A replication defect of pseudorabies virus induced by targeted α-helix distortion in the syntaxin-like bundle of glycoprotein H (V275P) is corrected by an adjacent compensatory mutation (V271A) Free

Abstract

Glycoprotein gH is essential for herpesvirus-induced membrane fusion during entry and cell-to-cell spread. Structural analyses of gH homologues revealed a conserved syntaxin-like bundle motif composed of three α-helices. Previous studies showed that targeted disruption of any of these helices strongly impaired maturation, cell surface expression and fusion activity of pseudorabies virus gH, as well as formation and spread of infectious virus. After passaging of one corresponding mutant (pPrV-gH-V275P) these replication defects were widely corrected by an adjacent spontaneous amino acid substitution (V271A). Although the doubly mutated gH was still non-functional in fusion assays, its targeted reinsertion into the cloned virus genome (pPrV-gH-V275P-V271A) led to a 200-fold increase in plaque sizes and 10 000-fold higher virus titres, compared with pPrV-gH-V275P. Thus, our results demonstrate that structural requirements for gH function in assays and virus replication are different, and that minor amounts of mature gH in virions are sufficient for productive replication.

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2015-08-01
2024-03-28
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References

  1. Atanasiu D., Saw W.T., Gallagher J.R., Hannah B.P., Matsuda Z., Whitbeck J.C., Cohen G.H., Eisenberg R.J. 2013; Dual split protein-based fusion assay reveals that mutations to herpes simplex virus (HSV) glycoprotein gB alter the kinetics of cell-cell fusion induced by HSV entry glycoproteins. J Virol 87:11332–11345 [View Article][PubMed]
    [Google Scholar]
  2. Backovic M., DuBois R.M., Cockburn J.J., Sharff A.J., Vaney M.C., Granzow H., Klupp B.G., Bricogne G., Mettenleiter T.C., Rey F.A. 2010; Structure of a core fragment of glycoprotein H from pseudorabies virus in complex with antibody. Proc Natl Acad Sci U S A 107:22635–22640 [View Article][PubMed]
    [Google Scholar]
  3. Baghian A., Huang L., Newman S., Jayachandra S., Kousoulas K.G. 1993; Truncation of the carboxy-terminal 28 amino acids of glycoprotein B specified by herpes simplex virus type 1 mutant amb1511-7 causes extensive cell fusion. J Virol 67:2396–2401[PubMed]
    [Google Scholar]
  4. Böhm S.W., Eckroth E., Backovic M., Klupp B.G., Rey F.A., Mettenleiter T.C., Fuchs W. 2015; Structure-based functional analyses of domains II and III of pseudorabies virus glycoprotein H. J Virol 89:1364–1376 [View Article][PubMed]
    [Google Scholar]
  5. Chowdary T.K., Cairns T.M., Atanasiu D., Cohen G.H., Eisenberg R.J., Heldwein E.E. 2010; Crystal structure of the conserved herpesvirus fusion regulator complex gH-gL. Nat Struct Mol Biol 17:882–888 [View Article][PubMed]
    [Google Scholar]
  6. Eisenberg R.J., Atanasiu D., Cairns T.M., Gallagher J.R., Krummenacher C., Cohen G.H. 2012; Herpes virus fusion and entry: a story with many characters. Viruses 4:800–832 [View Article][PubMed]
    [Google Scholar]
  7. Fuchs W., Klupp B.G., Granzow H., Osterrieder N., Mettenleiter T.C. 2002; The interacting UL31 and UL34 gene products of pseudorabies virus are involved in egress from the host-cell nucleus and represent components of primary enveloped but not mature virions. J Virol 76:364–378 [View Article][PubMed]
    [Google Scholar]
  8. Fuchs W., Backovic M., Klupp B.G., Rey F.A., Mettenleiter T.C. 2012; Structure-based mutational analysis of the highly conserved domain IV of glycoprotein H of pseudorabies virus. J Virol 86:8002–8013 [View Article][PubMed]
    [Google Scholar]
  9. Galdiero M., Whiteley A., Bruun B., Bell S., Minson T., Browne H. 1997; Site-directed and linker insertion mutagenesis of herpes simplex virus type 1 glycoprotein H. J Virol 71:2163–2170[PubMed]
    [Google Scholar]
  10. Garnier J., Gibrat J.F., Robson B. 1996; GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540–553 [View Article][PubMed]
    [Google Scholar]
  11. Johnson D.C., Huber M.T. 2002; Directed egress of animal viruses promotes cell-to-cell spread. J Virol 76:1–8 [View Article][PubMed]
    [Google Scholar]
  12. Klupp B.G., Mettenleiter T.C. 1999; Glycoprotein gL-independent infectivity of pseudorabies virus is mediated by a gD-gH fusion protein. J Virol 73:3014–3022[PubMed]
    [Google Scholar]
  13. Klupp B.G., Visser N., Mettenleiter T.C. 1992; Identification and characterization of pseudorabies virus glycoprotein H. J Virol 66:3048–3055[PubMed]
    [Google Scholar]
  14. Klupp B.G., Fuchs W., Weiland E., Mettenleiter T.C. 1997; Pseudorabies virus glycoprotein L is necessary for virus infectivity but dispensable for virion localization of glycoprotein H. J Virol 71:7687–7695[PubMed]
    [Google Scholar]
  15. Klupp B.G., Nixdorf R., Mettenleiter T.C. 2000; Pseudorabies virus glycoprotein M inhibits membrane fusion. J Virol 74:6760–6768 [View Article][PubMed]
    [Google Scholar]
  16. Klupp B., Altenschmidt J., Granzow H., Fuchs W., Mettenleiter T.C. 2008; Glycoproteins required for entry are not necessary for egress of pseudorabies virus. J Virol 82:6299–6309 [View Article][PubMed]
    [Google Scholar]
  17. Matsuura H., Kirschner A.N., Longnecker R., Jardetzky T.S. 2010; Crystal structure of the Epstein-Barr virus (EBV) glycoprotein H/glycoprotein L (gH/gL) complex. Proc Natl Acad Sci U S A 107:22641–22646 [View Article][PubMed]
    [Google Scholar]
  18. Mettenleiter T.C. 2002; Herpesvirus assembly and egress. J Virol 76:1537–1547 [View Article][PubMed]
    [Google Scholar]
  19. Mettenleiter T.C. 2003; Pathogenesis of neurotropic herpesviruses: role of viral glycoproteins in neuroinvasion and transneuronal spread. Virus Res 92:197–206 [View Article][PubMed]
    [Google Scholar]
  20. Nixdorf R., Klupp B.G., Karger A., Mettenleiter T.C. 2000; Effects of truncation of the carboxy terminus of pseudorabies virus glycoprotein B on infectivity. J Virol 74:7137–7145 [View Article][PubMed]
    [Google Scholar]
  21. Peeters B., de Wind N., Broer R., Gielkens A., Moormann R. 1992; Glycoprotein H of pseudorabies virus is essential for entry and cell-to-cell spread of the virus. J Virol 66:3888–3892[PubMed]
    [Google Scholar]
  22. Rauh I., Mettenleiter T.C. 1991; Pseudorabies virus glycoproteins gII and gp50 are essential for virus penetration. J Virol 65:5348–5356[PubMed]
    [Google Scholar]
  23. Schröter C., Klupp B.G., Fuchs W., Gerhard M., Backovic M., Rey F.A., Mettenleiter T.C. 2014; The highly conserved proline at position 438 in pseudorabies virus gH is important for regulation of membrane fusion. J Virol 88:13064–13072 [View Article][PubMed]
    [Google Scholar]
  24. Ungermann C., Langosch D. 2005; Functions of SNAREs in intracellular membrane fusion and lipid bilayer mixing. J Cell Sci 118:3819–3828 [View Article][PubMed]
    [Google Scholar]
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