1887

Abstract

A variant was selected from a clinical isolate of herpes simplex virus type 1 (HSV-1) during a single passage in the presence of a helicase–primase inhibitor (HPI) at eight times the IC. The variant was approximately 40-fold resistant to the HPI BAY 57-1293 and it showed significantly reduced growth in tissue culture with a concomitant reduction in virulence in a murine infection model. The variant contained a single mutation (Asn342Lys) in the UL5 predicted functional helicase motif IV. The Asn342Lys mutation was transferred to a laboratory strain, PDK cl-1, and the recombinant acquired the expected resistance and reduced growth characteristics. Comparative modelling and docking studies predicted the Asn342 position to be physically distant from the HPI interaction pocket formed by UL5 and UL52 (primase). We suggest that this mutation results in steric/allosteric modification of the HPI-binding pocket, conferring an indirect resistance to the HPI. Slower growth and moderately reduced virulence suggest that this mutation might also interfere with the helicase–primase activity.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.011221-0
2009-08-01
2024-12-08
Loading full text...

Full text loading...

/deliver/fulltext/jgv/90/8/1937.html?itemId=/content/journal/jgv/10.1099/vir.0.011221-0&mimeType=html&fmt=ahah

References

  1. Biswas S., Jennens L., Field H. J. 2007a; Single amino acid substitutions in the HSV-1 helicase protein that confer resistance to the helicase-primase inhibitor BAY 57–1293 are associated with increased or decreased virus growth characteristics in tissue culture. Arch Virol 152:1489–1500 [CrossRef]
    [Google Scholar]
  2. Biswas S., Smith C., Field H. J. 2007b; Detection of HSV-1 variants highly resistant to the helicase-primase inhibitor BAY 57-1293 at high frequency in two of ten recent clinical isolates of HSV-1. J Antimicrob Chemother 60:274–279 [CrossRef]
    [Google Scholar]
  3. Biswas S., Field H. J. 2008; Herpes simplex virus helicase-primase inhibitors: recent findings from the study of drug-resistance mutations. Antivir Chem Chemother 19:1–6 [CrossRef]
    [Google Scholar]
  4. Biswas S., Tiley L. S., Zimmermann H., Birkmann A., Field H. J. 2008a; Mutations close to functional motif IV in HSV-1 UL5 helicase that confer resistance to HSV helicase-primase inhibitors, variously affect virus growth rate and pathogenicity. Antiviral Res 80:81–85 [CrossRef]
    [Google Scholar]
  5. Biswas S., Kleymann G., Swift M., Tiley L. S., Lyall J., Aguirre-Hernández J., Field H. J. 2008b; A single drug-resistance mutation in HSV-1 UL52 primase points to a difference between two helicase-primase inhibitors in their mode of interaction with the antiviral target. J Antimicrob Chemother 61:1044–1047 [CrossRef]
    [Google Scholar]
  6. Brooks B. R., Bruccoleri R. E., Olafson B. D., States D. J., Swaminathan S., Karplus M. 1983; charmm: a program for macromolecular energy minimization and dynamics calculations. J Comput Chem 4:187–217 [CrossRef]
    [Google Scholar]
  7. de Bakker P. I. W., Bateman A., Burke D. F., Miguel R. N., Mizuguchi K., Shi J., Shirai H., Blundell T. L. 2001; homstrad: Adding sequence information to structure-based alignments of homologous protein families. Bioinformatics 17:748–749 [CrossRef]
    [Google Scholar]
  8. Dubbs D. R., Kit S. 1964; Mutant strains of herpes simplex deficient in thymidine kinase-inducing activity. Virology 22:493–502 [CrossRef]
    [Google Scholar]
  9. Field H. J., Wildy P. 1978; The pathogenicity of thymidine kinase-deficient mutants of herpes simplex virus in mice. J Hyg (Lond) 81:267–277 [CrossRef]
    [Google Scholar]
  10. Gorbalenya A. E., Koonin E. V. 1993; Helicases: amino acid sequence comparisons and structure–function relationships. Curr Opin Struct Biol 3:419–429 [CrossRef]
    [Google Scholar]
  11. Graves-Woodward K. L., Gottlieb J., Challberg M. D., Weller S. K. 1997; Biochemical analyses of mutations in the HSV-1 helicase-primase that alter ATP hydrolysis, DNA unwinding, and coupling between hydrolysis and unwinding. J Biol Chem 272:4623–4630 [CrossRef]
    [Google Scholar]
  12. Jones S., Thornton J. M. 1996; Principles of protein–protein interactions. Proc Natl Acad Sci U S A 93:13–20 [CrossRef]
    [Google Scholar]
  13. Jones G., Willett P., Glen R. C., Leach A. R., Taylor R. 1997; Development and validation of a genetic algorithm for flexible docking. J Mol Biol 267:727–748 [CrossRef]
    [Google Scholar]
  14. Kleymann G., Fischer R., Betz U. A., Hendrix M., Bender W., Schneider U., Handke G., Eckenber P., Hewlett G. other authors 2002; New helicase–primase inhibitors as drug candidates for the treatment of herpes simplex disease. Nat Med 8:392–398 [CrossRef]
    [Google Scholar]
  15. Laskowski R. A., MacArthur M. W., Moss D. S., Thornton J. M. 1993; procheck: A program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291 [CrossRef]
    [Google Scholar]
  16. Lee B., Richards F. M. 1971; The interpretation of protein structures: estimation of static accessibility. J Mol Biol 55:379–400 [CrossRef]
    [Google Scholar]
  17. Lee J. Y., Yang W. 2006; UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke. Cell 127:1349–1360 [CrossRef]
    [Google Scholar]
  18. Luthy R., Bowie J. U., Eisenberg D. 1992; Assessment of protein models with 3-dimensional profiles. Nature 356:83–85 [CrossRef]
    [Google Scholar]
  19. McDonald I. K., Thornton J. M. 1994; Satisfying hydrogen bonding potential in proteins. J Mol Biol 238:777–793 [CrossRef]
    [Google Scholar]
  20. Mizuguchi K., Deane C. M., Blundell T. L., Johnson M. S., Overington J. P. 1998; joy: protein sequence-structure representation and analysis. Bioinformatics 14:617–623 [CrossRef]
    [Google Scholar]
  21. Nagafuchi S., Oda H., Mori R., Taniguchi T. 1979; Mechanism of acquired resistance to herpes simplex virus infection as studied in nude mice. J Gen Virol 44:715–723 [CrossRef]
    [Google Scholar]
  22. Richmond T. J. 1984; Solvent accessible surface area and excluded volume in proteins. Analytical equations for overlapping spheres and implications for the hydrophobic effect. J Mol Biol 178:63–89 [CrossRef]
    [Google Scholar]
  23. Sali A., Blundell T. L. 1993; Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815 [CrossRef]
    [Google Scholar]
  24. Shi J., Blundell T. L., Mizuguchi K. 2001; fugue: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties. J Mol Biol 310:243–257 [CrossRef]
    [Google Scholar]
  25. Zhu L., Weller S. K. 1988; UL5, a protein required for HSV DNA synthesis: genetic analysis, overexpression in Escherichia coli , and generation of polyclonal antibodies. Virology 166:366–378 [CrossRef]
    [Google Scholar]
  26. Zhu L., Weller S. K. 1992; The six conserved helicase motifs of the UL5 gene product, a component of the herpes simplex virus type 1 helicase primase, are essential for its function. J Virol 66:469–479
    [Google Scholar]
/content/journal/jgv/10.1099/vir.0.011221-0
Loading
/content/journal/jgv/10.1099/vir.0.011221-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
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