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Abstract

RSV is the leading cause of infant hospitalizations and a significant cause of paediatric and geriatric morbidity worldwide. Recently, we reported that insulin-like growth factor 1 receptor (IGF1R) was a receptor for respiratory syncytial virus (RSV) in airway epithelial cells and that activation of IGF1R recruited the coreceptor, nucleolin (NCL), to the cell surface. Cilia and mucus that line the airways pose a significant barrier to viral and bacterial infection. The cortical actin cytoskeleton has been shown by others to mediate RSV entry, so we studied the roles of the RSV receptors and actin remodelling during virus entry. We found that IGF1R expression and phosphorylation were associated with the ability of RSV to infect cells. Confocal immunofluorescence imaging showed that actin projections, a hallmark of macropinocytosis, formed around viral particles 30 min after infection. Consistent with prior reports we also found that virus particles were internalized into early endosome antigen-1 positive endosomes within 90 min. Inhibiting actin polymerization significantly reduced viral titre by approximately ten-fold. Inhibiting PI3 kinase and PKCζ in stratified air-liquid interface (ALI) models of the airway epithelium had similar effects on reducing the actin remodelling observed during infection and attenuating viral entry. Actin projections were associated with NCL interacting with RSV particles resting on apical cilia of the ALIs. We conclude that macropinocytosis-like actin projections protrude through normally protective cilia and mucus layers of stratified airway epithelium that helps present the IGF1R receptor and the NCL coreceptor to RSV particles waiting at the surface.

Funding
This study was supported by the:
  • canadian institutes for health research (Award RN382934 - 418735)
    • Principle Award Recipient: DavidJ Marchant
  • 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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2023-11-28
2024-12-07
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References

  1. Griffiths C, Drews SJ, Marchant DJ. Respiratory syncytial virus: Infection, detection, and new options for prevention and treatment. Clin Microbiol Rev 2017; 30:277–319 [View Article] [PubMed]
    [Google Scholar]
  2. Feldman SA, Audet S, Beeler JA. The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate. J Virol 2000; 74:6442–6447 [View Article] [PubMed]
    [Google Scholar]
  3. Krusat T, Streckert HJ. Heparin-dependent attachment of respiratory syncytial virus (RSV) to host cells. Arch Virol 1997; 142:1247–1254 [View Article] [PubMed]
    [Google Scholar]
  4. Johnson SM, McNally BA, Ioannidis I, Flano E, Teng MN et al. Respiratory syncytial virus uses CX3CR1 as a receptor on primary human airway epithelial cultures. PLoS Pathog 2015; 11:e1005318 [View Article] [PubMed]
    [Google Scholar]
  5. Chirkova T, Lin S, Oomens AGP, Gaston KA, Boyoglu-Barnum S et al. CX3CR1 is an important surface molecule for respiratory syncytial virus infection in human airway epithelial cells. J Gen Virol 2015; 96:2543–2556 [View Article] [PubMed]
    [Google Scholar]
  6. Currier MG, Lee S, Stobart CC, Hotard AL, Villenave R et al. EGFR interacts with the fusion protein of respiratory syncytial virus strain 2-20 and mediates infection and mucin expression. PLoS Pathog 2016; 12:e1005622 [View Article] [PubMed]
    [Google Scholar]
  7. Krzyzaniak MA, Zumstein MT, Gerez JA, Picotti P, Helenius A. Host cell entry of respiratory syncytial virus involves macropinocytosis followed by proteolytic activation of the F protein. PLoS Pathog 2013; 9:e1003309 [View Article] [PubMed]
    [Google Scholar]
  8. Kurt-Jones EA, Popova L, Kwinn L, Haynes LM, Jones LP et al. Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat Immunol 2000; 1:398–401 [View Article] [PubMed]
    [Google Scholar]
  9. Behera AK, Matsuse H, Kumar M, Kong X, Lockey RF et al. Blocking intercellular adhesion molecule-1 on human epithelial cells decreases respiratory syncytial virus infection. Biochem Biophys Res Commun 2001; 280:188–195 [View Article] [PubMed]
    [Google Scholar]
  10. Tayyari F, Marchant D, Moraes TJ, Duan W, Mastrangelo P et al. Identification of nucleolin as a cellular receptor for human respiratory syncytial virus. Nat Med 2011; 17:1132–1135 [View Article] [PubMed]
    [Google Scholar]
  11. Holguera J, Villar E, Muñoz-Barroso I. Identification of cellular proteins that interact with Newcastle Disease Virus and human Respiratory Syncytial Virus by a two-dimensional virus overlay protein binding assay (VOPBA). Virus Res 2014; 191:138–142 [View Article] [PubMed]
    [Google Scholar]
  12. Griffiths CD, Bilawchuk LM, McDonough JE, Jamieson KC, Elawar F et al. Publisher Correction: IGF1R is an entry receptor for respiratory syncytial virus. Nature 2020; 583:615–619 [View Article] [PubMed]
    [Google Scholar]
  13. Gusscott S, Jenkins CE, Lam SH, Giambra V, Pollak M et al. IGF1R Derived PI3K/AKT signaling maintains growth in a subset of human T-cell acute lymphoblastic leukemias. PLoS One 2016; 11:e0161158 [View Article] [PubMed]
    [Google Scholar]
  14. Hua H, Kong Q, Yin J, Zhang J, Jiang Y. Insulin-like growth factor receptor signaling in tumorigenesis and drug resistance: a challenge for cancer therapy. J Hematol Oncol 2020; 13:64 [View Article] [PubMed]
    [Google Scholar]
  15. Peruzzi F, Prisco M, Dews M, Salomoni P, Grassilli E et al. Multiple signaling pathways of the insulin-like growth factor 1 receptor in protection from apoptosis. Mol Cell Biol 1999; 19:7203–7215 [View Article] [PubMed]
    [Google Scholar]
  16. Alonas E, Lifland AW, Gudheti M, Vanover D, Jung J et al. Combining single RNA sensitive probes with subdiffraction-limited and live-cell imaging enables the characterization of virus dynamics in cells. ACS Nano 2014; 8:302–315 [View Article] [PubMed]
    [Google Scholar]
  17. San-Juan-Vergara H, Sampayo-Escobar V, Reyes N, Cha B, Pacheco-Lugo L et al. Cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells. J Virol 2012; 86:1832–1843 [View Article] [PubMed]
    [Google Scholar]
  18. Churchill L, Chilton FH, Resau JH, Bascom R, Hubbard WC et al. Cyclooxygenase metabolism of endogenous arachidonic acid by cultured human tracheal epithelial cells. Am Rev Respir Dis 1989; 140:449–459 [View Article] [PubMed]
    [Google Scholar]
  19. Bilawchuk LM, Griffiths CD, Jensen LD, Elawar F, Marchant DJ. The susceptibilities of respiratory syncytial virus to nucleolin receptor blocking and antibody neutralization are dependent upon the method of virus purification. Viruses 2017; 9:207 [View Article] [PubMed]
    [Google Scholar]
  20. Jamieson KC, Wiehler S, Michi AN, Proud D. Rhinovirus induces basolateral release of IL-17C in highly differentiated airway epithelial cells. Front Cell Infect Microbiol 2020; 10:103 [View Article] [PubMed]
    [Google Scholar]
  21. Michi AN, Proud D. A toolbox for studying respiratory viral infections using air-liquid interface cultures of human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 2021; 321:L263–L280 [View Article] [PubMed]
    [Google Scholar]
  22. Elawar F, Griffiths CD, Zhu D, Bilawchuk LM, Jensen LD et al. A virological and phylogenetic analysis of the emergence of new clades of respiratory syncytial virus. Sci Rep 2017; 7:12232 [View Article] [PubMed]
    [Google Scholar]
  23. Hillyer P, Shepard R, Uehling M, Krenz M, Sheikh F et al. Differential responses by human respiratory epithelial cell lines to respiratory syncytial virus reflect distinct patterns of infection control. J Virol 2018; 92:15 [View Article] [PubMed]
    [Google Scholar]
  24. Li Y, Dinwiddie DL, Harrod KS, Jiang Y, Kim KC. Anti-inflammatory effect of MUC1 during respiratory syncytial virus infection of lung epithelial cells in vitro. Am J Physiol Lung Cell Mol Physiol 2010; 298:L558–63 [View Article] [PubMed]
    [Google Scholar]
  25. Rajan A, Piedra F-A, Aideyan L, McBride T, Robertson M et al. Multiple respiratory syncytial virus (RSV) strains infecting HEp-2 and A549 cells reveal cell line-dependent differences in resistance to RSV infection. J Virol 2022; 96:e0190421 [View Article] [PubMed]
    [Google Scholar]
  26. Firsching R, Buchholz CJ, Schneider U, Cattaneo R, ter Meulen V et al. Measles virus spread by cell-cell contacts: uncoupling of contact-mediated receptor (CD46) downregulation from virus uptake. J Virol 1999; 73:5265–5273 [View Article] [PubMed]
    [Google Scholar]
  27. Hovanessian AG, Puvion-Dutilleul F, Nisole S, Svab J, Perret E et al. The cell-surface-expressed nucleolin is associated with the actin cytoskeleton. Exp Cell Res 2000; 261:312–328 [View Article] [PubMed]
    [Google Scholar]
  28. Jin YJ, Cai CY, Zhang X, Zhang HT, Hirst JA et al. HIV Nef-mediated CD4 down-regulation is adaptor protein complex 2 dependent. J Immunol 2005; 175:3157–3164 [View Article] [PubMed]
    [Google Scholar]
  29. Stove V, Van de Walle I, Naessens E, Coene E, Stove C et al. Human immunodeficiency virus Nef induces rapid internalization of the T-cell coreceptor CD8alphabeta. J Virol 2005; 79:11422–11433 [View Article] [PubMed]
    [Google Scholar]
  30. Lei Y, Zhang J, Schiavon CR, He M, Chen L et al. SARS-CoV-2 spike protein impairs endothelial function via downregulation of ACE 2. Circ Res 2021; 128:1323–1326 [View Article] [PubMed]
    [Google Scholar]
  31. Paluck A, Osan J, Hollingsworth L, Talukdar SN, Saegh AA et al. Role of ARP2/3 complex-driven actin polymerization in RSV infection. Pathogens 2021; 11:26 [View Article] [PubMed]
    [Google Scholar]
  32. Mehedi M, McCarty T, Martin SE, Le Nouën C, Buehler E et al. Actin-related protein 2 (ARP2) and virus-induced filopodia facilitate human respiratory syncytial virus spread. PLoS Pathog 2016; 12:e1006062 [View Article] [PubMed]
    [Google Scholar]
  33. Jin J, Shen Y, Zhang B, Deng R, Huang D et al. In situ exploration of characteristics of macropinocytosis and size range of internalized substances in cells by 3D-structured illumination microscopy. Int J Nanomedicine 2018; 13:5321–5333 [View Article] [PubMed]
    [Google Scholar]
  34. Haigler HT, McKanna JA, Cohen S. Rapid stimulation of pinocytosis in human carcinoma cells A-431 by epidermal growth factor. J Cell Biol 1979; 83:82–90 [View Article] [PubMed]
    [Google Scholar]
  35. Sorkin A, Goh LK. Endocytosis and intracellular trafficking of ErbBs. Exp Cell Res 2008; 314:3093–3106 [View Article] [PubMed]
    [Google Scholar]
  36. Yamazaki T, Zaal K, Hailey D, Presley J, Lippincott-Schwartz J et al. Role of Grb2 in EGF-stimulated EGFR internalization. J Cell Sci 2002; 115:1791–1802 [View Article] [PubMed]
    [Google Scholar]
  37. Krndija D, Fairhead M. IGF1R undergoes active and directed centripetal transport on filopodia upon receptor activation. Biochem J 2019; 476:3583–3593 [View Article] [PubMed]
    [Google Scholar]
  38. Laird MHW, Rhee SH, Perkins DJ, Medvedev AE, Piao W et al. TLR4/MyD88/PI3K interactions regulate TLR4 signaling. J Leukoc Biol 2009; 85:966–977 [View Article] [PubMed]
    [Google Scholar]
  39. Marchant D, Singhera GK, Utokaparch S, Hackett TL, Boyd JH et al. Toll-like receptor 4-mediated activation of p38 mitogen-activated protein kinase is a determinant of respiratory virus entry and tropism. J Virol 2010; 84:11359–11373 [View Article] [PubMed]
    [Google Scholar]
  40. Rallabhandi P, Phillips RL, Boukhvalova MS, Pletneva LM, Shirey KA et al. Respiratory syncytial virus fusion protein-induced toll-like receptor 4 (TLR4) signaling is inhibited by the TLR4 antagonists Rhodobacter sphaeroides lipopolysaccharide and eritoran (E5564) and requires direct interaction with MD-2. mBio 2012; 3:e00218-12 [View Article] [PubMed]
    [Google Scholar]
  41. Rezende W, Ye X, Angelo LS, Carisey AF, Avadhanula V et al. The efficiency of P27 cleavage during in vitro respiratory syncytial virus (RSV) infection is cell line and RSV subtype dependent. J Virol 2023; 97:e0025423 [View Article]
    [Google Scholar]
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