Construction of a rationally designed human cytomegalovirus variant encoding a temperature-sensitive immediate-early 2 protein.
We generated a set of cysteine-to-glycine mutations and screened them to identify a temperature-sensitive allele of the human cytomegalovirus UL122 gene, which encodes the immediate-early 2 transcriptional activating protein. The mutant allele contains a single base pair substitution at amino acid 510. In transcription activation assays, the mutant protein activated the simian virus 40 early and human cytomegalovirus UL112 promoters at 32.5 degrees C but not at 39.5 degrees C. We constructed a mutant virus, BTNtsUL122, in which the wild-type UL122 locus is substituted with the mutant allele. The mutant produced progeny at 32.5 degrees C but not at 39.5 degrees C. Although the mutant virus accumulated immediate-early transcripts and proteins at the nonpermissive temperature, it did not produce any early (UL44 and UL54) and late (UL82) transcripts and it did not replicate its DNA. The mutant's defect at the nonpermissive temperature results, at least in part, from the inability of the temperature-sensitive immediate-early 2 protein to activate early viral promoters, whose products are required for DNA replication and progression into the late phase of the virus growth cycle.
Heider
Julie A
JA
Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014, USA.
Bresnahan
Wade A
WA
Shenk
Thomas E
TE
D019943:Amino Acid Substitution; D003545:Cysteine / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D014644:Genetic Variation; D005998:Glycine / Q000235:genetics; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008562:Membrane Glycoproteins; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D013696:Temperature; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014407:Tumor Cells, Cultured; D014759:Viral Envelope Proteins; D014764:Viral Proteins
D004279:DNA, Viral; C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D012367:RNA, Viral; D015534:Trans-Activators; C077406:UL115 protein, Human herpesvirus 5; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C071440:glycoprotein H, Cytomegalovirus; C499358:glycoprotein H, Human cytomegalovirus; C439068:glycoprotein O, cytomegalovirus; D003545:Cysteine; D005998:Glycine