Details

Autor(en) / Beteiligte

• HUFF, VICKI DOLORES

Titel

GENERATION AND USE OF A GLYCOPROTEIN B AMBER MUTANT OF HERPES SIMPLEX VIRUS TYPE 1

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

1987

Link zum Volltext

• ProQuest

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• Amber termination mutants contain a mutation in the coding region of a gene which creates a codon specifying the termination of protein synthesis. Translation of the mutant gene is prematurely terminated at the amber termination codon, resulting in a truncated protein. In the presence of an amber codon-suppressing tRNA, an amino acid is inserted at the amber codon and a protein of normal length is produced. The existence of effective suppressor systems in prokaryotes and thus the ability to propagate otherwise lethal mutants, has made termination mutants invaluable tools in prokaryotic genetics. Recent developments in site-directed mutagenesis and mammalian suppressor systems have made it feasible to generate amber mutants of animal viruses. To investigate ways to generate and use amber mutants in viral research, amber mutations were introduced into the herpes simplex virus type 1 (HSV-1) gene encoding the glycoprotein, gB. Located in the viral envelope, gB is involved in the entry of virus into host cells and is essential for the production of infectious virus particles. Amber mutations were introduced into cloned gB gene by oligonucleotide-directed mutagenesis and Xba I linker insertion. A virus, ambB1, was generated which contained an amber mutation in the cytoplasmic domain-encoding portion of the gB gene. Translation of the mutant gene was predicted to produce a gB molecule missing the final 41 amino acids, or 38% of the putative cytoplasmic domain. Surprisingly, AmbB1 was viable under non-suppressing conditions. Additionally, no alterations in viral plaque morphology was observed and the rate at which ambB1 virions entered host cells was normal. Analysis of cyanogen bromide peptides from ambB1 gB indicated that a truncated protein was synthesized. These data suggest that truncation of the final 41 amino acids of gB does not affect its function. This work demonstrates ways in which amber codons can be introduced at specific locations within a gene and the resulting mutants can be used to investigate protein function. This and other work, along with the recent development of effective suppressing host cells, will further the use of termination mutants in viral research.