E3 Ubiquitin Ligase Mindbomb 1 Facilitates Nuclear Delivery of Adenovirus Genomes
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E3 ubiquitin 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 Cancer 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 centrosome. 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 gene-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 ubiquitin ligase 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 proteins 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 humans. 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 protein 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 microtubule motors [email protected]. (6–8). Microtubules (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 human 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 genes 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