A cosmid-based system for constructing mutants of herpes simplex virus type 1

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Herpes simplex virus type 1 (HSV-1) DNA replication is complex.
• Understanding viral DNA origins is crucial for antiviral development.
• Cosmid vectors offer a method for handling large viral DNA fragments.

Purpose of the Study:

• To establish a cosmid-based system for generating and isolating defined herpes simplex virus type 1 mutants.
• To investigate the role of viral DNA replication origins (oriL) using cosmid clones.
• To construct and analyze mutants in essential and non-essential viral genes.

Main Methods:

• Preparation of overlapping cosmid libraries representing the entire HSV-1 genome.
• Excising intact cosmid inserts using PacI restriction endonuclease.
• Transfection of cosmid DNA into cultured cells to generate viral plaques.
• Construction of frameshift mutations in specific viral genes (UL2, UL44, UL33).

Main Results:

• Cosmid sets successfully generated infectious HSV-1, with variations in progeny depending on the presence of intact (ori+L) or deleted (ori-L) replication origins.
• Cosmids containing oriL in non-overlapping regions generated only ori-L virus.
• Mutants in non-essential genes (UL2, UL44) and an essential gene (UL33) were successfully constructed and isolated.

Conclusions:

• A cosmid-based system provides a powerful tool for the targeted isolation of large numbers of defined herpes simplex virus type 1 mutants.
• This system facilitates the study of viral DNA replication origins and gene function.
• The methodology aids in generating specific viral mutants for further research and potential therapeutic strategies.