Mutations in the NTP-binding motif of minute virus of mice (MVM) NS-1 protein uncouple ATPase and DNA helicase functions.
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.
Jindal
H K
HK
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Yong
C B
CB
Wilson
G M
GM
Tam
P
P
Astell
C R
CR
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D008915:Minute Virus of Mice / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010321:Parvoviridae / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators; D014158:Transcription, Genetic; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C081367:NS1 protein, minute virus of mice; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D015534:Trans-Activators; D017361:Viral Nonstructural Proteins; C045264:replication initiator protein; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004265:DNA Helicases