Mutants defective in herpes simplex virus type 2 ICP4: isolation and preliminary characterization

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Herpes simplex virus type 2 (HSV-2) is a significant human pathogen.
• The ICP4 protein is a crucial regulatory protein for HSV replication.
• Understanding HSV-2 ICP4 function is key to developing antiviral strategies.

Purpose of the Study:

• To isolate and characterize HSV-2 mutants with defects in the ICP4 gene.
• To investigate the role of HSV-2 ICP4 in viral replication using transformed cell lines.
• To assess the functional complementation between HSV-1 and HSV-2 ICP4 genes.

Main Methods:

• Biochemical transformation of Vero cells with the HSV-2 ICP4 gene.
• Isolation and characterization of HSV-2 mutants (hr259 and hr79) defective in ICP4.
• Growth assays and viral protein analysis in wild-type, mutant, and transformed cells.
• Marker rescue experiments to map mutations within the ICP4 gene.

Main Results:

• Two HSV-2 mutants, hr259 (ICP4 deletion) and hr79 (C-terminal ICP4 mutation), were isolated.
• Both mutants failed to grow in non-transformed Vero cells but replicated efficiently in cells expressing HSV-2 ICP4.
• hr259 produced no detectable ICP4 mRNA, while hr79 produced a truncated ICP4 mRNA.
• Both mutants grew well in cells expressing HSV-1 ICP4, and an HSV-1 ICP4 deletion mutant grew in HSV-2 ICP4-expressing cells.

Conclusions:

• The ICP4 gene of HSV-2 is essential for viral replication in non-permissive cells.
• Mutations affecting ICP4 mRNA start site or carboxy-terminal domain impair viral growth.
• There is functional cross-complementation between HSV-1 and HSV-2 ICP4 proteins, indicating conserved functions.