1887

Abstract

Reverse genetic systems have been used to introduce heterologous sequences into the rotavirus segmented double-stranded (ds)RNA genome, enabling the generation of recombinant viruses that express foreign proteins and possibly serve as vaccine vectors. Notably, insertion of SARS-CoV-2 sequences into the segment seven (NSP3) RNA of simian SA11 rotavirus was previously shown to result in the production of recombinant viruses that efficiently expressed the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the S1 region of the SARS-CoV-2 spike protein. However, efforts to generate a similar recombinant (r) SA11 virus that efficiently expressed full-length S1 were less successful. In this study, we describe modifications to the S1-coding cassette inserted in the segment seven RNA that allowed recovery of second-generation rSA11 viruses that efficiently expressed the ~120-kDa S1 protein. The ~120-kDa S1 products were shown to be glycosylated, based on treatment with endoglycosidase H, which reduced the protein to a size of ~80 kDa. Co-pulldown assays demonstrated that the ~120-kDa S1 proteins had affinity for the human ACE2 receptor. Although all the second-generation rSA11 viruses expressed glycosylated S1 with affinity for the ACE receptor, only the S1 product of one virus (rSA11/S1f) was appropriately recognized by anti-S1 antibodies, suggesting the rSA11/S1f virus expressed an authentic form of S1. Compared to the other second-generation rSA11 viruses, the design of the rSA11/S1f was unique, encoding an S1 product that did not include an N-terminal FLAG tag. Probably due to the impact of FLAG tags upstream of the S1 signal peptides, the S1 products of the other viruses (rSA11/3fS1 and rSA11/3fS1-His) may have undergone defective glycosylation, impeding antibody binding. In summary, these results indicate that recombinant rotaviruses can serve as expression vectors of foreign glycosylated proteins, raising the possibility of generating rotavirus-based vaccines that can induce protective immune responses against enteric and mucosal viruses with glycosylated capsid components, including SARS-CoV-2.

Funding
This study was supported by the:
  • Blatt Endowment
    • Principle Award Recipient: JohnThomas Patton
  • GIVAX,INC
    • Principle Award Recipient: JohnThomas Patton
  • Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Award R21AI44881)
    • Principle Award Recipient: JohnThomas Patton
  • 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.
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2023-10-13
2024-11-02
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References

  1. Matthijnssens J, Attoui H, Bányai K, Brussaard CPD, Danthi P et al. ICTV Virus Taxonomy Profile: Sedoreoviridae 2022. J Gen Virol 2022; 103:10 [View Article]
    [Google Scholar]
  2. Crawford SE, Ramani S, Tate JE, Parashar UD, Svensson L et al. Rotavirus infection. Nat Rev Dis Primers 2017; 3:17083 [View Article] [PubMed]
    [Google Scholar]
  3. Clark A, Black R, Tate J, Roose A, Kotloff K et al. Estimating global, regional and national rotavirus deaths in children aged <5 years: current approaches, new analyses and proposed improvements. PLoS One 2017; 12:e0183392 [View Article] [PubMed]
    [Google Scholar]
  4. Leshem E, Tate JE, Steiner CA, Curns AT, Lopman BA et al. Acute gastroenteritis hospitalizations among US children following implementation of the rotavirus vaccine. JAMA 2015; 313:2282–2284 [View Article] [PubMed]
    [Google Scholar]
  5. Burke RM, Tate JE, Kirkwood CD, Steele AD, Parashar UD. Current and new rotavirus vaccines. Curr Opin Infect Dis 2019; 32:435–444 [View Article] [PubMed]
    [Google Scholar]
  6. Folorunso OS, Sebolai OM. Overview of the development, impacts, and challenges of live-attenuated oral rotavirus vaccines. Vaccines 2020; 8:341 [View Article] [PubMed]
    [Google Scholar]
  7. Skansberg A, Sauer M, Tan M, Santosham M, Jennings MC. Product review of the rotavirus vaccines ROTASIIL, ROTAVAC, and Rotavin-M1. Hum Vaccin Immunother 2021; 17:1223–1234 [View Article] [PubMed]
    [Google Scholar]
  8. Trask SD, McDonald SM, Patton JT. Structural insights into the coupling of virion assembly and rotavirus replication. Nat Rev Microbiol 2012; 10:165–177 [View Article] [PubMed]
    [Google Scholar]
  9. Desselberger U. What are the limits of the packaging capacity for genomic RNA in the cores of rotaviruses and of other members of the Reoviridae?. Virus Res 2020; 276:197822 [View Article] [PubMed]
    [Google Scholar]
  10. Kanai Y, Komoto S, Kawagishi T, Nouda R, Nagasawa N et al. Entirely plasmid-based reverse genetics system for rotaviruses. Proc Natl Acad Sci U S A 2017; 114:2349–2354 [View Article] [PubMed]
    [Google Scholar]
  11. Komoto S, Fukuda S, Kugita M, Hatazawa R, Koyama C et al. Generation of infectious recombinant human rotaviruses from just 11 cloned cDNAs encoding the rotavirus genome. J Virol 2019; 93:e02207-18 [View Article] [PubMed]
    [Google Scholar]
  12. Kanda M, Fukuda S, Hamada N, Nishiyama S, Masatani T et al. Establishment of a reverse genetics system for avian rotavirus a strain PO-13. J Gen Virol 2022; 103:10 [View Article] [PubMed]
    [Google Scholar]
  13. Sánchez-Tacuba L, Feng N, Meade NJ, Mellits KH, Jaïs PH et al. An optimized reverse genetics system suitable for efficient recovery of simian, human, and murine-like rotaviruses. J Virol 2020; 94:e01294-20 [View Article] [PubMed]
    [Google Scholar]
  14. Kawagishi T, Nurdin JA, Onishi M, Nouda R, Kanai Y et al. Reverse genetics system for a human group A rotavirus. J Virol 2020; 94:e00963-19 [View Article] [PubMed]
    [Google Scholar]
  15. Hamajima R, Lusiany T, Minami S, Nouda R, Nurdin JA et al. A reverse genetics system for human rotavirus G2P[4]. J Gen Virol 2022; 103:12 [View Article] [PubMed]
    [Google Scholar]
  16. Diebold O, Gonzalez V, Venditti L, Sharp C, Blake RA et al. Using species A rotavirus reverse genetics to engineer chimeric viruses expressing SARS-CoV-2 spike epitopes. J Virol 2022; 96:e0048822 [View Article] [PubMed]
    [Google Scholar]
  17. Komoto S, Kanai Y, Fukuda S, Kugita M, Kawagishi T et al. Reverse genetics system demonstrates that rotavirus nonstructural protein NSP6 is not essential for viral replication in cell culture. J Virol 2017; 91:e00695-17 [View Article] [PubMed]
    [Google Scholar]
  18. Chang-Graham AL, Perry JL, Strtak AC, Ramachandran NK, Criglar JM et al. Rotavirus calcium dysregulation manifests as dynamic calcium signaling in the cytoplasm and endoplasmic reticulum. Sci Rep 2019; 9:10822 [View Article] [PubMed]
    [Google Scholar]
  19. Papa G, Venditti L, Arnoldi F, Schraner EM, Potgieter C et al. Recombinant rotaviruses rescued by reverse genetics reveal the role of NSP5 hyperphosphorylation in the assembly of viral factories. J Virol 2019; 94:e01110–e01119 [View Article] [PubMed]
    [Google Scholar]
  20. Falkenhagen A, Patzina-Mehling C, Gadicherla AK, Strydom A, O’Neill HG et al. Generation of simian rotavirus reassortants with VP4- and VP7-encoding genome segments from human strains circulating in Africa using reverse genetics. Viruses 2020; 12:201 [View Article] [PubMed]
    [Google Scholar]
  21. Falkenhagen A, Patzina-Mehling C, Rückner A, Vahlenkamp TW, Johne R. Generation of simian rotavirus reassortants with diverse VP4 genes using reverse genetics. J Gen Virol 2019; 100:1595–1604 [View Article] [PubMed]
    [Google Scholar]
  22. Criglar JM, Crawford SE, Zhao B, Smith HG, Stossi F et al. A genetically engineered rotavirus NSP2 phosphorylation mutant impaired in viroplasm formation and replication shows an early interaction between vNSP2 and cellular lipid droplets. J Virol 2020; 94:e00972-20 [View Article] [PubMed]
    [Google Scholar]
  23. Song Y, Feng N, Sanchez-Tacuba L, Yasukawa LL, Ren L et al. Reverse genetics reveals a role of rotavirus VP3 phosphodiesterase activity in inhibiting RNase L signaling and contributing to intestinal viral replication In Vivo. J Virol 2020; 94:e01952-19 [View Article] [PubMed]
    [Google Scholar]
  24. Nilsson EM, Sullivan OM, Anderson ML, Argobright HM, Shue TM et al. Reverse genetic engineering of simian rotaviruses with temperature-sensitive lesions in VP1, VP2, and VP6. Virus Res 2021; 302:198488 [View Article] [PubMed]
    [Google Scholar]
  25. Hundley F, Biryahwaho B, Gow M, Desselberger U. Genome rearrangements of bovine rotavirus after serial passage at high multiplicity of infection. Virology 1985; 143:88–103 [View Article] [PubMed]
    [Google Scholar]
  26. Hundley F, McIntyre M, Clark B, Beards G, Wood D et al. Heterogeneity of genome rearrangements in rotaviruses isolated from a chronically infected immunodeficient child. J Virol 1987; 61:3365–3372 [View Article] [PubMed]
    [Google Scholar]
  27. Shen S, Burke B, Desselberger U. Rearrangement of the VP6 gene of a group A rotavirus in combination with a point mutation affecting trimer stability. J Virol 1994; 68:1682–1688 [View Article] [PubMed]
    [Google Scholar]
  28. Ballard A, McCrae MA, Desselberger U. Nucleotide sequences of normal and rearranged RNA segments 10 of human rotaviruses. J Gen Virol 1992; 73 (Pt 3):633–638 [View Article] [PubMed]
    [Google Scholar]
  29. Gault E, Schnepf N, Poncet D, Servant A, Teran S et al. A human rotavirus with rearranged genes 7 and 11 encodes a modified NSP3 protein and suggests an additional mechanism for gene rearrangement. J Virol 2001; 75:7305–7314 [View Article] [PubMed]
    [Google Scholar]
  30. Patton JT, Taraporewala Z, Chen D, Chizhikov V, Jones M et al. Effect of intragenic rearrangement and changes in the 3’ consensus sequence on NSP1 expression and rotavirus replication. J Virol 2001; 75:2076–2086 [View Article] [PubMed]
    [Google Scholar]
  31. Barro M, Patton JT. Rotavirus nonstructural protein 1 subverts innate immune response by inducing degradation of IFN regulatory factor 3. Proc Natl Acad Sci U S A 2005; 102:4114–4119 [View Article] [PubMed]
    [Google Scholar]
  32. Morelli M, Dennis AF, Patton JT, Dermody TS. Putative E3 ubiquitin ligase of human rotavirus inhibits NF-κB activation by using molecular mimicry to target β-TrCP. mBio 2015; 6: [View Article]
    [Google Scholar]
  33. Arnold MM, Brownback CS, Taraporewala ZF, Patton JT. Rotavirus variant replicates efficiently although encoding an aberrant NSP3 that fails to induce nuclear localization of poly(A)-binding protein. J Gen Virol 2012; 93:1483–1494 [View Article] [PubMed]
    [Google Scholar]
  34. Gratia M, Sarot E, Vende P, Charpilienne A, Baron CH et al. Rotavirus NSP3 is a translational surrogate of the poly(A) binding protein-poly(A) complex. J Virol 2015; 89:8773–8782 [View Article] [PubMed]
    [Google Scholar]
  35. Piron M, Delaunay T, Grosclaude J, Poncet D. Identification of the RNA-binding, dimerization, and eIF4GI-binding domains of rotavirus nonstructural protein NSP3. J Virol 1999; 73:5411–5421 [View Article] [PubMed]
    [Google Scholar]
  36. Hatazawa R, Fukuda S, Kumamoto K, Matsushita F, Nagao S et al. Strategy for generation of replication-competent recombinant rotaviruses expressing multiple foreign genes. J Gen Virol 2021; 102: [View Article] [PubMed]
    [Google Scholar]
  37. Wei J, Radcliffe S, Lu M, Li Y, Cassaday J et al. A novel rotavirus reverse genetics platform supports flexible insertion of exogenous genes and enables rapid development of a high-throughput neutralization assay. bioRxiv 2023 [View Article]
    [Google Scholar]
  38. Komoto S, Fukuda S, Ide T, Ito N, Sugiyama M et al. Generation of recombinant rotaviruses expressing fluorescent proteins by using an optimized reverse genetics system. J Virol 2018; 92:e00588-18 [View Article] [PubMed]
    [Google Scholar]
  39. Kanai Y, Kawagishi T, Nouda R, Onishi M, Pannacha P et al. Development of stable rotavirus reporter expression systems. J Virol 2019; 93:e01774–18 [View Article]
    [Google Scholar]
  40. Pannacha P, Kanai Y, Kawagishi T, Nouda R, Nurdin JA et al. Generation of recombinant rotaviruses encoding a split NanoLuc peptide tag. Biochem Biophys Res Commun 2021; 534:740–746 [View Article] [PubMed]
    [Google Scholar]
  41. Philip AA, Herrin BE, Garcia ML, Abad AT, Katen SP et al. Collection of recombinant rotaviruses expressing fluorescent reporter proteins. Microbiol Resour Announc 2019; 8:e00523–19 [View Article]
    [Google Scholar]
  42. Philip AA, Perry JL, Eaton HE, Shmulevitz M, Hyser JM et al. Generation of recombinant rotavirus expressing NSP3-UnaG fusion protein by a simplified reverse genetics system. J Virol 2019; 93:e01616-19 [View Article] [PubMed]
    [Google Scholar]
  43. Philip AA, Patton JT. Expression of separate heterologous proteins from the rotavirus NSP3 genome segment using a translational 2A stop-restart element. J Virol 2020; 94:e00959-20 [View Article] [PubMed]
    [Google Scholar]
  44. Luke G, Escuin H, De Felipe P, Ryan M. 2a to the fore - research, technology and applications. Biotechnol Genet Eng Rev 2020; 26:223–260 [View Article]
    [Google Scholar]
  45. Philip AA, Patton JT. Generation of recombinant rotaviruses expressing human norovirus capsid proteins. J Virol 2022; 96:e0126222 [View Article] [PubMed]
    [Google Scholar]
  46. Kawagishi T, Sánchez-Tacuba L, Feng N, Costantini VP, Tan M et al. Mucosal and systemic neutralizing antibodies to norovirus induced in infant mice orally inoculated with recombinant rotaviruses. Proc Natl Acad Sci U S A 2023; 120:e2214421120 [View Article] [PubMed]
    [Google Scholar]
  47. Philip AA, Patton JT. Rotavirus as an expression platform of domains of the SARS-CoV-2 spike protein. Vaccines 2021; 9:449 [View Article] [PubMed]
    [Google Scholar]
  48. Duan L, Zheng Q, Zhang H, Niu Y, Lou Y et al. The SARS-CoV-2 spike glycoprotein biosynthesis, structure, function, and antigenicity: implications for the design of spike-based vaccine immunogens. Front Immunol 2020; 11:576622 [View Article] [PubMed]
    [Google Scholar]
  49. Huang Y, Yang C, Xu XF, Xu W, Liu SW. Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacol Sin 2020; 41:1141–1149 [View Article] [PubMed]
    [Google Scholar]
  50. Brouwer PJM, Caniels TG, van der Straten K, Snitselaar JL, Aldon Y et al. Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability. Science 2020; 369:643–650 [View Article] [PubMed]
    [Google Scholar]
  51. Liu L, Wang P, Nair MS, Yu J, Rapp M et al. Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike. Nature 2020; 584:450–456 [View Article] [PubMed]
    [Google Scholar]
  52. Rogers TF, Zhao F, Huang D, Beutler N, Burns A et al. Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model. Science 2020; 369:956–963 [View Article]
    [Google Scholar]
  53. Zost SJ, Gilchuk P, Chen RE, Case JB, Reidy JX et al. Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein. Nat Med 2020; 26:1422–1427 [View Article] [PubMed]
    [Google Scholar]
  54. Xiaojie S, Yu L, lei Y, Guang Y, Min Q. Neutralizing antibodies targeting SARS-CoV-2 spike protein. Stem Cell Research 2021; 50:102125 [View Article]
    [Google Scholar]
  55. Casalino L, Gaieb Z, Goldsmith JA, Hjorth CK, Dommer AC et al. Beyond shielding: the roles of glycans in the SARS-CoV-2 spike protein. ACS Cent Sci 2020; 6:1722–1734 [View Article] [PubMed]
    [Google Scholar]
  56. Medina-Enríquez MM, Lopez-León S, Carlos-Escalante JA, Aponte-Torres Z, Cuapio A et al. ACE2: the molecular doorway to SARS-CoV-2. Cell Biosci 2020; 10:148 [View Article] [PubMed]
    [Google Scholar]
  57. Philip AA, Dai J, Katen SP, Patton JT. Simplified reverse genetics method to recover recombinant rotaviruses expressing reporter proteins. J Vis Exp 2020; 158:e61039 [View Article] [PubMed]
    [Google Scholar]
  58. Arnold M, Patton JT, McDonald SM. Culturing, storage, and quantification of rotaviruses. Curr Protoc Microbiol 2009; 15:15C [View Article]
    [Google Scholar]
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