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Random screening for dominant-negative mutants of the cytomegalovirus nuclear egress protein M50

Brigitte Rupp1, Zsolt Ruzsics, Christopher Buser

  • 1Max von Pettenkofer Institut für Virologie, Ludwig-Maximilians-Universität München, Pettenkoferstrasse 9a, 80336 Munich, Germany.

Journal of Virology
|March 23, 2007
PubMed

Insights

Dominant-negative mutants in murine cytomegalovirus M50 gene were identified using a two-step screen. These mutants revealed a proline-rich motif essential for human cytomegalovirus capsid nuclear egress.

Area of Science:

  • Virology
  • Molecular Biology
  • Genetics

Background:

  • Dominant-negative (DN) mutants are crucial for assigning protein functions by inhibiting gene products.
  • Murine cytomegalovirus (MCMV) essential gene M50 serves as a model for functional analysis.

Purpose of the Study:

  • To develop and validate a two-step random screening procedure for identifying DN alleles.
  • To elucidate the function of the MCMV M50 protein and its human cytomegalovirus (HCMV) homolog UL50.

Main Methods:

  • A linker-scanning library was screened for loss-of-function mutants.
  • FLP-mediated ectopic insertion was used to test mutants for inhibition of virus reconstitution.
  • Conditional expression confirmed DN candidates and quantified inhibitory effects.

Main Results:

  • A two-step screening procedure successfully identified DN mutants of MCMV M50.
  • The study identified a morphogenesis block and constructed improved DN mutants.
  • A DN mutant of the homologous HCMV UL50 gene was constructed, predicting a novel functional site.

Conclusions:

  • A proline-rich motif in M50/UL50 is essential for cytomegalovirus capsid nuclear egress.
  • The developed screening method is effective for identifying DN alleles and functional sites in essential viral genes.

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