1887

Abstract

The fusion (F) protein is an important membrane glycoprotein necessary for cellular entry and replication of human metapneumovirus (hMPV). Selective prevention of -linked glycosylation may compromise the catalytic and fusion functions of the F protein. By using site-directed mutagenesis and reverse genetics, recombinant mutant viruses lacking one or two -linked glycosylation sites in the F protein were constructed. M1, which lacked glycosylation at position 57 of the F protein, had slightly compromised replication, whereas M2 and M4, which lacked glycosylation at position(s) 172 or 57 and 172, respectively, showed profound impairment of replication when compared with wild-type (WT) NL/1/00–GFP virus in both Vero E6 cells and mouse lungs. M2 was less fit than WT virus and in immunocompromised mouse lungs. The F proteins of WT and mutant viruses were similarly expressed on the infected cell membrane, while the activated fusion protein subunits, F1 of M2 and M4, were produced in lower quantities compared with those of WT and M1 virus. The mutated viruses lacking -linked glycosylation at position 353, either individually or together with other sites, could not be recovered. Thus, -linked glycosylation may be involved in the catalysis of the fusion protein from F0 to F1 and F2, which is critical for fusion function. Strategies targeting -linked glycosylation may be helpful for developing attenuated live vaccines or antiviral drugs for hMPV.

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2011-07-01
2024-03-28
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References

  1. Biacchesi S., Skiadopoulos M. H., Yang L., Lamirande E. W., Tran K. C., Murphy B. R., Collins P. L., Buchholz U. J. 2004; Recombinant human metapneumovirus lacking the small hydrophobic SH and/or attachment G glycoprotein: deletion of G yields a promising vaccine candidate. J Virol 78:12877–12887 [View Article][PubMed]
    [Google Scholar]
  2. Biacchesi S., Pham Q. N., Skiadopoulos M. H., Murphy B. R., Collins P. L., Buchholz U. J. 2005; Infection of nonhuman primates with recombinant human metapneumovirus lacking the SH, G, or M2-2 protein categorizes each as a nonessential accessory protein and identifies vaccine candidates. J Virol 79:12608–12613 [View Article][PubMed]
    [Google Scholar]
  3. Boivin G., Abed Y., Pelletier G., Ruel L., Moisan D., Côté S., Peret T. C., Erdman D. D., Anderson L. J. 2002; Virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups. J Infect Dis 186:1330–1334 [View Article][PubMed]
    [Google Scholar]
  4. Buchholz U. J., Finke S., Conzelmann K. K. 1999; Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J Virol 73:251–259[PubMed]
    [Google Scholar]
  5. Cattaneo R., Rose J. K. 1993; Cell fusion by the envelope glycoproteins of persistent measles viruses which caused lethal human brain disease. J Virol 67:1493–1502[PubMed]
    [Google Scholar]
  6. Chen X., Zhang Z. Y., Zhao Y., Liu E. M., Zhao X. D. 2010; Acute lower respiratory tract infections by human metapneumovirus in children in Southwest China: a 2-year study. Pediatr Pulmonol 45:824–831 [View Article][PubMed]
    [Google Scholar]
  7. Cimolai N., Taylor G. P., Mah D., Morrison B. J. 1992; Definition and application of a histopathological scoring scheme for an animal model of acute Mycoplasma pneumoniae pulmonary infection. Microbiol Immunol 36:465–478[PubMed] [CrossRef]
    [Google Scholar]
  8. de Graaf M., Herfst S., Schrauwen E. J., van den Hoogen B. G., Osterhaus A. D., Fouchier R. A. 2007; An improved plaque reduction virus neutralization assay for human metapneumovirus. J Virol Methods 143:169–174 [View Article][PubMed]
    [Google Scholar]
  9. Deffrasnes C., Hamelin M. E., Prince G. A., Boivin G. 2008; Identification and evaluation of a highly effective fusion inhibitor for human metapneumovirus. Antimicrob Agents Chemother 52:279–287 [View Article][PubMed]
    [Google Scholar]
  10. Deng R., Wang Z., Mirza A. M., Iorio R. M. 1995; Localization of a domain on the paramyxovirus attachment protein required for the promotion of cellular fusion by its homologous fusion protein spike. Virology 209:457–469 [View Article][PubMed]
    [Google Scholar]
  11. Ebata S. N., Côté M. J., Kang C. Y., Dimock K. 1991; The fusion and hemagglutinin-neuraminidase glycoproteins of human parainfluenza virus 3 are both required for fusion. Virology 183:437–441 [View Article][PubMed]
    [Google Scholar]
  12. Herfst S., de Graaf M., Schickli J. H., Tang R. S., Kaur J., Yang C. F., Spaete R. R., Haller A. A., van den Hoogen B. G. et al. 2004; Recovery of human metapneumovirus genetic lineages A and B from cloned cDNA. J Virol 78:8264–8270 [View Article][PubMed]
    [Google Scholar]
  13. Horvath C. M., Lamb R. A. 1992; Studies on the fusion peptide of a paramyxovirus fusion glycoprotein: roles of conserved residues in cell fusion. J Virol 66:2443–2455[PubMed]
    [Google Scholar]
  14. Jeffrey S. K. 2006; Epidemiology of human metapneumovirus. Clin Microbiol Rev 19:546–557[PubMed] [CrossRef]
    [Google Scholar]
  15. Johnson R. A., Prince G. A., Suffin S. C., Horswood R. L., Chanock R. M. 1982; Respiratory syncytial virus infection in cyclophosphamide-treated cotton rats. Infect Immun 37:369–373[PubMed]
    [Google Scholar]
  16. Miller S. A., Tollefson S., Crowe J. E. Jr, Williams J. V., Wright D. W. 2007; Examination of a fusogenic hexameric core from human metapneumovirus and identification of a potent synthetic peptide inhibitor from the heptad repeat 1 region. J Virol 81:141–149 [View Article][PubMed]
    [Google Scholar]
  17. Olszewska-Pazdrak B., Casola A., Saito T., Alam R., Crowe S. E., Mei F., Ogra P. L., Garofalo R. P. 1998; Cell-specific expression of RANTES, MCP-1, and MIP-1α by lower airway epithelial cells and eosinophils infected with respiratory syncytial virus. J Virol 72:4756–4764[PubMed]
    [Google Scholar]
  18. Ottolini M. G., Curtis S. R., Mathews A., Ottolini S. R., Prince G. A. 2002; Palivizumab is highly effective in suppressing respiratory syncytial virus in an immunosuppressed animal model. Bone Marrow Transplant 29:117–120 [View Article][PubMed]
    [Google Scholar]
  19. Schowalter R. M., Smith S. E., Dutch R. E. 2006; Characterization of human metapneumovirus F protein-promoted membrane fusion: critical roles for proteolytic processing and low pH. J Virol 80:10931–10941 [View Article][PubMed]
    [Google Scholar]
  20. Skiadopoulos M. H., Biacchesi S., Buchholz U. J., Amaro-Carambot E., Surman S. R., Collins P. L., Murphy B. R. 2006; Individual contributions of the human metapneumovirus F, G, and SH surface glycoproteins to the induction of neutralizing antibodies and protective immunity. Virology 345:492–501 [View Article][PubMed]
    [Google Scholar]
  21. Smith E. C., Popa A., Chang A., Masante C., Dutch R. E. 2009; Viral entry mechanisms: the increasing diversity of paramyxovirus entry. FEBS J 276:7217–7227 [View Article][PubMed]
    [Google Scholar]
  22. Tanabayashi K., Compans R. W. 1996; Functional interaction of paramyxovirus glycoproteins: identification of a domain in Sendai virus HN which promotes cell fusion. J Virol 70:6112–6118[PubMed]
    [Google Scholar]
  23. Tong S., Compans R. W. 1999; Alternative mechanisms of interaction between homotypic and heterotypic parainfluenza virus HN and F proteins. J Gen Virol 80:107–115[PubMed]
    [Google Scholar]
  24. van den Hoogen B. G., de Jong J. C., Groen J., Kuiken T., de Groot R., Fouchier R. A., Osterhaus A. D. 2001; A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med 7:719–724 [View Article][PubMed]
    [Google Scholar]
  25. Zhao X., Chen F. P., Megaw A. G., Sullender W. M. 2004; Variable resistance to palivizumab in cotton rats by respiratory syncytial virus mutants. J Infect Dis 190:1941–1946 [View Article][PubMed]
    [Google Scholar]
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