Linker insertion mutations in the herpes simplex virus type 1 UL28 gene: effects on UL28 interaction with UL15 and UL33 and identification of a second-site mutation in the UL15 gene that suppresses a lethal UL28 mutation.
The UL28 protein of herpes simplex virus type 1 (HSV-1) is one of seven viral proteins required for the cleavage and packaging of viral DNA. Previous results indicated that UL28 interacts with UL15 and UL33 to form a protein complex (terminase) that is presumed to cleave concatemeric DNA into genome lengths. In order to define the functional domains of UL28 that are important for DNA cleavage/packaging, we constructed a series of HSV-1 mutants with linker insertion and nonsense mutations in UL28. Insertions that blocked DNA cleavage and packaging were found to be located in two regions of UL28: the first between amino acids 200 to 400 and the second between amino acids 600 to 740. Insertions located in the N terminus or in a region located between amino acids 400 and 600 did not affect virus replication. Insertions in the carboxyl terminus of the UL28 protein were found to interfere with the interaction of UL28 with UL33. In contrast, all of the UL28 insertion mutants were found to interact with UL15 but the interaction was reduced with mutants that failed to react with UL33. Together, these observations were consistent with previous conclusions that UL15 and UL33 interact directly with UL28 but interact only indirectly with each other. Revertant viruses that formed plaques on Vero cells were detected for one of the lethal UL28 insertion mutants. DNA sequence analysis, in combination with genetic complementation assays, demonstrated that a second-site mutation in the UL15 gene restored the ability of the revertant to cleave and package viral DNA. The isolation of an intergenic suppressor mutant provides direct genetic evidence of an association between the UL28 and UL15 proteins and demonstrates that this association is essential for DNA cleavage and packaging.
Jacobson
Jennie G
JG
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, W1256 Biomedical Science Tower, Pittsburgh, PA 15261, USA. flhoma@pitt.edu
Yang
Kui
K
Baines
Joel D
JD
Homa
Fred L
FL
D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D002522:Chlorocebus aethiops; D042003:DNA Packaging / Q000235:genetics; D005816:Genetic Complementation Test; D018259:Herpesvirus 1, Human / Q000235:genetics*; D015151:Immunoblotting; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
C066670:UL33 protein, Human herpesvirus 1; D014764:Viral Proteins; C069185:DNA cleavage and packaging proteins, Herpesvirus