Characterization of linker insertion and point mutations in the NS-1 gene of minute virus of mice: effects on DNA replication and transcriptional activation functions of NS-1
| Autor: | Mario H. Skiadopoulos, Wey Liang Leong, Ralph Salvino, Emmanuel A. Faust |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
DNA Replication
Gene Expression Regulation Viral Transcription Genetic Blotting Western Molecular Sequence Data Restriction Mapping Eukaryotic DNA replication Viral Nonstructural Proteins Transfection Cell Line DNA replication factor CDT1 Mice Replication factor C Capsid Control of chromosome duplication Virology Animals Amino Acid Sequence Cloning Molecular Promoter Regions Genetic Replication protein A biology Base Sequence Viral Core Proteins Ter protein Genetic Complementation Test DNA replication Molecular biology Mutagenesis Insertional Phenotype DNA Viral Mutation biology.protein Minute Virus of Mice Mutagenesis Site-Directed Trans-Activators Origin recognition complex |
| Zdroj: | Virology. 188(1) |
| ISSN: | 0042-6822 |
| Popis: | The NS-1 gene of minute virus of mice encodes a multifunctional protein required for replication of the viral genome and for transcriptional regulation of the two MVM promoters. To study the localization of activities required for DNA replication and transactivation of the capsid gene promoter, insertion and point mutations were introduced into the NS-1 gene. The mutant NS-1 genes were expressed in COS-7 cells by using an SV 40 promoter driven NS-1 expression vector. The ability of the mutant proteins to complement a replication defective NS-1 mutant of the infectious MVM plasmid pMM984 and to activate transcription from the capsid gene promoter in chloramphenicol acetyl transferase expression assays was determined. Two point mutations Ser-249 to Ala and Lys-250 to Gin and a one amino acid insertion between Asp-606 and Leu-607 had no effect on viral DNA replication and transactivation activities. Six independent insertions of between 2 and 12 amino acids inhibited the DNA replication activity of NS-1 between 20-and at least 100-fold. There was no apparent correlation between the extent of inhibition of parvoviral DNA replication and the location of the mutations. The transcriptional activation function of NS-1 was inhibited between 1.5- and at least 20-fold and was therefore overall relatively less sensitive to mutagenesis than was its DNA replication function. An exception to this was a 5 amino acid insertion between Tyr-543 and Gin-544 that abolished transactivation as well as the ability of NS-1 to complement viral DNA replication. |
| Databáze: | OpenAIRE |
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