Transient expression of deletion mutants of the herpes simplex virus thymidine kinase-encoding gene in mouse fibroblast cells

Previous studies have shown that at least three polypeptides of 43, 39 and 38 kDa are translated from separate AUG codons of the thymidine kinase (TK) encoding mRNA of herpes simplex virus type 1. In addition, small tk-specific transcripts initiated within the tk coding region were observed. However, functional activity of these three proteins and their role in establishing of the TK+ cell phenotype is not yet clear. In order to locate the 5' boundary of the gene encoding functionally active TK, we constructed a set of deletion mutants with truncated 5' ends and examined their ability to provide a TK+ phenotype after microinjection into nuclei of LTK- cells. The results demonstrate that nucleotide sequences upstream from the second ATG codon can be removed without affecting the TK+ phenotype. Deletion of the second start codon and its downstream region inactivates the TK function. Those deletion mutants which contain only the third ATG codon are TK-. Thus, the 38-kDa polypeptide that initiates at the third start codon is not endowed with the TK+ activity. Constructs containing deletions up to nt +210 and lacking all 5'-end canonical and aberrant transcription control regions, as well as first start codon, can provide the TK+ function.