Key Points
- Identify the specific nucleotide sequences and regulatory domains responsible for directing the temporal expression of early (beta) versus late (gamma 2) genes in herpes simplex virus 1.
- Engineered recombinant herpes simplex virus 1 strains containing chimeric constructs that replaced regions of the early thymidine kinase (tk) gene with specific regulatory sequences from a late (gamma 2) gene.
- Evaluated gene expression phenotypes based on timing of viral gene activation (early vs. late) and expression sensitivity in the presence of phosphonoacetate, an inhibitor of viral DNA synthesis.
- Replacement of tk nucleotides -200 to +51 with gamma 2 nucleotides -77 to +104 conferred all characteristic attributes of late genes, including full sensitivity to phosphonoacetate.
- A chimeric gene fusing tk upstream of nucleotide -16 to gamma 2 downstream of -12 exhibited a hybrid phenotype, showing expression early and late in infection alongside partial resistance to phosphonoacetate.
- Insertion of gamma 2 nucleotides +17 to +104 into the 5' transcribed noncoding domain of tk enabled late gene expression, demonstrating that gamma 2 regulation requires sequences downstream from the TATAA box.
Structured PICO
PPopulationVero cells and human 143TK- cells infected with wild-type herpes simplex virus 1 (HSV-1) and recombinant viruses
IInterventionConstruction of recombinant viruses with chimeric tk genes (replacing regions of the thymidine kinase gene with specific domains of a gamma 2 gene)
CComparatorWild-type HSV-1(F) and other recombinant constructs
OOutcomeThymidine kinase (TK) enzyme activity and mRNA expression levels (early vs late infection, and sensitivity to phosphonoacetate)surrogate
In HSV-1, the sequence conferring late (gamma 2) gene regulation is located downstream from the TATAA box, contrasting with early (beta) genes where regulatory domains are mainly upstream.