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

1994, Journal of Biological Chemistry

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 NTPbinding/ATPase domain. W e show that all mutants were unable to support replication of MVM DNA in a transient DNA replication assay. Furthermore, all mutants, except for the K40SS 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-bindingJATPase domain severely reduced helicase activity in vitro. However, affinity labeling experiments indicate that none of these mutations, except for K469T, impaired "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. NS-1 is the major nonstructural protein encoded by the autonomous parvovirus, minute virus of mice (MVM).l It is a n 83-kDa phosphorylated nucleoprotein (Cotmore and Tattersall, 19861, multifunctional in both replication and transcription. NS-1 is essential for replication (Tullis et al., 1988). Current models propose that MVM DNA replication initiates by conversion of parental single-stranded viral DNA into a dimer replicative form. The dimer can be resolved to unit length replicative form molecules which function in the synthesis of progeny genomes (Astell et al., 1985; Tattersall and Cotmore, 1990). As predicted (Astell et al., 19851, NS-1 has been found covalently attached to the 5' ends of resolved concatemer junction fragments in infected cells, indicating that it acts as the site-specific nickase (Cotmore and Tattersall, 1992). Moreover, recombinant NS-1 purified from insect cells displays both helicase Grant MA-6728. H. K. J., C. B. Y., and G. M. W. contributed equally to * This work was supported by Medical Research Council of Canada these studies. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of a Medical Research Council Studentship.