Initiation of herpes simplex virus thymidine kinase polypeptides

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• The herpes simplex virus type 1 thymidine kinase (HSV1 tk) gene is used as a reporter transgene.
• Ectopic expression in mouse testis leads to N-terminally truncated translation products (P2, P3) due to mRNA 5'-end deficiencies.

Purpose of the Study:

• To elucidate the precise initiation codons responsible for the synthesis of truncated HSV1 tk polypeptides (P2, P3, P4) in mouse testis.
• To investigate the role of specific ATG and non-ATG codons in HSV1 tk translation initiation.

Main Methods:

• Generation of modified HSV1 tk genes with single intact ATG codons (ATG1, ATG46, ATG60) or modified non-ATG initiation codons.
• Transfection of cells with modified tk genes and analysis of protein products using SDS-PAGE.
• Assessment of Ganciclovir sensitivity and thymidine kinase (TK) activity in transfected cells.

Main Results:

• Full-length P1 and P2 produced when only ATG1 was intact.
• P2 and P3 produced when only ATG46 was intact, indicating P3 initiates at ATG46.
• P2 and P4 produced when only ATG60 was intact, indicating P4 initiates at ATG60.
• P2 production ceased when putative non-ATG initiation codons were modified, suggesting P2 initiates at a non-ATG codon.
• P1 and P3 possess TK activity, while P4 likely lacks it.

Conclusions:

• P3 is initiated at ATG46, P4 at ATG60, and P2 at a non-ATG codon.
• The study clarifies the complex translation initiation of HSV1 tk in a specific biological context.
• Understanding these initiation events is crucial for accurate interpretation of reporter gene expression and TK activity.