Mutations in the NTP-binding motif of minute virus of mice (MVM) NS-1 protein uncouple ATPase and DNA helicase functions

• Published in: The Journal of biological chemistry 1994-02, Vol.269 (5), p.3283-3289
• Main Authors: JINDAL, H. K, YONG, C. B, WILSON, G. M, TMA, P, ASTELL, C. R
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - metabolism; Amino Acid Sequence; Animals; Bacterial Proteins - genetics; Base Sequence; Binding Sites; Biological and medical sciences; Cell Line; DNA Helicases - metabolism; DNA Replication; DNA-Binding Proteins - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Viral; minute virus of mice; Minute Virus of Mice - enzymology; Minute Virus of Mice - genetics; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Moths; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Parvoviridae - genetics; Protein Structure, Secondary; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Replication; Sequence Deletion; Sequence Homology, Amino Acid; Trans-Activators; Transcription, Genetic; Transfection; Viral Nonstructural Proteins - biosynthesis; Viral Nonstructural Proteins - genetics; Viral Nonstructural Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)41860-6

The NS-1 protein of minute virus of mice (MVM) is required for viral DNA replication and transcriptional regulation. To define the domain structure of NS-1, we have generated point mutations in its putative NTP-binding/ATPase domain. We show that all mutants were unable to support replication of MVM DNA in a transient DNA replication assay. Furthermore, all mutants, except for the K405S substitution, were able to transactivate the P38 promoter in transient transfection experiments. NS-1 proteins bearing COOH-terminal deletions of 29 and 33 amino acid residues were also transcriptionally inert. Biochemical analysis of recombinant NS-1 expressed in insect cells shows that mutations in the putative NTP-binding/ATPase domain severely reduced helicase activity in vitro. However, affinity labeling experiments indicate that none of these mutations, except for K469T, impaired NTP-binding activity. Finally, all point mutants retained significant levels of ATPase activity, except for the E444Q mutant (1%). These findings suggest that the replication and transcription activities of NS-1 reside in separate functional domains. In addition, NS-1 proteins with mutations in the putative nucleotide binding fold have lost helicase activity, whereas most retain nucleotide binding and ATPase functions, suggesting that the mutations have uncoupled the ATPase and helicase activities.