Temperature-sensitive mutants in herpes simplex virus type 1 ICP4 permissive for early gene expression

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Herpes simplex virus type 1 (HSV-1) immediate-early protein ICP4 is crucial for viral gene regulation.
• Temperature-sensitive (ts) mutants in the ICP4 gene typically overproduce immediate-early proteins and underproduce early/late proteins.

Purpose of the Study:

• To genetically and molecularly characterize two unique HSV-1 regulatory mutants, ts48 and ts303, affecting the ICP4 gene.
• To investigate the specific impact of these mutations on viral gene expression, particularly early gene function.

Main Methods:

• Complementation analysis and marker rescue experiments to genetically map mutations.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to analyze protein synthesis at permissive and non-permissive temperatures.
• Trans-induction assays using chloramphenicol acetyltransferase (CAT) reporter gene under early gene control.
• Analysis of viral DNA synthesis.

Main Results:

• Mutations in ts48 and ts303 were mapped to the 3' portion of the ICP4 coding sequence.
• At non-permissive temperatures, ts48 and ts303 overproduced ICP4 and ICP27 but allowed synthesis of early proteins (ICP6, ICP8, gB).
• Trans-induction assays showed ts48 and ts303 could induce CAT activity similar to wild-type, indicating intact early gene regulation.
• Viral DNA synthesis was partially restored (15-50% of wild-type) in ts48 and ts303 infected cells.

Conclusions:

• Mutants ts48 and ts303 possess defects in a regulatory event occurring after immediate-early and early gene expression.
• These mutants exhibit distinct phenotypes compared to other ICP4 mutants, suggesting a specific role for the C-terminal region of ICP4 in later stages of viral gene regulation and DNA replication.