B normalized to nuclear area and hexon foci, respectively, per field of view (FOV) and averaged across five FOV. (D) Representative images showing distribution of AdV capsids (green: anti-hexon) relative to centrosome (red: anti-pericentrin) merged with DRAQ5 nuclear stain (blue) in MIB1 KO clone 2-1 and its WT MIB1-reconstituted counterpart infected with JR34 (as above) in the presence or absence of LMB (20 nM) and/or MG132 (30 μM). (Scale bar: 10 μm, maximum projected.) (E) Quantification of distribution of AdV capsids relative to MTOC depicted in D as the mean FITC intensity (anti-hexon) over the MTOC region (anti-pericentrin) averaged across five FOV.

Sarbanes et al. PNAS | 5of12 E3 ubiquitin ligase Mindbomb 1 facilitates nuclear delivery of adenovirus genomes https://doi.org/10.1073/pnas.2015794118 Downloaded by guest on September 28, 2021 and APEX2-mKate underwent APEX2 labeling. Staining for the At the same time, proteins enriched in proximity to the mutant introduced FLAG-APEX2-tagged protein revealed that both WT C985S MIB1 reflected cytoskeleton components (adj. P value = MIB1-APEX2 and C985S MIB1-APEX2 localized predominantly 0.0201) by GO term analysis (Fig. 4C). Of note, these proteins to a single point within each cell. This was corroborated by strong included NUP85, more commonly referred to as pericentrin, (H2O2 dependent) streptavidin stain for biotinylated proteins in which we had used as a marker for the centrosome in our IF the region surrounding the APEX2-tagged protein (Fig. 4A). The experiments. We concluded that WT and C985S MIB1 possess observation of this singular per cell aligned with reported distinct differences in their proximal proteomes—with WT MIB1 roles for MIB1 as a key component of centriolar satellites, sub- associating preferentially with RNPs while C985S MIB1 remains structures in the vicinity of the centrosome involved in regulation more tightly associated with certain cytoskeletal and centrosome of homeostasis and ciliogenesis (34, 35). We confirmed components. the dominant localization of FLAG-APEX2-tagged WT and C985S MIB1 to the centrosome in AdV-infected cells by cos- The MIB1 Ubiquitinated Proteome Is Enriched in RNP Factors and taining for FLAG-tagged MIB1 constructs and pericentrin (SI Depleted in Cytoskeletal Factors. While proximity-labeling pro- Appendix,Fig.S6B). By contrast, APEX2-tagged mKate and bi- teomics had characterized MIB1’s cellular context during AdV otin label was found throughout the cell with no discrete infection, we were also interested in proteins that intersected localization (Fig. 4A). more directly with MIB1 in its role as an E3 ubiquitin ligase. To To obtain proximal proteomes for each of these constructs characterize possible ubiquitination targets of MIB1 during virus in AdV-infected cells, MIB1 KO cells reconstituted with WT infection, we turned to a complementary proteomics approach. MIB1-APEX2, C985S MIB1-APEX2, and APEX2-mKate were Tandem ubiquitin binding entities (TUBEs) can bind to diverse infected with 200 virus particles/cell (p/cell) of AdV5rep+-GFP ubiquitin linkage types (K48, K63, mono, poly, etc.) and have followed by labeling at 70 min postinfection (mpi), been widely employed to isolate endogenous ubiquitinated pro- streptavidin isolation of biotinylated proteins, and liquid chro- teins from total cell lysates (38). Using this approach, we hoped matography-tandem mass spectrometry (LC-MS/MS). Corrobo- to identify those proteins enriched for ubiquitination in WT rating the localization observed by IF, proteins in proximity to MIB1-expressing cells relative to both cells lacking MIB1 or cells both WT and C985S MIB1 in infected cells were significantly reconstituted with the mutant C985S MIB1 in which all MIB1- enriched (twofold or greater) in centrosome (adjusted [adj.] P mediated ubiquitination events should be absent. MIB1 KO − − value = 6.62 × 10 8 and 1.1 × 10 6 for WT and C985S MIB1, cells, and KO cells reconstituted with either WT MIB1 or C985S respectively) and, furthermore, centriolar satellite components MIB1 were infected with AdV5rep+-GFP (200 p/cell) and col- (e.g., CCDC14, CEP131, KIAA0753, OFD1, PCM1, PIBF1, and lected at 1 hpi. Lysates were incubated with TUBE Dynabeads, − − SSX2IP) (adj. P value = 9.57 × 10 9 and 2.29 × 10 6 for WT and and the isolated proteins were submitted for LC-MS/MS. C985S MIB1, respectively) (Fig. 4B). Interestingly, the Golgi We identified 9 proteins that were significantly (P < 0.05) subcompartment and coated vesicles were additional functional enriched by greater than twofold in both the comparison be- categories enriched proximal to both WT and C985S MIB1 tween WT MIB1 versus KO cells and between WT MIB1 versus relative to the mKate control (SI Appendix, Table S1). No AdV5 C985S MIB1 following TUBE incubation. These were considered virion capsid proteins were detected in any of the samples. strong potential candidates of direct ubiquitination by MIB1. Several of these MIB1 target candidates, including STAU2 and The MIB1 Proximal Proteome Is Ubiquitination-Dependent and DRG1, have been reported to localize to the spindle or centro- Enriched in RNP-Related Factors. We next compared the proximal some and possess MT-binding domains (39, 40). This increased proteomes of WT MIB1 against the mutant C985S MIB1 in confidence in the capacity of this ubiquitination-based but prox- AdV-infected cells directly (Fig. 4C). We had hypothesized that imity-blind MS approach to detect plausible targets of centro- in WT MIB1-expressing cells, rapid release kinetics and/or sub- some- and satellite-localized MIB1. Reducing our stringency to sequent degradation of targets by the proteasome might result in proteins significantly enriched by more than twofold in a single a depletion of direct ubiquitination targets in the vicinity of WT group comparison (WT MIB1 over MIB1 KO cells or WT MIB1 MIB1. Direct targets might then be enriched in proximity to the over C985S MIB1) but enriched by greater than twofold in both C985S mutant that, while unable to carry out ubiquitination, yielded an additional list of 22 proteins more strongly observed should retain the capacity to bind substrates. However, we also upon TUBE isolation in the presence of WT MIB1 (SI Appendix, considered the possibility that MIB1’s protein partners and lo- Table S2). GO analysis of this expanded protein list (31 proteins: calization may in fact depend upon its ability to carry out ubiq- orange) revealed enrichment of “RNA binding” (8/31 proteins, uitination of a specific target protein(s). adj. P value = 0.0028), “RNA transport” (6/31 proteins, adj. P Despite an overarching shared association with centriolar sat- value = 0.001), and “ribonucleoprotein complex” (10/31 proteins, − ellites and when compared against the mKate con- adj. P value = 4.12 × 10 5) categories (Fig. 4D). The emergence of trol, comparison of WT MIB1 versus the mutant C985S MIB1 these functional categories corroborated the observation of RNA- revealed key differences in their proximal proteomes (Fig. 4C). related proteins in proximity to WT MIB1 from our proximity- Among those proteins enriched in proximity to WT relative to labeling dataset. C985S MIB1 were a number of well-characterized cytoplasmic We also noted, in parallel, 73 proteins that were depleted RNA granule components. By (GO) terms analysis, upon TUBE incubation in cells expressing WT MIB1 over both we identified significant enrichment of RNA-binding (adj. P MIB1 KO and C985S-reconstituted cells (fold change [FC] < −2 value = 0.0003) and RNA transport genes (adj. P value = 0.0375). in both, P < 0.05 in either) (SI Appendix, Table S2). GO terms CAPRIN1, ATXN2, and MSI2 are components of stress granules, analysis of this set of proteins revealed enrichment for functional cytoplasmic aggregates of stalled translational machinery that categories corresponding to either “cytoskeleton” (29/73 proteins, − form in response to cellular stresses (36). UBQLN2, enriched in adj. P value = 2.1 × 10 7)or“proteasome-mediated ubiquitin-de- proximity to WT MIB1, although not an RNA-binding protein, pendent protein catabolism” (16/73 proteins, adj. P value = 1.1 × − has been shown to associate with stress granules and act as a 10 8)(Fig.4D). This also corroborated our proximity-labeling data bridge between stress granules and the ubiquitin–proteasome in which the C985S mutant displayed enhanced proximity to a subset pathway (37). EDC4 (classified among the significantly enriched of cytoskeletal proteins. Due to the mild, nondenaturing conditions category “cytoplasmic ribonucleoprotein granule”)isadefining of TUBE incubation, we hypothesize that these proteins might re- factor within constitutively present RNA processing bodies, or flect broader ubiquitin-dependent changes in the binding partners of P-bodies, sites of RNA storage and degradation (36). TUBE-isolated MIB1-ubiquitinated proteins, specifically, binding

6of12 | PNAS Sarbanes et al. https://doi.org/10.1073/pnas.2015794118 E3 ubiquitin ligase Mindbomb 1 facilitates nuclear delivery of adenovirus genomes Downloaded by guest on September 28, 2021 ABFLAG-APEX2 Streptavidin merge

Proximal proteome (APEX2) -H202 10 MIB1 +WT MIB1-APEX2

CEP131 PCM1 CCDC14 5 AKAP9 OFD1 PIBF1 CDK5RAP2 SSX2IP BICD1 CSPP1 KIAA0753 +WT MIB1-APEX2 KIAA0101 CEP350 PCM1 0 CEP192 C985S/mKate log2(FC) +H202

-5 +C985S-APEX2

-5 0 5 10 WT MIB1/mKate log2(FC) APEX2- mKate

C Proximal proteome (APEX2) D Ubiquitinated proteome (TUBEs) MICROBIOLOGY HNRNPK ABI1 5.0 SUCLG1 PRKRA NSUN2 3 RANBP9 STAU2 SRPK1 NUP85 EXOSC10 SMNDC1 FMR1 2.5 H1FX SNRPA1 NUDCD2 HDAC2 ZCCHC3 ZC3H11A KHSRP EIF3C RAN FUBP1 UPF1 2 ANKRD26 PRRC2C CCT4 ATXN2 SERBP1 API5 YY1 HAUS6 0.0 PRKAR2A RBMX SOX2 EIF4E2 BAIAP2L1 VPS4B KIF1A ANXA11 CAPRIN1SRP14 MYH9 -log(p-value) WDR62 LARP4 MSI2 TUBB EML4 PPP5C -2.5 TUBB6 1 +WT MIB1/+C985S log2(FC) BAG2 AURKB VIM TUBB4B TUBA4A TUBB4A PAWR TUBA1C -5.0 TUBB3

0 HSPB1

-30 3 6 -6 -30 3 6 WT MIB1/C985S log2(FC) +WT MIB1/KO log2(FC)

Fig. 4. Proteomic approaches confirm MIB1 localization to centriolar satellites in AdV-infected Hap1 cells and reveal MIB1 ubiquitination-dependent as- sociation with RNPs. (A) Uninfected MIB1 KO clone 2-1 reconstituted with FLAG-APEX2–tagged WT MIB1, C985S MIB1, or mKate were incubated with biotin-

phenol for 1 h and pulse labeled with H2O2 followed by fixation and staining with AF488-coupled streptavidin for biotinylated proteins (green), the indicated FLAG-APEX2–tagged construct (red: anti-FLAG), and DRAQ5 nuclear stain (blue). (Scale bar: 10 μm, maximum projected.) (B and C) For LC-MS/MS, MIB1 KO clone 2-1 reconstituted with FLAG-APEX2 fused to either mKate, WT MIB1, or C985S MIB1 were incubated with biotin-phenol and infected with AdV5rep+-GFP

(MOI = 200p/cell), pulse-labeled with H2O2 at 70 min pi, and immediately collected for lysate preparation and streptavidin pulldown to isolate biotinylated proteins (n = 3). (B) Proximal protein enrichment (FC) over APEX2-mKate is plotted as log2 of the ratio of protein abundance for WT MIB1/mKate (x axis) and C985S MIB1/mKate (y axis). GO terms analysis of proteins twofold or greater (P value <0.05) in the WT MIB1/mKate comparison (blue) shows enrichment of centriolar satellite proteins (green, red rim), as a subset of centrosome proteins (red). (C) Ubiquitination-dependent proximal proteome determined by direct

comparison of APEX2-tagged WT MIB1 and C985S MIB1 (unique peptides ≥1) plotted as log2 of the ratio of protein abundance for WT MIB1/C985S MIB1 (x axis) and −log(P value) (y axis). GO terms analysis of those proteins enriched by twofold or greater (orange) or depleted by twofold or greater (blue) in proximity to WT MIB1 relative to C985S MIB1 (P value <0.05) reveals enrichment of RNA-binding proteins (red with gene names labeled) and depletion of cytoskeletal proteins (purple with gene names labeled). (D) Identification of differentially ubiquitinated proteins between MIB1 KO clone 2-1 and KO reconstituted with either WT MIB1 or C985S MIB1 infected with AdV5rep+-GFP (MOI = 200 p/cell) (n = 3). At 1 hpi, cells were harvested and endogenous

ubiquitinated proteins were isolated using TUBE-Dynabeads followed by LC-MS/MS and plotted as log2 of the ratio of protein abundance for WT MIB1/MIB1 KO (x axis) and WT MIB1/C985S MIB1 (y axis). GO terms analysis of those proteins enriched twofold or greater in both WT MIB1/KO and WT MIB1/C985S (P value <0.05 in either) (orange) or depleted by twofold or greater in WT MIB1/KO and WT MIB1/C985S (P value <0.05 in either) (blue) reflect RNA binding (red with all gene names labeled) or cytoskeleton (purple with select proteins significant to both WT MIB1/KO and WT MIB1/C985S condition labeled by gene name), respectively.

Sarbanes et al. PNAS | 7of12 E3 ubiquitin ligase Mindbomb 1 facilitates nuclear delivery of adenovirus genomes https://doi.org/10.1073/pnas.2015794118 Downloaded by guest on September 28, 2021 partners whose association might be abrogated following ubiquiti- AdV serotypes were dependent on MIB1 for virus delivery and nation of a given target by WT MIB1. Again no viral capsid proteins replication. Sequence diversity of capsid and capsid-associated were detected. proteins between these serotypes as well as the lack of APEX2 proximity labeling or TUBE isolation of capsid proteins lead us to Discussion favor a cellular protein as the target of MIB1 ubiquitination. In this study, we carried out a genome-wide gene-trap screen of However, the possibility that MIB1 acts through a conserved motif AdV infection and identified an AdV host factor, the E3 ubiq- across serotypes or through combinatorial action on multiple uitin ligase MIB1. Using fluorescence microscopy, we deter- targets cannot be excluded. By identifying those proteins enriched mined that MIB1 is required for the final step in AdV entry: upon TUBE incubation in WT MIB1-expressing cells, we have capsid disassembly and vDNA release at the nuclear pore. In the generated a list of MIB1 target candidates that may next be pro- absence of MIB1, capsids successfully reach the nucleus but fail bed individually for their effect on AdV infectivity and DNA to deposit their vDNA within (Fig. 5). As an E3 ubiquitin ligase, release. MIB1 acts by ubiquitinating specific substrates resulting in either In the meantime, our unbiased proteomic approaches have degradation by the proteasome or alteration of signaling/locali- uncovered an intriguing balance between functional classes of zation (34, 41). Using a series of point mutants we determined proteins—RNPs and cytoskeleton—that appear regulated by that virus infection depends upon MIB1’s ubiquitination activity. MIB1 and may therefore be altered in MIB1 KO cells with con- Our subsequent observation that the proteasome is similarly sequences for AdV DNA delivery. Comparing the proximal pro- required for vDNA release upon capsid arrival at the nucleus, teomes of WT MIB1 relative to the C985S mutant revealed that suggests that ubiquitination by MIB1 mediates AdV genome while a broad association with centriolar satellites is shared, WT delivery through proteasomal degradation of a negative regula- MIB1 is enriched in proximity to a number of well-characterized tor of AdV infection (Fig. 5). In this respect, AdVs now emerge RNA-binding proteins while depleted for certain cytoskeletal as nucleus-targeting viruses, similar to influenza virus and herpes components. Isolation of ubiquitinated proteins from MIB1 KO simplex virus (HSV), that require ubiquitin and/or the protea- cells and KO cells reconstituted with WT MIB1 or C985S MIB1 some for successful vDNA delivery to the nucleus (42, 43). recapitulated even more strikingly this MIB1 ubiquitination-de- During preparation of this manuscript, work now published by pendent enrichment of RNP-related proteins and commensurate Bauer et al. similarly identified and characterized MIB1 as a host depletion of cytoskeletal factors. factor for AdV infection required for AdV genome delivery (44). An existing role for MIB1 in the regulation of RNPs has Using electron microscopy, they show that in MIB1 KO cells, emerged very recently in the context of spermatogenesis in AdV capsids arrive at the NPC but remain paralyzed at the pore, (45). C. elegans spermatids lack a nuclear intact and unable to release the vDNA into the nucleus. Their envelope and instead delineate nuclear and cytoplasmic com- observation of transient interactions between MIB1 and NPC- partments via a dense halo of RNA and RNA-binding proteins docked virions by live-cell imaging suggests that the critical MIB1 that encompass chromatin and centrosome. In C. elegans sper- ubiquitination event(s) occurs at this site. Having validated MIB1 matids harboring mutations in MIB1, this perinuclear RNA halo as a component of centriolar satellites by proximity-labeling pro- is absent and large tubules are observed in its place (45, 46). An teomics, we posit that MIB1 interactions near the nuclear pore overabundance of in these mutants is also observed (47). could occur as part of these centrosomal substructures dynami- Our findings imply that this RNP-regulatory role for MIB1 may cally sampling the region between centrosome and nuclear extend to mammalian somatic cells. Taken together, these ob- membrane (Fig. 5). servations not only suggest a conserved role for MIB1 in regu- The specific target(s) of MIB1 ubiquitination relevant to AdV lating a balance between RNPs and filamentous structures but infection has remained elusive. We had determined that different furthermore, specifically at the boundary between cytoplasm and

WT MIB1 KO WT MTOC centriolar +MG132 satellite

proteasome

AdV capsid capsid disassembly NPC no capsid no capsid disassembly disassembly

nuclear no vDNA deposition no vDNA deposition deposition 1.25 hpi of vDNA

Fig. 5. Schematic model of MIB1’s role in AdV genome deposition following capsid arrival at the nuclear pore. In WT cells (Left), MIB1, an E3 ubiquitin ligase, and centriolar satellite component, facilitates successful capsid disassembly and vDNA deposition into the nucleus through the ubiquitination and protea- somal degradation of an as-yet-unidentified target protein (white oval). In MIB1 KO cells (Middle), the MIB1 target is no longer ubiquitinated and degraded resulting in a block on capsid disassembly and genome release. Similarly, upon treatment of WT cells with MG132 (Right), the ubiquitinated MIB1 target, no longer able to be degraded by the proteasome, persists and inhibits capsid disassembly and genome release (graphic created with BioRender.com).

8of12 | PNAS Sarbanes et al. https://doi.org/10.1073/pnas.2015794118 E3 ubiquitin ligase Mindbomb 1 facilitates nuclear delivery of adenovirus genomes Downloaded by guest on September 28, 2021 nucleus. This is precisely the interface at which MIB1 appears to 62251; 1:1,000) was used for nuclear stain. Drugs were used at concentrations act during AdV infection. indicated in figure legends and include MG132 (Sigma: M7449), leptomycin B We hypothesize that MIB1’s role in AdV genome delivery may (LMB) (Millipore: L2913), and the deubiquitinating enzyme (DUB)-inhibitor PR- intersect with its emerging role in perinuclear RNP regulation. 619 (LifeSensors: SI9619). RNPs have, in fact, already been implicated in the relevant step Cell Culture. Hap1 cells (obtained from T.R.B.) were cultured in Iscove’s of AdV capsid docking and DNA delivery at the NPC. Trotman modified Dulbecco’s medium (IMDM, Gibco) supplemented to contain 10% et al. observed that rabbit reticulocyte lysates inhibited capsid fetal bovine serum (FBS) and 1% nonessential amino acids (NEAAs) and binding to nuclear envelopes in vitro due to the presence of an utilized for the haploid gene-trap screen as well as for followup validation − − inhibitory RNP whose components remain unknown (13). Might and mechanistic studies into MIB1. A549, HeLa, and human STAT1 / fibro- any of the RNP granule-associated proteins identified through blasts (immortalized by SV40 large T antigen) (for evaluation of MIB1 phe- our proteomic approaches overlap with components of this notype in different cellular backgrounds), HEK293 (production of replication- mystery RNP? We posit that MIB1 might mediate a reorgani- incompetent AdV), and derivative Lenti-X 293T cells (production of lentiviruses) ’ zation or local enrichment of this RNP at the nuclear envelope were maintained in Dulbecco s modified Eagle medium (DMEM, Gibco) sup- plemented to contain 10% FBS and 1% NEAAs. to assist in the forcible displacement of capsids from the nuclear pore (perhaps by competing for direct binding with or by si- Haploid Genetic Screen. Gene-trap retrovirus was produced in 293T cells as multaneously displacing Nup214). This would be compatible previously described (18). To generate the mutagenized library, 1 × 108 Hap1 with Trotman et al.’s observations in which direct addition of this cells were infected for 3 consecutive days in the presence of protamine sulfate RNP from a spatially disrupted lysate would preclude capsid (8 mg/mL) and passaged for 5 d prior to freezing. For the screen itself, a library binding in the first place. of 1 × 108 mutagenized cells was infected with AdV5rep+-GFP at a multiplicity In C. elegans, the mib-1 mutant phenotype is phenocopied by of infection (MOI) of 1,000 virus particles/cell. The resistant colonies were ex- 7 mutations in a number of endogenous RNA interference (RNAi) panded for 15 d and ∼1 × 10 cells were used for gDNA isolation. factors, including homologs of defining components of mam- malian RNPs known as P-bodies (48). Mammalian P-bodies are Sequence Analysis of Gene-Trap Insertion Sites. Gene-trap insertion sites in AdV5-positive cells were identified by sequencing the gDNA flanking the sites of mRNA sequestration and degradation and have even been gene-trap insertion site as previously described (52). Genomic DNA was shown to migrate along MTs and associate with the centrosome subjected to a linear amplification PCR using a biotinylated primer followed (49). As such, P-bodies could be prime candidates for interaction by single-stranded DNA linker ligation, PCR, and subsequent sequencing with MIB1 as a component of centriolar satellites. Indeed, recent using the HiSEq. 2000 platform (Illumina). The acquired reads were mapped work by others to define the proximal proteome of centriolar to the (hg19) using the Bowtie alignment tool allowing for satellites using a number of different baits (including MIB1), as a single mismatch, and insertion sites located in Refseq genes were identi- MICROBIOLOGY well as our own proximity-labeling data, have repeatedly uncov- fied. For each Refseq gene, the longest transcript was chosen and over- ered P-body components in proximity to WT MIB1 (50, 51). lapping gene regions were disregarded, since orientation bias cannot be unambiguously assigned in these areas. The number of disruptive mutations Recent studies monitoring the dynamics of P-bodies have (i.e., in sense with transcriptional orientation) and nondisruptive (i.e., in observed a phenomenon in which P-bodies that traffic to the sur- antisense with transcriptional orientation) mutations per individual gene face of the nuclear membrane are rapidly redirected away again was counted and genes significantly enriched for disruptive mutations fol- (49). These dynamics imply a MT switch triggered upon arrival at lowing AdV5 infection were determined using a binomial test and corrected the surface of the nucleus reminiscent of that proposed by Strunze for multiple testing using a Benjamini–Hochberg false discovery rate (FDR). et al. for AdV capsids (12). In their model, the simultaneous Significantly enriched genes with over 40 integrations in the AdV dataset coupling of AdV capsids to kinesin-1 on periphery-directed MTs (National Center for Biotechnology Information [NCBI] Single Read Archive and to import factors transiting through the pore generates a [SRA] accession no: SRP269890 dataset SRX8663593) were compared to a shearing force required for capsid disruption and DNA release. reference dataset of WT Hap1 cells (NCBI SRA accession no: SRP058962 dataset SRX1045464) that was analyzed similarly. Genes already significantly We propose that AdV capsids may achieve this MT-mediated enriched in the WT dataset were subtracted from the identified AdV host disruption by intersecting with a retrafficking pathway of P-bodies factor genes. regulated by MIB1. Simultaneous live-cell particle tracking of fluorescently labeled P-bodies and AdV capsids will help clarify if Plasmid Generation. The MIB1 sequence was PCR amplified off of p3HA-hMIB1 and how the dynamics of these species are intertwined and altered plasmid (gift from Vanessa Redecke, St. Jude Children’s Research Hospital, in the absence of MIB1. Memphis, TN [Addgene plasmid #33317; http://www.addgene.org/33317; The work described here encompassing the identification and RRID: Addgene_33317]) (53) and subcloned into the pDONOR Gateway characterization of MIB1 as a host factor of AdV infection has vector. The MIB1 open (ORF) was then transferred via Gate- paved the way for these types of mechanistic studies which will way cloning into the lentiviral pLX304 backbone and subsequently used for lentivirus production. MIB1 point mutants were generated using site-di- continue to yield new insights into the fascinating complexity of rected mutagenesis of the WT MIB1 construct with Phusion High-Fidelity PCR viral genome delivery. Master Mix (Thermo Fisher Scientific). The plasmid was then digested with DpnI to remove template plasmid input and PCR purified using the Qiaquick Materials and Methods PCR Purification Kit (Qiagen). Domain mutants were generated via ampli- Antibodies and Chemicals. Primary antibodies used for Western blot include fication of WT MIB1 sequence with primer-mediated incorporation of a rabbit anti-MIB1 (Abcam: 124929 1:2,000), rabbit anti-CAR (Santa Cruz: sc- 3×FLAG tag sequence at the 5′ end to facilitate detection by Western blot. 15405; 1:1,000), and mouse anti-FLAG M2 (Sigma: F3165; 1:2,000). Primary antibodies for IF experiments include mouse anti-hexon (AdV5) (Develop- Lentivirus Production and Transduction. Lentivirus stocks were generated in ment Studies Hybridoma: TC21-9C12.C9; 1:200), rabbit anti-FLAG (Cell Sig- Lenti-X 293T cells (Takara Bio Inc.: 632180) by cotransfection of plasmids naling Technologies: 14793S; 1:1,000), and rabbit anti-pericentrin (Abcam: expressing 1) the ORF or sgRNA of interest, 2) HIV gag-pol, and 3) the ve- ab4448; 1:1,000). Infections with influenza A virus and Zika virus (ZIKV), and sicular stomatitis virus glycoprotein (VSV-G) in a ratio of 0.55:0.35:0.1. For cell surface expression of CAR, were quantified by flow cytometry using blasticidin-selectable lentiviral vectors, we used the pLX304 lentiviral back- mouse anti- (NP) (Millipore: MAB8251; 1:1,000), mouse anti- bone in which the V5 tag was removed, and for puromycin-selectable len- flavivirus group antigen 4G2 (Millipore: MAB10216; 1:500), and PE-conjugated tiviral vectors, we used the lentiCRISPRv2 lentiviral backbone (gift from Feng rabbit anti-CAR (Sino Biological: 10799-R271-P), respectively. Secondary anti- Zheng, Broad Institute of MIT and Harvard, Cambridge, MA). Cells were bodies were Alexa Fluor 488-, Alexa Fluor 594-conjugated anti-mouse and transfected using Lipofectamine 2000 (Invitrogen) and media were replaced anti-rabbit IgG (Thermo Fisher Scientific), Alexa Fluor 680-conjugated to with DMEM containing 3% FBS at 6 h posttransfection. Supernatants were streptavidin (Thermo Fisher Scientific), goat anti-rabbit and anti-mouse IgG collected at 24 and 48 h, clarified by centrifugation at 2,850 × g, filtered conjugated to horseradish peroxidase (HRP), and mouse anti–β-actin conju- using 0.45-μm syringe filters, and stored at −80 °C. For lentiviral transduction, gated to HRP (Sigma: A3854; 1:10,000). DRAQ5 (Thermo Fisher Scientific: cells were seeded into six-well plates at a density of 4 × 105 cells/well and

Sarbanes et al. PNAS | 9of12 E3 ubiquitin ligase Mindbomb 1 facilitates nuclear delivery of adenovirus genomes https://doi.org/10.1073/pnas.2015794118 Downloaded by guest on September 28, 2021 transduced with lentiviral pseudoparticles by spinoculation at 930 × g for 5′-AGTCCGCTCCCATGTCAAAG-3′) and Ad35 (HAd35prF131: 5′-TTTCATTTTTCG‐ 60 min at 37 °C in medium containing 3% FBS, 20 mM Hepes, and 4 μg/mL CGCACGGT-3′, HAd35prR131: 5′-ACCCCAAAGACGGCCTAATG-3′). polybrene. Transduced cells were selected at 24 h posttransduction with either For infection with JR34 AdV5 for imaging of FLAG-pVII DNA release, cells 2.5 μg/mL blasticidin or 1 μg/mL puromycin. were seeded at 2 × 105 cells per well onto a glass coverslip pretreated with poly-L- in 24-well plates. The next day, cells were washed once with Generation and Validation of CRISPR KO Clones. sgRNA for CRISPR editing cold Opti-MEM, wash was replaced with 200 μL Opti-MEM, and placed on ice targeting MIB1 (sgRNA #1: 5′-CCGGAATAACCGGGTGATGG-3′ and sgRNA #2: for 10 min to cool before spiking in 50 μL of JR34 virus at the indicated MOI 5′-CACTTCCCGGTGTAGTAATT-3′ targeting exon 1 and exon 2, respectively) (typically 2,000 p/cell). Infection was synchronized on ice for 45 min and cells were designed using http://crispor.tefor.net/ (54) and cloned into pX330 or were washed once with cold Opti-MEM and incubated with warmed IMDM lentiCRISPRv2 plasmid for subsequent transfection. MIB1 CRISPR KOs in Hap1 at 37 °C to initiate infection. For drug treatment experiments unless other- cells were generated by transfection with the relevant guides in pX330 and wise stated, cells were pretreated at 37 °C for 30 min prior to virus addition, cultured for 4 d prior to single-cell seeding into 96-well plates. MIB1 KOs in and drug was included during the 45-min synchronization on ice and over all other cell lines (in HeLa, A549, STAT1−/− cells) were generated by trans- the course of infection. ducing with lentiviruses generated from lentiCRISPRv2 containing the rele- vant guides and selected for 3 d with puromycin (1 μg/mL). Transduced cells Immunofluorescence. Unless otherwise described, cells were fixed in 4% PFA were seeded into a 96-well plate at a dilution of 0.7 cells/well to obtain prepared in PBS at RT for 10 min followed by blocking with PBTG (PBS single-cell clonal populations. Clones were expanded and screened for pro- containing 10% normal goat serum, 1% BSA, 0.1% Triton X-100) at RT for 1 tein knockout by Western blot analysis. Genomic DNA was extracted using to 2 h. Cells were then incubated with primary antibodies diluted in PBTG the Qiagen DNeasy Blood and Tissue Kit (QIAGEN: 69504) and used as a (concentrations above) at 4 °C overnight. Following four washes with PBS template for amplification of an ∼800- to 1,000-bp region flanking the PAM with 0.1% Tween-20 (PBST), cells were stained with Alexa Fluor-conjugated site. Discrete bands were gel extracted, cloned into the pCR4-TOPO vector secondary antibodies in PBTG (concentrations above) at RT for 1 h. Following using the TOPO TA Cloning Kit (Invitrogen: 45-0030), and Sanger sequenced. one wash with PBST, cells were incubated with nuclear stain DRAQ5 diluted Relative proliferation of WT and verified KO clones was measured using the in PBS at RT for 15 min, followed by washes with PBST and a final wash with Cell Titer-Glo Luminescent Cell Viability Assay (Promega: G7570) according PBS. Coverslips were mounted on SuperFrost Plus Microscope Slides (Thermo to manufacturer’s instructions. Fisher Scientific: 12-550-15) using ProLong Gold antifade reagent (Invi- trogen: P36934). Images were acquired using a 63× oil immersion objective Virus Production and Infections. Replication-incompetent AdV5rep−-GFP (al- on an inverted Zeiss Axiovert 200 spinning disk confocal microscope using ternatively Ad5CiG for CMV-driven CAT-IRES-GFP reporter), WT AdV2, AdV7, solid-state 491, 561, and 644 lasers (Spectral Applied) for excitation for col- × AdV35, Ad5CiG-F7 (AdV7 fiber), and Ad5CiG-F35 (AdV35 fiber) viruses were lection with an Andor iXon 512 512 electron multiplying charge-coupled provided by E.F.-P. and prepared as previously described (25, 55). Replica- device camera through MetaMorph software. Acquired images were ana- rep+ lyzed as described using Fiji software (64). tion-competent AdV5 (AdV5 -GFP in which E4-ORF3 ORF has been replaced by EGFP) was generously provided by Patrick Hearing, Stony Brook · University, Stony Brook, NY. The protein VII-FLAG-tagged JR34 adenovirus Western Blot. Cell lysates were prepared in RIPA buffer (50 mM Tris HCl [pH (E1/E3 deleted) was a generous gift from Robin J. Parks, Ottawa Health 8.0], 150 mM NaCl, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxy- Research Institute, Ottawa, Ontario, Canada (30). cholate, 1% Nonidet P-40 with addition of cOmplete Mini EDTA (ethyl- enediaminetetraacetic acid)-free protease inhibitor tablet; Roche), incubated The generation of viral stocks for additional viruses tested has been on ice for 30 min and clarified at 14,000 × g for 20 min at 4 °C. Protein previously described: hPIV3-GFP (56) (based on strain JS), YFV-Venus (57) (de- concentration was determined by bicinoninic (BCA) protein assay (Thermo rived from YF17D-5′C25Venus2AUbi), VEEV-GFP (58) (derived from pTC83-GFP Fisher Scientific: 23227) and samples were resolved on 4 to 12% Bis-Tris gels infectious clone), VacV-GFP (59) (based on strain: Western Reserve), HSV-1 (Invitrogen) followed by transfer onto nitrocellulose 0.45 μm (Amersham US11-GFP (60) (based on strain: Patton), VSV-GFP (61) (based on strain: Indi- Protran: GE10600002) membrane. Membranes were blocked with 5% milk in ana), LCMV-GFP (62) (based on strain: Armstrong), and influenza A/WSN/33 PBST and incubated with primary antibody at 4 °C overnight in 5% milk PBST. virus (generously provided by Peter Palese, Mount Sinai School of Medicine, Membranes were washed three times with PBST and incubated with sec- New York, NY). ZIKV (PRVABC59 obtained from the Centers for Disease Con- ondary antibody. For chemiluminescent readout, membranes were incubated trol and Prevention [CDC], Ft. Collins, CO) was amplified in Hap1 cells and ti- with HRP-conjugated secondary antibody and exposed using SuperSignal trated by plaque assay on Huh-7.5 cells. Lenti-GFP virus was generated in Lenti-X West Pico PLUS Chemiluminescent Substrate or SuperSignal West Femto 293T cells as described. Coxsackievirus CVB-3-GFP (63) (derived from infectious Maximum Sensitivity Substrate (Thermo Fisher Scientific: 34577 and 34095) clone pMKS1-GFP) was amplified in HeLa cells and titrated by TCID50 (median by film. tissue culture infectious dose 50). All GFP-reporter virus infections and quantification were performed using Ubiquitinated Proteome Isolation Using TUBES. Hap1 cells were seeded at 6 × the doses and timepoints reported in the figure legends for quantification 106 cells per 10-cm dish (quadruplicate per condition). For infection, cells × by flow cytometry. For the infections, cells were seeded at a density of 2.5 were washed once with Opti-MEM, wash was replaced with 3 mL of Opti- 4 × 4 10 to 5 10 cells/well in 24-well plates. The next day, cells were washed MEM, and placed on ice for 10 min before spiking in 200 p/cell of AdV5rep+- μ once with Opti-MEM (Gibco) and adsorbed with 200 L of virus inoculum GFP prepared in Opti-MEM. Following synchronization for 45 min at 4 °C, prepared in Opti-MEM at 37 °C. After 1.5 h, inoculum was removed, cells inoculum was removed and cells were washed with cold Opti-MEM. Pre- were washed with Opti-MEM, fresh medium was added, and cells were in- warmed Opti-MEM was added and cells were incubated at 37 °C. At 1 hpi cubated at 37 °C. At the final timepoint dependent on viral replication ki- cells were placed on ice, washed with PBS, and lysed by adding 500 μL TUBE netics, cells were lifted with Accumax cell dissociation medium (eBioscience: lysis buffer (50 mM Tris·HCl [pH 7.5], 0.15 M NaCl, 1 mM EDTA, 1% Nonidet 00-4666-56) and fixed with 4% paraformaldehyde (PFA). Cells were pelleted P-40, 10% glycerol) freshly supplemented with protease inhibitor tablet at 930 × g for 5 min at 4 °C, resuspended in cold phosphate-buffered saline (Roche) and 50 μM DUB inhibitor PR-619 (LifeSensors: SI9619) and stored (PBS) containing 3% FBS and stored at 4 °C until analysis using a LSRII flow at −80 °C. In preparation for incubation with TUBE Dynabeads, lysates were cytometer (BD Biosciences). FlowJo software (Treestar) was used to obtain thawed and clarified by centrifugation at maximum speed (21,000 × g)ina the percentage of GFP-expressing cells and their mean fluorescent intensity table-top centrifuge at 4 °C. Supernatants were transferred to fresh tubes, (MFI). For non-GFP reporter viruses (influenza A virus and ZIKV), cells were protein concentrations were quantified by BCA protein assay, and lysate washed once in PBS (+2% FBS), permeabilized in 1×BD perm (BD Biosciences: input was normalized to 2 mg of protein lysate in a 500-μL volume for each 554723) for 1 h at room temperature (RT) followed by overnight incubation condition for addition to the TUBE beads. with primary antibody. Cells were then washed an additional three times A total of 100 μL of control magnetic beads (LifeSensors: UM400M) and with BD perm, incubated with secondary antibody for 1 h at RT, washed TUBE2 magnetic beads (LifeSensors: UM402M) per sample were prepared in three times with BD perm, and once with PBS (+2% FBS), and transferred separate low-fluid-retention tubes by washing three times with TBST to fluorescence-activated cell sorting tubes for flow cytometry as for (20 mM Tris·HCl [pH 8.0], 0.15 M NaCl, 0.1% Tween-20). To remove proteins reporter viruses. that nonspecifically bind to the TUBE beads, the 500 μL of normalized input Alternate WT AdV serotypes of AdV2, AdV7, and AdV35 were quantified lysate was added to the control beads and nutated for 1 h at 4 °C. To isolate by qPCR for vDNA at 72 hpi using the following primers: Ad2 (HAd2prF181: 5′- ubiquitinated proteins, this supernatant was transferred directly to the GCAAACGCTCTGCAACAAGA-3′, HAd2prR181: 5’-CGATCAGCTCGCTCATGA prepared TUBE2 beads and nutated for 3 h at 4 °C. After incubation, beads CT-3′), Ad7 (HAd7AprF167: 5′-GACTCTGAGCGACGATCTGG-3′, HAd7AprR167: were washed three times with TBST, twice with TBS, and transferred to fresh

10 of 12 | PNAS Sarbanes et al. https://doi.org/10.1073/pnas.2015794118 E3 ubiquitin ligase Mindbomb 1 facilitates nuclear delivery of adenovirus genomes Downloaded by guest on September 28, 2021 tubes before two final washes with TBS. Supernatants were removed and sequencing grade trypsin (Promega). Digestion was stopped by acidification the beads were stored dry at −20 °C prior to analysis by mass spectrometry. using trifluoroacetic acid (TFA, Thermo Fisher Scientific), and peptides were purified by in-house constructed reversed-phase micropurification tips. Proximity Labeling. WT and C985S MIB1 constructs were C-terminally tagged APEX2 mass spectrometry. Decanted agarose beads were subjected to on-bead with a FLAG-tagged APEX2 protein via Gibson assembly of overlapping PCR- digestion by the addition of 1 μg sequencing grade trypsin (Promega). The amplified MIB1 fragment and FLAG-APEX2 fragments into the recipient supernatant was withdrawn, evaporated to dryness and dissolved in 50 mM pLX304 lentivirus expression vector followed by site-directed mutagenesis to AMBIC (Fluka Chemicals), 10 mM DTT (EMD Chemicals) in water, and disulfide introduce the V943F mutation. APEX2-mKate was PCR amplified off of bonds were reduced for 1 h at room temperature with vigorous shaking. IAA plasmid pcDNA3 APEX2-NES (a gift from Alice Ting, Stanford University, (Sigma) was added to 20 mM and alkylation proceeded for 1 h at RT in the Stanford, CA, Addgene plasmid #49386; http://www.addgene.org/49386; dark. Samples were further digested for 6 h at RT using trypsin. Digestion was RRID: Addgene_49386) (65) and cloned into the pLX304 backbone. Lentivi- stopped by acidification using TFA (Thermo Fisher Scientific), and peptides were ruses from these three constructs were used to reconstitute Hap1 MIB1 KO purified by in-house constructed reversed-phase micropurification tips. clone 2-1 as described. LC-MS/MS. Solvent A was 0.1% formic acid in water and solvent B was 0.1% For proteomics, cells were seeded at 1.5 × 107 cells per 15-cm dish in qua- formic acid, 80% acetonitrile (ACN) in water. All LC-MS solvents are of LC-MS druplicate per condition. On the day of infection, cells were preincubated with grade and purchased from Thermo Fisher Scientific. Liquid chromatography 500 μM biotin-phenol (BP: sold as biotin tyramide, Iris Biotech: LS-3500.1000) at was performed by a Dionex 3000 Ultimate HPLC (high-performance liquid 37 °C for 30 min. Media were then replaced with cold Opti-MEM containing BP chromatography) equipped with a NCS3500RS nano- and microflow pump rep+ (500 μM) and 500 μLofAdV5 -GFP (MOI = 200 p/cell) or plain Opti-MEM (for (Dionex). Peptides were loaded onto a 100 μm × 20 mm Acclaim PepMap C18 uninfected controls) was spiked into the medium. Cells were incubated at 4 °C trap column (Thermo Fisher Scientific) at 3 μL/min. Separation was achieved for synchronization of infection. At 40 min postadsorption, inoculum was using a 75 μm × 120 mm pulled-emitter nanocolumn (Nikkyo Technos). replaced with 25 mL of prewarmed IMDM (+BP) and plates were incubated at Solvent B went from 1 to 38% over 90 min, followed by a sharp 1-min in- 37 °C for 75 min. At 70 min pi, MIB1-proximal proteins were labeled by a 1-min crease to 90% where it was kept for 8 min. Peptides were analyzed using a incubation by spiking in H2O2 (Thermo Fisher Scientific: H325) to 100 mM, Q-Exactive HF mass spectrometer (Thermo Fisher Scientific). Data were immediately followed by media removal and addition of 10 mL of a freshly recorded in positive mode with top 20 data-dependent acquisition. MS1 “ ” prepared quencher solution (10 mM sodium ascorbate [Spectrum Chemical: resolution was set to 60,000 and an MS2 resolution of 30,000. Automatic S1349], 5 mM Trolox [Sigma: 238813] in PBS) for 2 min at RT. Solution was gain control targets of 3e6 (MS1) and 2e5 (MS2) were applied. aspirated and cells were incubated with another 10 mL of quencher and col- lected into 15-mL conical tubes on ice by scraping. Cells were then pelleted at Data Analysis. Data were analyzed with MaxQuant v. 1.6.6.0 (66). Spectra were 1,200 × g, washed with 1 mL of quencher, and transferred to 1.2-mL Eppen- queried against the human proteome (downloaded from .org on Feb- dorf tubes. Cells were pelleted at 5,000 × g, supernatants were removed, and ruary 12, 2019) concatenated with a custom adenovirus database and common pellets were stored at −80 °C until further processing. contaminants. A false discovery rate of 1% at both peptide and protein levels was To enrich proximity-labeled proteins, pellets were lysed in 1 mL of RIPA- MICROBIOLOGY applied. Further statistical analysis was performed within the Perseus framework Quench (Quench prepared in RIPA buffer and supplemented with protease- (67). Significant changes were determined using the Student’s t test (P < 0.05). GO inhibitor tablet), and disrupted with pipetting. After a 15-min incubation on – ice, samples were sonicated for 15 s and clarified by centrifugation at 15,000 × terms enrichment analysis was performed using STRING protein protein inter- g at 4 °C. Meanwhile, 400 μL of High-Capacity Neutravidin Agarose Resin bead action networks functional enrichment analysis (https://string-db.org). slurry (Thermo Fisher Scientific: 29284) was transferred to low-fluid retention 1.5-mL tubes (National Scientific: CN1700L-BP) and washed twice in 1 mL RIPA Data Availability. Sequencing data from the haploid genetic screen for both buffer prior to addition of proximity-labeled samples. Supernatants from AdV5-selected cells (NCBI SRA accession no. SRP269890 dataset SRX8663593) clarified samples were added to the neutravidin beads and nutated overnight and unselected WT Hap1 reference (NCBI SRA accession no. SRP058962 at 4 °C. Supernatants were removed and beads were washed twice with RIPA dataset SRX1045464) are deposited on the NCBI SRA. The mass spectrometry buffer, once with 1 M potassium chloride (KCl), once with 0.1 M sodium car- proteomics data have been deposited to the ProteomeXchange Consortium

bonate (Na2CO3), once with freshly prepared 2 M urea in 10 mM Tris·HCl (pH via the PRIDE partner repository (https://www.ebi.ac.uk/pride/) with the 8.0), and three times in 10 mM Tris·HCl (pH 8.0) with transfer to a fresh tube dataset identifier PXD020542. Processed data/analysis can be viewed within before the final wash. Samples were eluted either directly in lysis buffer with supplementary excel file Dataset S1. reducing agent for Western blot or beads were stored in 500 μLofTris·HCl at −20 °C prior to mass spectrometry. Ten percent input and 10% post-IP ACKNOWLEDGMENTS. We thank the following investigators for contribut- fractions for each condition were analyzed by Western blot to ensure H2O2 ing viral molecular clones, viral stocks, and cell lines: P. Hearing (replication- labeling and streptavidin-mediated depletion. competent AdV5-GFP), R. J. Parks (AdV5 JR34), I. Mohr (HSV-1-GFP), P. L. For IF readout, cells were seeded onto glass coverslips treated with poly- Collins (hPIV-3-GFP), I. Frolov (VEEV-GFP), J. K. Rose (VSV-GFP), J. C. de la Torre (LCMV-GFP), P. Palese (influenza A virus), J. L. Whitton (CVB-3-GFP), P. L-lysine in 24-well plates. The following day, cells were preincubated with BP Traktman (VacV-GFP), the CDC (ZIKV), and J. L. Casanova (human STAT1−/− and infection and H O labeling steps were carried out as described above 2 2 fibroblasts). Thanks to the resource centers at The Rockefeller University: the except cells were fixed by addition of 4% PFA immediately following the technical advice of S. Mazel, S. Semova, A. Keprova, and S. Han at the Flow first quencher incubation and processed for IF as described. Cytometry Resource Center and the expertise of the Proteomics Resource Center and the technical advice of A. North, K. Cialowicz, and C. Pyrgaki Proteomics Collection and Analysis. at the Bio-Imaging Facility. Thanks to B. Razooky for image analysis advice. Sample digestion. We are also grateful for the administrative and/or experimental support of Ubiqutin isolation mass spectrometry. Beads were incubated with 8 M urea in S. M. Pecoraro Di Vittorio, G. Santiago, A. O’Connell, M. E. Castillo, A. Web- 50 mM ammonium bicarbonate (AMBIC, Fluka Chemicals: 09830) for 1 h at RT. son, and S. Shirley. Thanks to A. W. Ashbrook and L. C. Aguado for valuable The supernatant was withdrawn and the process was repeated. The two feedback on the manuscript and to all in the C.M.R. laboratory whose advice supernatants were pooled and disulfide bonds were reduced by additon of over the years helped guide this project. This work was supported by a grant from the Robertson Foundation (to H.-H.H.). The project was also supported DTT (dithiothreitol; EMD Chemicals) to 10 mM final concentration and in- in part by grant R01AI143295 (to C.M.R.) from the National Institute of Al- cubation for 1 h at RT with vigorous shaking. Iodoaceamide (IAA, Sigma) was lergy and Infectious Disease, NIH. S.L.S. was supported by a David Rockefeller added to 20 mM and alkylation proceeded for 1 h at RT in the dark. Samples Graduate Student Fellowship and Rockefeller University Women and Science were diluted to reduce urea concentration to 3.5 M and digested using Lysyl Fellowship. T.R.B. is supported by the Oncode Institute and the Cancer Ge- Endopeptidase (Lys-C, Wako Chemicals) overnight at RT. The following day nomics Center (GCG.nl) and a Vici grant from the Netherlands Organization samples were further diluted to 1.5 M urea and digested with 1 μg for Scientific Research (NWO; 016.170.033).

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