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A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation
Published on: April 2, 2012
Temperature-dependent conformational changes in herpes simplex virus ICP4 that affect transcription activation
1Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Journal of Virology
|February 14, 2003
Summary
Mutations in herpes simplex virus type 1 ICP4 protein impair its activator function and viral growth. These altered ICP4 proteins bind DNA but exhibit conformational changes affecting transcription activation and gene expression.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Herpes simplex virus type 1 (HSV-1) infected cell protein 4 (ICP4) is crucial for viral replication and transcription.
- The C-terminal domain of ICP4 mediates interactions essential for its role as a transcriptional activator.
- Understanding ICP4's structure-function relationship is key to deciphering HSV-1 pathogenesis.
Purpose of the Study:
- To investigate the functional significance of conserved, surface-exposed amino acid regions in the C-terminus of HSV-1 ICP4.
- To characterize the impact of specific mutations on ICP4's transcriptional activator function and viral growth.
- To elucidate the molecular mechanisms underlying ICP4's role in regulating viral gene expression.
Main Methods:
- Oligonucleotide mutagenesis was employed to introduce alanine substitutions in conserved regions of HSV-1 ICP4 (residues 1000-1200).
- Mutant ICP4 proteins were analyzed in transient transfection assays and within the viral genome.
- Phenotypic analysis included viral growth assays at nonpermissive temperatures and electrophoretic mobility shift assays (EMSAs) to assess DNA binding and complex formation.
Main Results:
- Seven ICP4 mutants exhibited varying degrees of functional impairment.
- Mutants M1, M3, and M7 displayed temperature-sensitive growth defects, overproducing immediate-early (IE) proteins at the nonpermissive temperature.
- While all mutants retained DNA-binding ability, EMSAs revealed altered DNA-protein complex mobility, suggesting conformational changes in mutant ICP4 at the nonpermissive temperature.
Conclusions:
- Mutations in specific C-terminal regions of ICP4 lead to conformational alterations that impair transcriptional activation and viral growth.
- The observed defects in IE protein regulation and DNA-protein complex mobility highlight the complex regulatory role of ICP4.
- These findings underscore the importance of ICP4's structural integrity for efficient viral replication and gene expression modulation.
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