key: cord-0332483-r030seib
authors: Bauer, Michael; Gomez-Gonzalez, Alfonso; Suomalainen, Maarit; Hemmi, Silvio; Greber, Urs F.
title: The E3 ubiquitin ligase Mind bomb 1 enhances nuclear import of viral DNA by inactivating a virion linchpin protein that suppresses exposure of virion pathogen-associated molecular patterns
date: 2020-08-29
journal: bioRxiv
DOI: 10.1101/2020.08.29.242826
sha: 56c3357456606e48a5cda8d0f6eaf67d65df27dd
doc_id: 332483
cord_uid: r030seib

In eukaryotic cells, genomes from incoming DNA viruses mount two opposing reactions, viral gene expression and innate immune response, depending on genome exposure (uncoating) to either RNA-polymerases or DNA sensors. Here we show that adenovirus particles contain a tunable linchpin protein with a dual function: response to host cues for scheduled DNA release into the nucleus, and innate immunity suppression by preventing unscheduled DNA release. Scheduled DNA release required the proteasome and ubiquitination of the viral core protein V. Cells lacking the E3 ligase Mind bomb 1 (Mib1) were resistant to wild-type adenovirus infection. Viruses lacking protein V or bearing non-ubiquitinable protein V, however, readily infected Mib1 knockout cells, yet were less infectious than wild-type virus. Their genomes were poorly imported into the nucleus and remained uncoated in the cytosol, thereby enhancing chemokine and interferon production through the DNA sensor cGAS. Our data uncover how the ubiquitin-proteasome system controls the function of a virion linchpin protein suppressing pathogen-associated molecular patterns and triggers viral DNA uncoating at the nuclear pore complex for nuclear import and infection.

Pathogen-associated molecular patterns (PAMPs) activate the immune system as early as pathogens enter a cell, which facilitates adaptive immunity and coordinated cell defense 1, 2 . PAMPs comprise a range of features, including DNA, double-stranded RNA, lipopolysaccharides or cytosolic glycans. They are decoded by pattern recognition receptors (PRRs), for example Toll-like receptors, DNA-and RNA-sensors such as cyclic GMP-AMP synthase (cGAS), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), retinoid acid-inducible gene I (RIG-I)-like receptors (RLRs), or cytosolic lectins, which can lead to inflammatory responses 3, 4, 5, 6 . The innate immune response is limited in extent and duration by both self-regulation and by the intruding pathogen. The work here uncovers that double-stranded DNA-PAMPs from incoming adenovirus (AdV) particles are effectively sequestered away from cytosolic sensors by the action of a virion protein functioning as a linchpin that secures the integrity of the incoming particles. This security mechanism prevents a massive cytokine response induced by unscheduled disassembly of the incoming capsid in the cytoplasm and exposure of the viral genome to the DNA sensor cGAS. The function of this linchpin can be relieved by targeted ubiquitination of protein V and allows viral genome release at the nuclear pore complex (NPC) for nuclear import and infection.

Ubiquitination tunes a wide range of cellular processes, including DNA-damage response, protein trafficking or autophagy, and generally results in proteasomal degradation or signal transduction 7 .

Ubiquitination starts with the linkage of a single ubiquitin peptide 76 amino acids in length to the amino group of a lysine residue (K) via the C-terminal glycine (G) of ubiquitin 8, 9 . This process comprises three distinct catalysts, E1 ubiquitin activation, E2 ubiquitin conjugation and E3 ubiquitin ligation enzymes 9 . The latter control the substrate specificity in the ubiquitin transfer reaction and define the type of ubiquitin linkage 10 . Proteins with ubiquitin binding domains recognize ubiquitinated proteins, and specialized ubiquitin hydrolases remove ubiquitin 11 . Ubiquitin itself can be modified with ubiquitin by isopetide linkage at one of its lysine residues or at the N-terminal methionine residue, which is the basis of ubiquitin chain signaling through large chemical diversity, and affects almost any protein in a mammalian cell. Branched poly-ubiquitins with K11-or K48-presence of MG132 strongly reduced the levels of capsid-free vDNA, indicating that proteasomal activity is required specifically for the release of vDNA from capsids docked at the NPC.

Adenovirus core protein V is ubiquitinated during entry and removed from the virion at the NPC Colocalization of EGFP-tagged Mib1 and viral capsids at the nuclear envelope occurs frequently as shown before 32 . We used an affinity purification mass spectrometry (MS) based strategy to test the hypothesis that a viral capsid protein serves as a ubiquitination substrate of Mib1. HeLa cells were infected with AdV-C5 at high multiplicity of infection (MOI) for 2 h, lysed under strong denaturing conditions, and digested with trypsin protease. Trypsin cleaves the polypeptide chain C-terminal of K and arginine (R) residues, and leaves a di-glycine (Gly, G) remnant on the ε-amino group of ubiquitinated K residues, thus creating a peptide motif specifically recognized by a monoclonal antibody 47, 48 . Immunoprecipitates of ubiquitinated cellular and viral peptides analyzed by liquid chromatography tandem MS (LC-MS/MS) contained peptides from over 5000 cellular proteins, as well as four viral proteins: penton base, IIIa, V, and VI ( Fig. 2A, 2B, 2C) . The virion proteins were not previously described to be ubiquitinated, except for protein VI 49 . Unlike penton base, IIIa and VI 26, 50 , a large fraction of protein V remains inside the virus particle until capsid disassembly at the NPC 51 . We therefore investigated the role of protein V and the ubiquitination of K178 and K188 during the virus entry, and explored the dynamics of GFP-tagged protein V in incoming AdV-C2-GFP-V particles. Atto647-labeled AdV-C2-GFP-V particles (bearing a red fluorophore Atto647 and a green GFP-V) reached the nucleus of mScarlet-Mib1 expressing Mib1-KO cells (HeLa-sgMib1) some 30 min pi. Approximately 18 min after docking at the NPC, GFP-V puncta were rapidly discharged from the capsids (Movie 1, Fig. 2D ). Of note, we never observed translocation of protein V puncta into the nucleus, while vDNA is either imported into the nucleus or misdelivered to the cytosol 32, 52, 53 . GFP-V puncta were found to dissociate from capsids in control HeLa-sgNT, but not in HeLa-sgMib1 cells (Fig. 2E) . It is therefore likely that the sudden loss of protein V from capsids at the NPC represents the final capsid disassembly step and accompanies the viral genome release.

To determine how protein V affects the entry of the virion into host cells, we generated an AdV-C5 mutant deleted of the protein V coding region (AdV-C5-∆V) (Fig. 3A) . Particles isolated by cesium chloride gradient centrifugation and analyzed by SDS-PAGE and QuickBlue staining showed lack of protein V but no differences in the major virion proteins, including hexon, penton base, IIIa, fiber (IV), VI, and VII (Fig. 3B) . Proteins VIII, IX, X, IVa2 and protease were not visualized in these gels.

Sanger sequencing confirmed that the protein V coding region was removed from vDNA (Fig. S2A) .

Full analyses of the viral genome by next-generation sequencing revealed no other mutations in the viral genome (Fig. S2B, and supporting material) . The integrity of the virions was further demonstrated by transmission electron microscopy of negatively stained specimens (Fig. 3C) .

Protein V promotes the assembly of newly formed viral particles 54 , and AdV particles lacking protein V have been reported to form smaller plaques than wt AdV-C5, with the caveat that this old deletion mutant had many other mutations besides the deletion of V 55 . To test if protein V was involved in the viral entry, we performed a single-round infection assay with AdV-C5-∆V and parental AdV-C5 wild-type and measured the immediate early viral protein E1A 20 h pi. Inocula were adjusted to the number of particles bound per cell, as described earlier 56 . To reach a certain level of E1A expression, a significantly higher number of AdV-C5-∆V particles was required per cell, as compared to wild-type, indicating that particles lacking protein V were less infectious than wild-type ( Fig. 3D) . To investigate entry more closely, we performed analyses of single fluorescent particles using confocal light microscopy. These experiments revealed no difference in viral escape from the endosomes or trafficking to the nucleus between AdV-C5-∆V and wild-type particles (Fig. 3E, 3F) .

However, the analyses of incoming viral genomes tagged with EdC nucleosides 32, 52 revealed significant differences between AdV-C5-∆V and wild-type infections. While nearly all genomes were released from both wild-type and AdV-C5-∆V capsids 3 h pi (Fig. 4A, 4B) , the majority of wild-type virus genomes were delivered to the nucleus, yet most of the AdV-C5-∆V genomes were in the cytoplasm (Fig. 4A, 4C ). This defect in nuclear import of AdV-C5-∆V vDNA likely explains the low 8 infectivity of these particles. We next tested whether the absence of the ubiquitination substrate protein V would affect the infection of Mib1-KO cells. Strikingly, infection of Mib1-KO cells with AdV-C5-∆V but not AdV-C5-∆IX was readily possible, while wild-type infection was blocked (Fig. 4D) . A higher number of released vDNA free from capsids was found in Mib1-KO cells, indicating that Mib1 was not involved in releasing vDNA from the AdV-C5-∆V particles (Fig. 4E, 4F) .

AdV-C5-∆V are more thermo-sensitive than wild-type particles and release their genome in the cytoplasm before reaching the nucleus

The observation that AdV-C5-∆V particles released their genome prematurely and did not require a ubiquitination cue from Mib1 suggested that they were less stable than wild-type particles. We assessed the thermal stability of the particles in vitro by measuring the fluorescence increase of the DNA intercalating dye DiYO-1, a highly sensitive end point assay for the disruption of the capsid shell 51, 57 . At temperatures below 40 °C, both wild-type and AdV-C5-∆V particles were largely intact, as indicated by a low level of DiYO-1 fluorescence, but AdV-C5-∆V readily reached half maximal fluorescence at 42° C while wild-type virus remained intact up to about 47 °C (Fig. 5A) . Accordingly, the infectivity of AdV-C5-∆V particles was more thermo-sensitive than wild-type virus (Fig. 5B) . This notion was reinforced by single virus particle analyses in cells, where more than 40% of the AdV-C5-∆V particles lost their genome in the presence of the uncoating inhibitor leptomycin B (LMB) (Fig. 5C, 5D ). Both wild-type and AdV-C5-∆V infections were completely sensitive to LMB (Fig.   S3A ). LMB is a widely used nuclear export inhibitor 58 , precludes the detachment of virions from microtubules near the nuclear envelope and thereby blocks virion attachment to the NPC and prevents nuclear import of viral DNA and infection 59, 60 . The treatment of cells with protein degradation inhibitors, such as DBeQ blocking the p97 AAA-ATPase or MLN9708 against the proteasome, did not inhibit the premature release viral DNA from AdV-C5-∆V (Fig S3B, S3C) .

Likewise, the depletion of microtubules with nocodazole had no effect (Fig S3D, S3E) . Collectively, the results showed that the AdV-C5-∆V particles were less stable than wild-type, and suggested that unscheduled release from AdV-C5-∆V might be due to intrinsic particle instability in a crowded cytosol rather than a specific host anti-viral effect.

The results so far suggested that the viral DNA core-associated protein V serves a ubiquitin responsive linchpin in the release of the viral DNA from the incoming capsid. We next assessed whether the ubiquitination of protein V at K178 and K188 was important for AdV infection, and created three AdV-C5 mutants, in which either one or both of these lysine residues was replaced by arginine, a basic amino acid that cannot serve as a ubiquitin acceptor site (Fig. S4A) . HeLa-sgNT and sgMib1 cells infected with both single mutants and the double mutant (K178R, K188R, K178/188R) all showed strongly Mib1-dependent infections (Fig. S4B) . Importantly, the doseresponse curves with the mutants were virtually identical to the wild-type, indicating that the mutant particles had the same specific infectivity as the wild-type (Fig S4C) . We surmised that other lysines than K178 and K188 are ubiquitinated in the double mutant, and that these modifications were not detected by our MS analyses of protein V in the wild-type particles.

To abrogate any canonical ubiquitination of protein V, we changed all 26 lysine residues of protein V to arginines. Purified AdV-C5-V-KR particles incorporated the V-KR protein at comparable amounts as the parental AdV-C5 protein V, as shown by SDS-PAGE and QuickBlue protein staining (Fig.   6A ). While the V-KR band appeared to migrate at a slightly faster than the wild-type protein V, there was no difference in any of the other capsid proteins resolved in the gel. Yet, the infectivity of AdV-C5-V-KR particles in HeLa cells was strongly impaired compared to the parental AdV-C5, comparable to the AdV-C5-ΔV mutant (Fig. 6B) . The AdV-C5-V-KR particles released some of their vDNA but did not efficiently import this vDNA into the nucleus (Fig. 6C, 6D) . In accordance, AdV-C5-V-KR infected HeLa-sgMib1 cells with greater efficiency than AdV-C5, demonstrating that this virussimilar to AdV-C5-ΔVis less dependent on Mib1 for infection (Fig. 6E) . The data showed that preventing ubiquitination at K178 and K188 had no effect on vDNA uncoating and infection, but replacing all lysine residues with arginine strongly reduced infection and dependence on Mib1.

To test whether ubiquitination of Lys178 and Lys188 is sufficient to revert the phenotype of the V-KR mutant and restore the infection dependence on Mib1, we reverted the arginine residues to lysine at positions 178 and 188. The resulting mutant particles AdV-C5-V-KRrev efficiently incorporated the V-RKrev protein (Fig 6A) . The AdV-C5-V-KRrev particles exhibited more efficient nuclear import of vDNA and higher infectivity than V-KR but were still less infectious than wild-type AdV-C5 (Fig. 6B-D) . Nevertheless, AdV-C5-V-KRrev was strongly restricted in Mib1-KO cells, similar to AdV-C5 but unlike the AdV-C5-V-KR virus, suggesting that ubiquitination of protein V at K178 and K188 specifies the infection dependence on Mib1 (Fig. 6E) .

The results so far showed that tampering with the function of protein V reduces infectivity and increases the amounts of incoming vDNA in the cytoplasm. We next tested if the protein V mutants affected the induction of innate immune responses in the non-transformed, GM-CSF dependent, macrophage-like MPI-2 cell line 61, 62 . Cells were infected with equal amounts of virus particles and mRNA levels of various cytokines were measured by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) at 5 and 10 h pi. Strikingly, virus particles lacking protein V led to higher levels of interleukin-1α (Il1a), Ccl2, Cxcl2, and Ccl5 mRNAs than wild-type (Fig. 7A) . These results were confirmed by single cell analyses of the Ccl2 mRNA using RNA FISH assay with branched DNA signal amplification (Fig. S5A) . Although V-KRrev and V-KR particles enhanced these cytokines compared to wild-type infection, their induction was not as pronounced as with AdV-C5-ΔV, in direct correlation with the levels of cytosolic vDNA. The results were corroborated by measuring type I IFN released from the infected MPI-2 cells. The assay used mouse embryonic fibroblasts expressing firefly luciferase under the control of the endogenous type I IFN inducible Mx2 promoter (MEF-Mx2-Luc-βKO) 62 . Strikingly, infection with AdV-C5-ΔV led to a significantly higher IFN-β secretion than the wild-type virus. AdV-C5-V-KR and the V-KRrev increased IFN-β secretion as well, but not as prominently as AdV-C5-ΔV (Fig. 7B) .

A major DNA-PAMP sensor and transducer in eukaryotic cells is the cGAS/STING branch 6 , which detects also AdV DNA 63, 64 . Upon DNA binding cGAS produces cGAMP which binds the adaptor protein STING and leads to the activation of TBK1. Active TBK1 phosphorylates IRF3 which then dimerizes, translocates to the nucleus, and promotes the expression of type I IFN. Significantly, the knockdown of cGAS by small hairpin (sh) RNA 64 reduced the induction of Ccl2 and Ccl5 mRNA in MPI-2 cells as compared to control shEGFP cells ( Fig. 7C and Fig. S5B) . The results were validated by ELISA assays showing that AdV-C5-ΔV increased the protein levels of CCL2 and CCL5 to much higher levels than the wild-type, notably in a cGAS-dependent manner (Fig. 7D, 7E and   Fig. S5 ). Taken together, these results indicate that the presence of protein V in AdV particles reduced the levels of PAMPs in the cytoplasm and confined the innate response against the incoming virus (Fig. 8) . Since protein V occurs in no other AdV genus than Mastadenovirus, and is highly conserved in Mastaadenoviruses 65, 66, 67 , we speculate that the viruses lacking protein V evolved other mechanisms to secure their capsid DNA before arriving at the nucleus.

Understanding how cells control virion stability is key to pathology and advances the field of synthetic virology, for example for the development of customized gene delivery vehicles and vaccines. Human AdVs and their interactions with cells are a highly advanced model of virus-host interactions at all levels, ranging from single cell infection, immunity, persistent and acute human disease to therapy and vaccination 35, 56, 68, 69 . Human AdV particles are composed of major and minor capsid proteins conferring structural and accessory functions, such as DNA confinement, particle stability or membrane rupture 68, 70, 71 . The latter is mediated by protein VI with an amphipathic helix interacting with sphingolipids 72, 73, 74, 75, 76, 77, 78, 79 . Virions penetrated to the cytosol are leaky containers, and their DNA is accessible to dyes, as shown by click chemistry 52 . They detach from nuclear envelope-proximal microtubules, bind to the NPC filament protein Nup214, and uncoat their genome through the involvement of kinesin motors and microtubules 25, 60, 80, 81, 82 .

Our data here show how an internal linchpin protein safe-guards the virions trafficking in the cytoplasm from premature DNA release, and how the NPC-docked virions are primed for disassembly by the E3 ubiquitin ligase Mib1. The mechanism identified here is distinct from the one inactivating antibody-coated virus particles 83 . AdVs loaded with antibodies may enter cells, for example in individuals with preexisting anti-AdV antibodies, and the cytosolic particles become subject to inactivation through recognition of the immunoglobulins by the E3 ubiquitin ligase TRIM21 and subsequent proteasomal degradation 84 . This catastrophic disassembly process exposes vDNA in the cytosol and raises innate immunity and inflammation. The question how native AdV capsids hold their DNA and resist the cytoplasmic crowding has remained unresolved, however. This is remarkable since these non-opsonized particles have not only shed a number of stabilizing proteins, such as IIIa, VIII, VI, pentons and fibers 50, 85 , but are also subject to pulling forces from dynein and kinesin motors during cytoplasmic transport on microtubules 86, 87, 88, 89, 90 .

Here we show that protein V secures the viral genome in the capsid, and that its release is mediated by the E3 ligase Mib1. This process facilitates nuclear import of the viral genome. Genetic ablation of protein V reduced the efficiency of nuclear import of vDNA and thereby decreased the infectivity of the particles. However, the absence of protein V did not compromise the disassembly of the capsid or the release of the vDNA. In fact, our results show that the absence of protein V renders the particles less stable, leading to premature disassembly of the capsid before reaching the NPC.

Accordingly, AdV particles lacking protein V were less thermostable than wild-type particles, possibly because about 160 copies of protein V act as a capsid glue by connecting the vDNA and protein VI 55, 91, 92, 93 . Protein V balances the negative charges on vDNA together with proteins VII, X/µ, and IVa2, akin to a histone H1-like protein 94 . During entry, protein V quantitatively dissociates from the particles at the NPC 51 . This result and the observation that salt-treated vDNA cores isolated from purified virions release protein V 95 suggest that protein V binding to the vDNA core can be tuned.

We identified the cell tuner of protein V, the E3 ligase Mib1, and showed that the efficiency of nuclear translocation of the AdV genome depends on the ubiquitination of protein V. Ubiquitination analyses by affinity purification mass spectrometry identified two lysine residues on protein V that carried a di-glycine (di-G) remnant after tryptic digest. Di-G remnants on the ε-amino group of lysines derive from digestion of proteins conjugated to ubiquitin or ubiquitin-like modifiers, such as ISG15 and NEDD8, which cannot be distinguished from ubiquitin by MS 48, 96 . It is unlikely that the di-G remnants on protein V were derived from another modification than ubiquitin, since Kim et al. reported that by our detection methodology >94% of K-ε-GG sites are ubiquitinated and only 6% due to NEDD8ylation or ISG15ylation 47 . In fact, we believe that lysine residues additional to K178 and K188 on protein V are ubiquitinated during entry. In agreement with this notion, mass spectrometry and shotgun proteomics of purified AdV-C5 gave no evidence for ubiquitination of protein V residues (data not shown), consistent with lack of protein V ubiquitination evidence from liquid chromatography-high resolving mass spectrometry (LC-MS) of purified AdV-C2 particles 97 , implying that ubiquitination of protein V occurs during entry and can be used as a toggle switch.

Protein V potentially provides a direct ubiquitination substrate for Mib1. Yet, not all viral genomes were released from AdV-C5-ΔV virions in the Mib1-KO cells suggesting that another factor besides protein V may also be ubiquitinated and involved in Mib1-priming of vDNA uncoating. We speculate that this factor is a cellular protein that stabilizes the viral capsid against disruption by kinesinmediated pulling forces, which had been implicated in capsid disruption at the NPC 25 . It is plausible that ubiquitination and proteasomal degradation of this factor occurs at the NPC since the proteasome inhibitor MG132 prevented the vDNA release from the NPC-docked capsid upon induction of Mib1 expression in Mib1-KO cells. The role of the proteasome in AdV capsid disassembly at the NPC represents a novel function and can now be studied in more detail. It extends the role of the proteasome beyond binding, internalization and trafficking of other viruses 31, 98, 99, 100, 101 , and increases the therapeutic potential of proteasome inhibitors. Developing proteasomal inhibitors against vDNA uncoating may also be beneficial in reducing cytokine response and inflammation. Remarkably, genetic perturbations of protein V in AdV-C5-ΔV, AdV-C5-V-KR and AdV-C5-V-KRrev increased the levels of viral genomes in the cytosol as a result of unscheduled vDNA release or misdelivery. The AdV-C5-ΔV particles led to the largest overall induction of innate cytokines, strongly dependent on the presence of the cGAS sensor. We speculate that the non-ubiquinatable V-KR protein partially shields misdelivered vDNA from cellular sensors. Misdelivery of vDNA is, however, not unique to AdV. For example, more than half of human immunodeficiency virus-1 (HIV-1) reverse-transcribed genomes were found to be capsid-free in the cytosol of primary human macrophages 102 . It remains to be explored how other viruses secure and shield their vDNA from cellular sensors, or activate cytosolic DNA sensors 103, 104 .

HeLa-ATCC, A549, HDF-TERT, and HER911 cells were maintained in Dulbecco's Modified Eagle's Medium (GIBCO) supplemented with non-essential amino acids (Thermo Fisher) and 10% fetal calf serum (FCS, GIBCO). During infection experiments, the medium was additionally supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were grown at 37 °C in a 5% CO2 atmosphere for no longer than 20 passages. HeLa-sgNT and HeLa-sgMib1 cells have been previously described 32 . HeLa-sgMib1 cells expressing mScarlet-Mib1 were generated by lentiviral transduction followed by selection with 2 µg/ml puromycin. MPI-2 cells were maintained in RPMI medium (Sigma) supplemented with 10% fetal calf serum and 10 ng/ml granulocyte macrophage colony-stimulating factor (GM-CSF, Miltenyi Biotec) 61, 62 .

All AdVs were grown in A549 cells and purified over two cesium chloride gradients as previously described 50, 105 . AdV-C5 (wt300) has been previously described 106 . AdV-C5-ΔIX was kindly provided by R. Hoeben (Leiden University Medical Center, The Netherlands) 107 . AdV-C2-GFP-V was used as described 51 . Capsid-labeled viruses were generated as described 26, 108 . Genomelabeled AdV was produced by growing the virus in A549 cells in the presence of 2.5 µM EdC (5ethynyl-2'-deoxycytidine, Jena Biosciences) as described 52 

, and AdV-C5-V-K178/188R were generated by recombineering from the pKSB2 bacmid which contains the entire AdV-C5 wt300 genome 106, 109 .

In a first step, the galK cassette was amplified by PCR from the pGalK plasmid using primers that carried 45 bp homology sequences directly up-or downstream of the protein V coding region (see Table S1 , primers GalK_f and GalK_r). The PCR product was purified by gel extraction and digested with DpnI (Promega) for 1 h to remove residual template DNA before a second round of purification. Electrocompetent E. coli SW102 cells harboring the AdV-C5 containing bacmid (pKSB2)

were then electroporated with the purified PCR construct. Positive clones were verified by sequencing and underwent a second electroporation reaction. In the case of AdV-C5-ΔV, this was done with a dsDNA oligonucleotide (dV_f and dV_r, Table S1 ) consisting of the left and right homology sequences (Fig. 4A ). For the AdV-C5-V-KR mutant, the recombination substrate was a synthesized modified protein V DNA sequence in which all lysine codons were replaced by arginine codons flanked the by left and right homology arms (Table S1 ; synthesized by Thermo Fisher Scientific). The resulting AdV-C5-V-KR bacmid then served as the template for constructing the AdV-C5-V-KRrev mutant. Bacmid DNA from positive clones was extracted, digested with PacI to release the AdV genome, and transfected into HER911 cells 110 . Rescued viruses were plaquepurified, expanded, and verified by sequencing.

Ten thousand cells were seeded in a black 96-well imaging plate. On the following day, the virus was diluted in infection medium (DMEM supplemented with 2% FCS, non-essential amino acids, pen/strep) to reach an infection of about 40%. The culture supernatant was aspirated and 100 µl of diluted virus suspension was added to the cells. The cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10-15 min at RT. Remaining PFA was quenched with 25 mM NH4Cl diluted in PBS for 5-10 min, followed by permeabilization with 0.5% Triton X-100 in PBS for 3-5 min. Cells were stained with rabbit anti-protein VI 26 or mouse anti-E1A clone M73 (Millipore, 05-599) diluted in blocking buffer (10% goat serum in PBS) for 1 h at 4 °C. After three washes of 4 min each in PBS, cells were stained with secondary antibody (goat anti-rabbit-AlexaFluor 488 or goat anti-mouse-AlexaFluor 488, Thermo Fisher) diluted in blocking buffer containing 1 µg/ml DAPI for 30 min at RT.

After three more washes of 4 min in PBS, cells were imaged in a Molecular Devices high-throughput microscope (IXM-XL or IXMc) in widefield mode with a 20x objective. For quantification of infection with CellProfiler 111 , nuclei were segmented according to the DAPI signal, and the intensity of the infection marker over the nuclear mask was measured.

Virus input was normalized to the number of viral particles that bound to the cells, which was determined in a binding assay. To this end, HeLa cells grown on cover slips in a 24-well plate were incubated with virus for 1 h on ice, after which the virus inoculum was washed and away and cells were immediately fixed with 4% PFA. Bound viral particles were stained with mouse anti-Hexon 9C12 antibody 112 and goat anti-mouse-AlexaFluor 488. Nuclei were stained with DAPI and cell outlines with AlexaFluor 647-conjugated succinimidyl ester (Thermo Fisher). Samples were imaged using a Leica SP8 confocal laser scanning microscope (cLSM). Three-dimensional stacks were recorded and the number of particles that bound to cells were quantified in maximum projections using CellProfiler. HeLa cells in a 96-well plate were then incubated with a 1:2 dilution series of virus starting from 50 bound particles per cell. After 1 h on ice, virus inoculum was removed, and fresh medium was added. Cells were fixed at 20 hpi and stained for E1A as described above. MOI for infection assays was based on infectious particles in the paerticular assays. For example, MOI 0.5 indicated that 50% of the cells were infected at the time of fixation.

A Leica SP8 cLSM was used in all experiments, in which single viral particles and genomes were imaged. Imaging was performed with a 63x magnification oil objective with a numerical aperture of 1.40 and a zoom factor of 2, with a pixel size of 0.181 μm. z-stacks were captured with a step size of 0.5 μm to capture the entire cell, and the size of the pinhole was 1 Airy unit. Leica hybrid detectors (HyD) were used for each channel.

Purified virus particles were lysed in SDS-PAGE lysis buffer (200 mM Tris pH 6.8, 10% glycerol, 5 mM EDTA, 0.02% bromophenol blue, 5% SDS, 50 mM DTT) and boiled for 5 min at 95 °C. Samples were then loaded onto a 10% SDS-PAGE gel and transferred to a PVDF membrane (Amersham).

After blocking with blocking solution (5% milk powder in 20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.5), the membrane was incubated with primary and secondary antibodies diluted in blocking solution at 4 °C overnight or 1 h at RT, with 4 washes of TBST in between. HRP-coupled secondary antibody was detected using the ECL reagent (GE Healthcare). Primary antibodies used in Western blotting were rabbit polyclonal anti-Hexon and rabbit polyclonal anti-protein V (both kind gifts from Ulf Pettersson). Secondary antibody used in Western blotting was goat anti-rabbit-HRP (Cell Signaling, 7074). Chemiluminescence signals were detected using an ImageQuant LAS 4000 system. Alternatively, after gel electrophoresis separated proteins were stained in the gel using Coomassie or QuickBlue protein stain (LubioScience).

Cells grown on cover slips were infected with genome-labeled AdV for various timepoints. Where specified, cells were incubated with specific inhibitors (50 nM LMB, 5 µM DBeQ, 10 nM MLN9708, 10 µM Nocodazole) 1 h prior, during and after virus inoculation at 37 °C. For microtubule disruption, cells were incubated in addition for 1 h on ice in the presence of 10 µM Nocodazole 1h before virus inoculation. After fixation, quenching, and permeabilization, samples were stained for incoming capsids with the 9C12 anti-hexon antibody. After primary and secondary antibody incubation, the cover slips were inverted onto a 30 μl droplet of click reaction mix for 2 h at RT. The freshly prepared click reaction mix consisted of 10 μM AlexaFluor 488-conjugated azide (Thermo Fisher Scientific), 1 mM CuSO4, and 10 mM sodium ascorbate in the presence of 1 mM THPTA (Sigma) and 10 mM aminoguanidine (Sigma) in PBS. Samples were stained with DAPI and AlexaFluor 647-conjugated succinimidyl ester and imaged with a Leica SP8 cLSM as described above. Nuclei and single viral genomes and/or capsids were segmented according to the corresponding signal using CellProfiler. Genomes were classified as capsid-positive based on their corresponding Hexon signal. Nuclear genomes were those that overlapped with the nuclear mask that was created based on the DAPI signal. Percentage of nuclear genomes is set in relation to all capsid-free genomes.

Eight thousand HeLa-sgMIB1 cells expressing mScarlet-MIB1 were seeded in a 10-well CELLview slide (Greiner Bio-One) with a 175 μm thick cover glass embedded in its bottom. After two days, the cells were incubated with AdV-C2-GFP-V-atto647 51 at 37 °C for 30 min. Unbound virus was washed away, fresh medium without phenol-red was added to the cells, and live imaging was started on a Visitron CSU-W1 spinning disk microscope consisting of a Nikon Eclipse T1 microscope and a Yokogawa confocal scanning unit W1 with a stage top incubation system at 37 °C and 5% CO2. Zstacks consisting of four steps with a step size of 1.4 μm were acquired every 10 s for up to 30 min with a 100x oil objective (NA 1.4) and a pinhole of 50 μm. The focus was maintained with a perfect focus system (PFS).

Di-glycine immunoprecipitation (IP) and MS analysis was performed essentially as described 113 .

HeLa-sgNT cells were seeded in three 15 cm dishes to a confluency of ca. 90%. Cells were pretreated for 2 h with 10 μM MG132 (Sigma Aldrich) before addition of 250 μg of AdV-C5. After incubation at 37 °C for 2 h, cells were washed two times with PBS and trypsinized. Cells were pelleted by centrifugation at 500 xg at 4 °C for 4 min, resuspended in PBS, and centrifuged again. respectively. Peptides were injected into the MS at a flow rate of 300 nl/min and were separated using a 120 min gradient of 2% to 35% buffer B. The MS was set to acquire full-scan MS spectra (300-1700 m/z) at a resolution of 60,000. A top 12 method was used for data-dependent acquisition (DDA) mode.

Raw files were analyzed using the Proteome Discoverer software, v2.1 (Thermo Fisher). Parent ion and tandem mass spectra were searched against the UniProtKB Homo sapiens and Human Adenovirus C5 databases using the SEQUEST algorithm. For the search, the enzyme specificity 20 was set to trypsin with a maximum of two missed cleavage sites. The precursor mass tolerance was set to 10 ppm and the fragment mass tolerance to 0.02 Da. Carbamidomethylation of cysteines was set as a fixed modification; N-terminal acetylation, oxidation of methionine, and di-glycine lysines were searched as dynamic modifications. The datasets were filtered on posterior error probability to achieve 1% false discovery rate on protein and peptide level.

Graphite coated electron microscopy grids were treated with five microliters of glycerol free purified virus for five minutes. Grids were washed three times with distillated water and stained for thirty seconds with ten microliters of a 2% uranyl acetate solution. Samples were imaged in a FEI CM100 electron microscope at 80 keV.

Streptolysin O (SLO) penetration assay was essentially performed as described 114 AlexaFluor 488 in blocking buffer containing DAPI. Cells were imaged in an IXMc high-throughput microscope, and infection was quantified based on the protein VI signal over segmented nuclei.

Virus input was normalized to the number of viral particles that bound to MPI-2 cells, which was determined in a binding assay as described above. Four hundred and fifty thousand MPI-2 wt, shcGAS, or shEGFP cells were seeded in a 24-well plate. On the following day, the virus was diluted in cold binding medium to reach 350 bound virus particles and added to the cells for 30 min at 37 °C. Inocula were removed, cells were washed two times with cold binding medium, and fresh growth medium was added for a total of 5 or and CCL5 protein levels were quantified using a sandwich ELISA procedure (Sigma, Cat# RAB0055 & RAB0077) following the manufacturer's instructions. Absorbance at 450 nm was measured in a Tecan Infinite M200 plate reader.

Thirty thousand MEF-Mx2-Luc-βKO cells were seeded per well in a 96-well plate 62 . On the following day, MPI-2 derived supernatants were diluted 1/10 and 1/100 in fresh media and added to the cells for 20 hours. In parallel, recombinant mouse IFNβ (kindly provided by Peter Staeheli) was serially diluted and used as a standard to assess IFNβ Units per ml. Twenty hours post inoculation SN were discarded, cells were washed once with PBS and lysed in 40µl of 1 x CCLR buffer (Promega) for 10 min on a rocking plate at RT. Twenty five microliters from each lysate were transferred to a white Nunclon 96-well plate (ThermoFisher). Thirty microliters of Luciferase Assay Reagent (LAR, Promega) were added to the wells using a TECAN plate reader with injection unit, followed by shaking for 2 seconds and integration of the luminescence signal over 10 seconds. 

All graphs display mean ± standard deviation (SD) unless stated otherwise. Statistical tests used are indicated in the figure legends. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001).

Oligo name DNA sequence (homology sequences in italics) 

Innate immunity to virus infection

Systems vaccinology

Pathogen recognition and inflammatory signaling in innate immune defenses

Intracellular NOD-like receptors in host defense and disease

The role of 'eat-me' signals and autophagy cargo receptors in innate immunity

Regulation of cGAS-and RLR-mediated immunity to nucleic acids

The ubiquitin code

Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools

Ubiquitin modifications

RING-type E3 ligases: master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination

Mechanisms of Deubiquitinase Specificity and Regulation

Diversity of degradation signals in the ubiquitin-proteasome system

Ubiquitination, ubiquitin-like modifiers, and deubiquitination in viral infection

The familiar and the unexpected in structures of icosahedral viruses

Mechanisms of virus uncoating

How viruses enter animal cells

Principles of Virus Uncoating: Cues and the Snooker Ball

Uncoating of non-enveloped viruses

A spring-loaded mechanism for the conformational change of influenza hemagglutinin

Structure of influenza haemagglutinin at the pH of membrane fusion

Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions

Low pH-triggered beta-propeller switch of the low-density lipoprotein receptor assists rhinovirus infection

BAP31 and BiP are essential for dislocation of SV40 from the endoplasmic reticulum to the cytosol

Biophysical properties of single rotavirus particles account for the functions of protein shells in a multilayered virus

Kinesin-1-mediated capsid disassembly and disruption of the nuclear pore complex promote virus infection

Drifting motions of the adenovirus receptor CAR and immobile integrins initiate virus uncoating and membrane lytic protein exposure

Maturation of adenovirus primes the protein nano-shell for successful endosomal escape

Editorial: Physical Virology and the Nature of Virus Infections

RNAi screening reveals proteasome-and Cullin3-dependent stages in vaccinia virus infection

Influenza A virus uses the aggresome processing machinery for host cell entry

TIM-1 Ubiquitination Mediates Dengue Virus Entry

The E3 Ubiquitin Ligase Mind Bomb 1 Controls Adenovirus Genome Release at the Nuclear Pore Complex

A review of 65 years of human adenovirus seroprevalence

tolerability, and immunogenicity of a recombinant adenovirus type-5

COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial

Adenoviruses -Infection, pathogenesis and therapy

State-of-the-art human adenovirus vectorology for therapeutic approaches

Adenovirus: Epidemiology, Global Spread of Novel Serotypes, and Advances in Treatment and Prevention

Adenovirus persistence, reactivation, and clinical management

Interaction of adenovirus with antibodies, complement, and coagulation factors

Human adenovirus infections: update and consideration of mechanisms of viral persistence. Current opinion in infectious diseases 31

The UPR sensor IRE1alpha and the adenovirus E3-19K glycoprotein sustain persistent and lytic infections

The E3 ubiquitin ligase Mib1 regulates Plk4 and centriole biogenesis

A new cellular stress response that triggers centriolar satellite reorganization and ciliogenesis

Mapping a dynamic innate immunity protein interaction network regulating type I interferon production

Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta

Tethering of an E3 ligase by

regulates the abundance of centrosomal KIAA0586/Talpid3 and promotes ciliogenesis

Systematic and quantitative assessment of the ubiquitin-modified proteome

Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling

A capsid-encoded PPxY-motif facilitates adenovirus entry

Stepwise dismantling of adenovirus 2 during entry into cells

Stepwise loss of fluorescent core protein V from human adenovirus during entry into cells

Tracking viral genomes in host cells at single-molecule resolution

Misdelivery at the Nuclear Pore Complex-Stopping a Virus Dead in Its Tracks

Adenoviral protein V promotes a process of viral assembly through nucleophosmin 1

Thermostability/infectivity defect caused by deletion of the core protein V gene in human adenovirus type 5 is rescued by thermo-selectable mutations in the core protein X precursor

Cell-to-cell and genome-to-genome variability of Adenovirus transcription tuned by the cell cycle. bioRxiv

Fluorescence Tracking of Genome Release during Mechanical Unpacking of Single Viruses

CRM1 is responsible for intracellular transport mediated by the nuclear export signal

Nuclear targeting of adenovirus type 2 requires CRM1-mediated nuclear export

The nuclear export factor CRM1 controls juxta-nuclear microtubule-dependent virus transport

GM-CSF-dependent macrophage lines are a unique model to study tissue macrophage functions

Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor

Adenovirus Detection by the cGAS/STING/TBK1 DNA Sensing Cascade

Key Role of the Scavenger Receptor MARCO in Mediating Adenovirus Infection and Subsequent Innate Responses of Macrophages

Genetic content and evolution of adenoviruses

Adenoviruses across the animal kingdom: a walk in the zoo

Molecular evolution of human adenoviruses

Lessons learned from adenovirus

Imaging the adenovirus infection cycle

The adenovirus capsid: major progress in minor proteins

Looking inside adenovirus

Adenovirus protein VI mediates membrane disruption following capsid disassembly

Atomic Structures of Minor Proteins VI and VII in the Human Adenovirus

The adenovirus major core protein VII is dispensable for virion assembly but is essential for lytic infection

Dynamic competition for hexon binding between core protein VII and lytic protein VI promotes adenovirus maturation and entry

Adenovirus major core protein condenses DNA in clusters and bundles, modulating genome release and capsid internal pressure

Adenovirus Entry: From Infection to Immunity

Functional genetic and biophysical analyses of membrane disruption by human adenovirus

Co-option of Membrane Wounding Enables Virus Penetration into Cells

Import of adenovirus DNA involves the nuclear pore complex receptor CAN/Nup214 and histone H1

Adenovirus Type-5 Entry and Disassembly Followed in Living Cells by FRET, Fluorescence Anisotropy, and FLIM

Nuclear import of adenovirus DNA involves direct interaction of hexon with an N-terminal domain of the nucleoporin Nup214

Antibody and DNA sensing pathways converge to activate the inflammasome during primary human macrophage infection

TRIM21 mediates antibody inhibition of adenovirus-based gene delivery and vaccination

The first step of adenovirus type 2 disassembly occurs at the cell surface, independently of endocytosis and escape to the cytosol

Microtubuledependent plus-and minus end-directed motilities are competing processes for nuclear targeting of adenovirus

A stochastic model for microtubule motors describes the in vivo cytoplasmic transport of human adenovirus

Imaging, Tracking and Computational Analyses of Virus Entry and Egress with the Cytoskeleton

Role of kinesins in directed adenovirus transport and cytoplasmic exploration

Role of cytoplasmic dynein and kinesins in adenovirus transport

Structure and composition of the adenovirus type 2 core

Interactions among the three adenovirus core proteins

Isolation and characterization of the DNA and protein binding activities of adenovirus core protein V

Distribution of DNA-condensing protein complexes in the adenovirus core

The structure of nucleoprotein cores released from adenovirions

Systematic approaches to identify E3 ligase substrates

Post translational modifications in adenovirus type 2

Epsin 1 is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virus

The ubiquitin/proteasome system mediates entry and endosomal trafficking of Kaposi's sarcoma-associated herpesvirus in endothelial cells

Viral takeover of the host ubiquitin system

Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors

Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid

Innate immune recognition of DNA: A recent history. Virology 479-480C

Cytosolic sensing of viruses

The role of the adenovirus protease on virus entry into cells

The adenovirus type 5 E1A transcriptional control region contains a duplicated enhancer element

Adenovirus type 5 virions can be assembled in vivo in the absence of detectable polypeptide IX

Virus assembly and disassembly: the adenovirus cysteine protease as a trigger factor

Simple and highly efficient BAC recombineering using galK selection

Characterization of 911: a new helper cell line for the titration and propagation of early region 1-deleted adenoviral vectors

CellProfiler: image analysis software for identifying and quantifying cell phenotypes

Postentry neutralization of adenovirus type 5 by an antihexon antibody

Large-scale identification of ubiquitination sites by mass spectrometry

A direct and versatile assay measuring membrane penetration of adenovirus in single cells

Analyzing real-time PCR data by the comparative C(T) method