MUTATIONS IN THE NTP-BINDING MOTIF OF MINUTE VIRUS OF MICE (MVM) NS-1PROTEIN UNCOUPLE ATPASE AND DNA HELICASE FUNCTIONS

The NS-1 protein of minute virus of mice (MVM) is required for viral D NA replication and transcriptional regulation. To define the domain st ructure 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. Furtherm ore, all mutants, except for the K405S substitution, were able to tran sactivate the P38 promoter in transient transfection experiments. NS-1 proteins bearing COOH-terminal deletions of 29 and 33 amino acid resi dues were also transcriptionally inert. Biochemical analysis of recomb inant NS-1 expressed in insect cells shows that mutations in the putat ive NTP-binding/ATPase domain severely reduced helicase activity in vi tro. However, affinity labeling experiments indicate that none of thes e mutations, except for K469T, impaired NTP-binding activity. Finally, all point mutants retained significant levels of ATPase activity, exc ept for the E444Q mutant (1%). These findings suggest that the replica tion and transcription activities of NS-1 reside in separate functiona l domains. In addition, NS-1 proteins with mutations in the putative n ucleotide binding fold have lost helicase activity, whereas most retai n nucleotide binding and ATPase functions, suggesting that the mutatio ns have uncoupled the ATPase and helicase activities.