A second-site revertant of a defective herpes simplex virus ICP4 protein with restored regulatory activities and impaired DNA-binding properties.
A mutant of herpes simplex virus type 1, vi12, encodes a DNA-binding- and transactivation-deficient ICP4 polypeptide. Because of the mutation, the vi12 virus does not grow on Vero cells but must be propagated on cells that express complementing levels of wild-type ICP4 (E5 cells). A pseudorevertant of vi12, designated pri12, was isolated on the basis of the restored ability to replicate on Vero cells. In addition to the original i12 insertion mutation at amino acid 320, the ICP4 molecule expressed from pri12 possesses an alanine to valine substitution at amino acid 342 within the ICP4 gene. The infectivity of pri12 on Vero cells as measured by burst size is elevated by 5 orders of magnitude relative to that observed for vi12, reflecting the restored ability of the mutant ICP4 molecule possessing the alanine to valine substitution to activate transcription and thus support viral replication. Despite the restored regulatory activities of the pri12 ICP4 molecule, the ability of the pseudorevertant ICP4 molecule to form a high-affinity, specific interaction with the consensus binding site was still impaired relative to that of wild-type ICP4. This observation suggests that the in vitro-measured DNA-binding properties of ICP4 may not reflect the functional interactions occurring in vivo that mediate transcriptional activation.
Shepard
A A
AA
Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
DeLuca
N A
NA
D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D003673:Defective Viruses / Q000235:genetics* / Q000378:metabolism; D005838:Genotype; D017874:Immediate-Early Proteins; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D018139:Simplexvirus / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014709:Vero Cells; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics* / Q000378:metabolism
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D054334:Viral Regulatory and Accessory Proteins; C048925:herpes simplex virus, type 1 protein ICP4