1887

Abstract

Modified vaccinia Ankara (MVA) is an attenuated strain of vaccinia virus (VACV), a dsDNA virus that replicates its genome in the cytoplasm and as a result is canonically sensed by the cyclic GMP-AMP synthase (cGAS) and its downstream stimulator of interferon genes (STING). MVA has a highly restricted host range due to major deletions in its genome including inactivation of immunomodulatory genes, only being able to grow in avian cells and the hamster cell line BHK21. Here we studied the interplay between MVA and the cGAS/STING DNA in this permissive cell line and determined whether manipulation of this axis could impact MVA replication and cell responses. We demonstrate that BHK21 cells retain a functional cGAS/STING axis that responds to canonical DNA sensing agonists, upregulating interferon stimulated genes (ISGs). BHK21 cells also respond to MVA, but with a distinct ISG profile. This profile remains unaltered after CRISPR/Cas9 knock-out editing of STING and ablation of cytosolic DNA responses, indicating that MVA responses are independent of the cGAS/STING axis. Furthermore, infection by MVA diminishes the ability of BHK21 cells to respond to exogenous DNA suggesting that MVA still encodes uncharacterised inhibitors of DNA sensing. This suggests that using attenuated strains in permissive cell lines may assist in identification of novel host-virus interactions that may be of relevance to disease or the therapeutic applications of poxviruses.

Funding
This study was supported by the:
  • Lorna and Yuti Chernajowsky Biomedical Trust
    • Principle Award Recipient: CarlosMaluquer de Motes
  • Biotechnology and Biological Sciences Research Council (Award BB/T006501/1)
    • Principle Award Recipient: CarlosMaluquer de Motes
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001755
2022-05-18
2024-12-02
Loading full text...

Full text loading...

/deliver/fulltext/jgv/103/5/jgv001755.html?itemId=/content/journal/jgv/10.1099/jgv.0.001755&mimeType=html&fmt=ahah

References

  1. Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and its eradication. Geneva: World Health Organization; 1988 http://whqlibdoc.who.int/smallpox/9241561106.pdf
  2. Mahnel H, Mayr A. Experiences with immunization against orthopox viruses of humans and animals using vaccine strain MVA. Berl Munch Tierarztl Wochenschr 1994; 107:253–256 [PubMed]
    [Google Scholar]
  3. Mayr A, Hochstein-Mintzel V, Stickl H. Abstammung, Eigenschaften und Verwendung des attenuierten Vaccinia-Stammes MVA. Infection 1975; 3:6–14 [View Article]
    [Google Scholar]
  4. Mayr A, Stickl H, Müller HK, Danner K, Singer H. The smallpox vaccination strain MVA: marker, genetic structure, experience gained with the parenteral vaccination and behavior in organisms with a debilitated defence mechanism (author’s transl). Zentralbl Bakteriol B 1978; 167:375–390 [PubMed]
    [Google Scholar]
  5. Sutter G, Moss B. Nonreplicating vaccinia vector efficiently expresses recombinant genes. Proc Natl Acad Sci U S A 1992; 89:10847–10851 [View Article] [PubMed]
    [Google Scholar]
  6. Sutter G, Wyatt LS, Foley PL, Bennink JR, Moss B. A recombinant vector derived from the host range-restricted and highly attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza virus. Vaccine 1994; 12:1032–1040 [View Article] [PubMed]
    [Google Scholar]
  7. Gilbert SC. Clinical development of Modified Vaccinia virus Ankara vaccines. Vaccine 2013; 31:4241–4246 [View Article] [PubMed]
    [Google Scholar]
  8. Meyer H, Sutter G, Mayr A. Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence. J Gen Virol 1991; 72 (Pt 5):1031–1038 [View Article] [PubMed]
    [Google Scholar]
  9. Antoine G, Scheiflinger F, Dorner F, Falkner FG. The complete genomic sequence of the modified vaccinia Ankara strain: comparison with other orthopoxviruses. Virology 1998; 244:365–396 [View Article] [PubMed]
    [Google Scholar]
  10. Meisinger-Henschel C, Schmidt M, Lukassen S, Linke B, Krause L et al. Genomic sequence of chorioallantois vaccinia virus Ankara, the ancestor of modified vaccinia virus Ankara. J Gen Virol 2007; 88:3249–3259 [View Article] [PubMed]
    [Google Scholar]
  11. Sutter G, Wyatt LS, Foley PL, Bennink JR, Moss B. A recombinant vector derived from the host range-restricted and highly attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza virus. Vaccine 1994; 12:1032–1040 [View Article] [PubMed]
    [Google Scholar]
  12. Bratke KA, McLysaght A, Rothenburg S. A survey of host range genes in poxvirus genomes. Infect Genet Evol 2013; 14:406–425 [View Article] [PubMed]
    [Google Scholar]
  13. Oliveira GP, Rodrigues RAL, Lima MT, Drumond BP, Abrahão JS. Poxvirus host range genes and virus-host spectrum: a critical review. Viruses 2017; 9:11 [View Article] [PubMed]
    [Google Scholar]
  14. Liu R, Mendez-Rios JD, Peng C, Xiao W, Weisberg AS et al. SPI-1 is a missing host-range factor required for replication of the attenuated modified vaccinia Ankara (MVA) vaccine vector in human cells. PLoS Pathog 2019; 15:e1007710 [View Article] [PubMed]
    [Google Scholar]
  15. Peng C, Moss B. Repair of a previously uncharacterized second host-range gene contributes to full replication of modified vaccinia virus Ankara (MVA) in human cells. Proc Natl Acad Sci U S A 2020; 117:3759–3767 [View Article] [PubMed]
    [Google Scholar]
  16. Sun L, Wu J, Du F, Chen X, Chen ZJ. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 2013; 339:786–791 [View Article] [PubMed]
    [Google Scholar]
  17. Ablasser A, Goldeck M, Cavlar T, Deimling T, Witte G et al. cGAS produces a 2’-5’-linked cyclic dinucleotide second messenger that activates STING. Nature 2013; 498:380–384 [View Article] [PubMed]
    [Google Scholar]
  18. Zhou W, Whiteley AT, de Oliveira Mann CC, Morehouse BR, Nowak RP et al. Structure of the Human cGAS-DNA Complex Reveals Enhanced Control of Immune Surveillance. Cell 2018; 174:300–311 [View Article] [PubMed]
    [Google Scholar]
  19. Xie W, Lama L, Adura C, Tomita D, Glickman JF et al. Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic activity and liquid-phase condensation. Proc Natl Acad Sci U S A 2019; 116:11946–11955 [View Article] [PubMed]
    [Google Scholar]
  20. Wu J, Sun L, Chen X, Du F, Shi H et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 2013; 339:826–830 [View Article] [PubMed]
    [Google Scholar]
  21. Ishikawa H, Barber GN. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 2008; 455:674–678 [View Article] [PubMed]
    [Google Scholar]
  22. Tanaka Y, Chen ZJ. STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway. Sci Signal 2012; 5:ra20 [View Article] [PubMed]
    [Google Scholar]
  23. Gui X, Yang H, Li T, Tan X, Shi P et al. Autophagy induction via STING trafficking is a primordial function of the cGAS pathway. Nature 2019; 567:262–266 [View Article] [PubMed]
    [Google Scholar]
  24. Liu D, Wu H, Wang C, Li Y, Tian H et al. STING directly activates autophagy to tune the innate immune response. Cell Death Differ 2019; 26:1735–1749 [View Article] [PubMed]
    [Google Scholar]
  25. Yamashiro LH, Wilson SC, Morrison HM, Karalis V, Chung J-YJ et al. Interferon-independent STING signaling promotes resistance to HSV-1 in vivo. Nat Commun 2020; 11:3382 [View Article] [PubMed]
    [Google Scholar]
  26. El-Jesr M, Teir M, Maluquer de Motes C. Vaccinia virus activation and antagonism of cytosolic DNA sensing. Front Immunol 2020; 11:568412 [View Article] [PubMed]
    [Google Scholar]
  27. Eaglesham JB, McCarty KL, Kranzusch PJ. Structures of diverse poxin cGAMP nucleases reveal a widespread role for cGAS-STING evasion in host-pathogen conflict. Elife 2020; 9:e59753 [View Article]
    [Google Scholar]
  28. Hernáez B, Alonso G, Georgana I, El-Jesr M, Martín R et al. Viral cGAMP nuclease reveals the essential role of DNA sensing in protection against acute lethal virus infection. Sci Adv 2020; 6:38 [View Article] [PubMed]
    [Google Scholar]
  29. Eaglesham JB, Pan Y, Kupper TS, Kranzusch PJ. Viral and metazoan poxins are cGAMP-specific nucleases that restrict cGAS-STING signalling. Nature 2019; 566:259–263 [View Article] [PubMed]
    [Google Scholar]
  30. Maluquer de Motes C. Poxvirus cGAMP nucleases: Clues and mysteries from a stolen gene. PLoS Pathog 2021; 17:e1009372 [View Article] [PubMed]
    [Google Scholar]
  31. Dai P, Wang W, Cao H, Avogadri F, Dai L et al. Modified vaccinia virus Ankara triggers type I IFN production in murine conventional dendritic cells via a cGAS/STING-mediated cytosolic DNA-sensing pathway. PLoS Pathog 2014; 10:e1003989 [View Article] [PubMed]
    [Google Scholar]
  32. Georgana I, Sumner RP, Towers GJ, Maluquer de Motes C. Virulent poxviruses inhibit DNA sensing by preventing STING activation. J Virol 2018; 92:10 [View Article] [PubMed]
    [Google Scholar]
  33. Döring M, De Azevedo K, Blanco-Rodriguez G, Nadalin F, Satoh T et al. Single-cell analysis reveals divergent responses of human dendritic cells to the MVA vaccine. Sci Signal 2021; 14:697 [View Article] [PubMed]
    [Google Scholar]
  34. Barnowski C, Ciupka G, Tao R, Jin L, Busch DH et al. Efficient Induction of Cytotoxic T Cells by Viral Vector Vaccination Requires STING-dependent DC functions. Front Immunol 2020; 11:1458 [View Article] [PubMed]
    [Google Scholar]
  35. Zhong C, Liu F, Hajnik RJ, Yao L, Chen K et al. Type I interferon promotes humoral immunity in viral vector vaccination. J Virol 2021; 95:e0092521 [View Article] [PubMed]
    [Google Scholar]
  36. Carroll MW, Moss B. Host range and cytopathogenicity of the highly attenuated MVA strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman mammalian cell line. Virology 1997; 238:198–211 [View Article] [PubMed]
    [Google Scholar]
  37. Zivcec M, Safronetz D, Haddock E, Feldmann H, Ebihara H. Validation of assays to monitor immune responses in the Syrian golden hamster (Mesocricetus auratus). J Immunol Methods 2011; 368:24–35 [View Article] [PubMed]
    [Google Scholar]
  38. Concordet J-P, Haeussler M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res 2018; 46:W242–W245 [View Article] [PubMed]
    [Google Scholar]
  39. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 2014; 343:84–87 [View Article] [PubMed]
    [Google Scholar]
  40. Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 1997; 15:871–875 [View Article] [PubMed]
    [Google Scholar]
  41. Rice AP, Roberts BE. Vaccinia virus induces cellular mRNA degradation. J Virol 1983; 47:529–539 [View Article] [PubMed]
    [Google Scholar]
  42. Falkner FG, Moss B. Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors. J Virol 1988; 62:1849–1854 [View Article]
    [Google Scholar]
  43. Staib C, Drexler I, Ohlmann M, Wintersperger S, Erfle V et al. Transient host range selection for genetic engineering of modified vaccinia virus Ankara. Biotechniques 2000; 28:1137–1142 [View Article] [PubMed]
    [Google Scholar]
  44. Sánchez-Puig JM, Blasco R. Isolation of vaccinia MVA recombinants using the viral F13L gene as the selective marker. Biotechniques 2005; 39:665–666 [View Article] [PubMed]
    [Google Scholar]
  45. Gowripalan A, Smith SA, Tscharke DC. Selection of vaccinia virus recombinants using CRISPR/Cas9. Bio Protoc 2021; 11:e4270 [View Article] [PubMed]
    [Google Scholar]
  46. Ricci PS, Schäfer B, Kreil TR, Falkner FG, Holzer GW. Selection of recombinant MVA by rescue of the essential D4R gene. Virol J 2011; 8:529 [View Article] [PubMed]
    [Google Scholar]
  47. Samuelsson C, Hausmann J, Lauterbach H, Schmidt M, Akira S et al. Survival of lethal poxvirus infection in mice depends on TLR9, and therapeutic vaccination provides protection. J Clin Invest 2008; 118:1776–1784 [View Article] [PubMed]
    [Google Scholar]
  48. Waibler Z, Anzaghe M, Ludwig H, Akira S, Weiss S et al. Modified vaccinia virus Ankara induces Toll-like receptor-independent type I interferon responses. J Virol 2007; 81:12102–12110 [View Article] [PubMed]
    [Google Scholar]
  49. Price PJR, Luckow B, Torres-Domínguez LE, Brandmüller C, Zorn J et al. Chemokine (C-C Motif) receptor 1 is required for efficient recruitment of neutrophils during respiratory infection with modified vaccinia virus Ankara. J Virol 2014; 88:10840–10850 [View Article] [PubMed]
    [Google Scholar]
  50. Ludwig H, Suezer Y, Waibler Z, Kalinke U, Schnierle BS et al. Double-stranded RNA-binding protein E3 controls translation of viral intermediate RNA, marking an essential step in the life cycle of modified vaccinia virus Ankara. Journal of General Virology 2006; 87:1145–1155 [View Article] [PubMed]
    [Google Scholar]
  51. Backes S, Sperling KM, Zwilling J, Gasteiger G, Ludwig H et al. Viral host-range factor C7 or K1 is essential for modified vaccinia virus Ankara late gene expression in human and murine cells, irrespective of their capacity to inhibit protein kinase R-mediated phosphorylation of eukaryotic translation initiation factor 2. Journal of General Virology 2009; 91:470–482 [View Article] [PubMed]
    [Google Scholar]
  52. Unterholzner L, Sumner RP, Baran M, Ren H, Mansur DS et al. Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7. PLoS Pathog 2011; 7:e1002247 [View Article] [PubMed]
    [Google Scholar]
  53. Stuart JH, Sumner RP, Lu Y, Snowden JS, Smith GL et al. Vaccinia virus protein C6 inhibits type I IFN signalling in the nucleus and binds to the transactivation domain of STAT2. PLoS Pathog 2016; 12:e1005955 [View Article] [PubMed]
    [Google Scholar]
  54. Meade N, Furey C, Li H, Verma R, Chai Q et al. Poxviruses evade cytosolic sensing through disruption of an mTORC1-mTORC2 regulatory circuit. Cell 2018; 174:1143–1157 [View Article] [PubMed]
    [Google Scholar]
  55. Meade N, King M, Munger J, Walsh D. mTOR dysregulation by vaccinia virus F17 controls multiple processes with varying roles in infection. J Virol 2019; 93:15 [View Article] [PubMed]
    [Google Scholar]
  56. Yang N, Wang Y, Dai P, Li T, Zierhut C et al. Vaccinia E5 is a major inhibitor of the DNA sensor cGAS. bioRxiv 2021 [View Article]
    [Google Scholar]
  57. Oliveira M, Rodrigues DR, Guillory V, Kut E, Giotis ES et al. Chicken cGAS senses fowlpox virus infection and regulates macrophage effector functions. Front Immunol 2020; 11:613079 [View Article] [PubMed]
    [Google Scholar]
  58. Albarnaz JD, Torres AA, Smith GL. Modulating vaccinia virus immunomodulators to improve immunological memory. Viruses 2018; 10:E101 [View Article] [PubMed]
    [Google Scholar]
  59. Ho TY, Mealiea D, Okamoto L, Stojdl DF, McCart JA. Deletion of immunomodulatory genes as a novel approach to oncolytic vaccinia virus development. Mol Ther Oncolytics 2021; 22:85–97 [View Article] [PubMed]
    [Google Scholar]
  60. Shin J, Hong S-O, Kim M, Lee H, Choi H et al. Generation of a novel oncolytic vaccinia virus using the IHD-W strain. Hum Gene Ther 2021; 32:517–527 [View Article] [PubMed]
    [Google Scholar]
  61. Wasilenko ST, Stewart TL, Meyers AFA, Barry M. Vaccinia virus encodes a previously uncharacterized mitochondrial-associated inhibitor of apoptosis. Proc Natl Acad Sci U S A 2003; 100:14345–14350 [View Article] [PubMed]
    [Google Scholar]
  62. Fagan-Garcia K, Barry M. A vaccinia virus deletion mutant reveals the presence of additional inhibitors of NF-kappaB. J Virol 2011; 85:883–894 [View Article] [PubMed]
    [Google Scholar]
  63. Sumner RP, Maluquer de Motes C, Veyer DL, Smith GL. Vaccinia virus inhibits NF-κB-dependent gene expression downstream of p65 translocation. J Virol 2014; 88:3092–3102 [View Article] [PubMed]
    [Google Scholar]
  64. Mansur DS, Maluquer de Motes C, Unterholzner L, Sumner RP, Ferguson BJ et al. Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence. PLoS Pathog 2013; 9:e1003183 [View Article] [PubMed]
    [Google Scholar]
  65. Albarnaz JD, Ren H, Torres AA, Shmeleva EV, Melo CA et al. Molecular mimicry of NF-κB by vaccinia virus protein enables selective inhibition of antiviral responses. Nat Microbiol 2022; 7:154–168 [View Article] [PubMed]
    [Google Scholar]
/content/journal/jgv/10.1099/jgv.0.001755
Loading
/content/journal/jgv/10.1099/jgv.0.001755
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error