THE NS1 POLYPEPTIDE OF THE MURINE PARVOVIRUS MINUTE VIRUS OF MICE BINDS TO DNA-SEQUENCES CONTAINING THE MOTIF [ACCA](2-3)

Citation
Sf. Cotmore et al., THE NS1 POLYPEPTIDE OF THE MURINE PARVOVIRUS MINUTE VIRUS OF MICE BINDS TO DNA-SEQUENCES CONTAINING THE MOTIF [ACCA](2-3), Journal of virology, 69(3), 1995, pp. 1652-1660
Citations number
55
Categorie Soggetti
Virology
Journal title
ISSN journal
0022538X
Volume
69
Issue
3
Year of publication
1995
Pages
1652 - 1660
Database
ISI
SICI code
0022-538X(1995)69:3<1652:TNPOTM>2.0.ZU;2-D
Abstract
A DNA fragment containing the minute virus of mice 3' replication orig in was specifically coprecipitated in immune complexes containing the virally coded NS1, but not the NS2, polypeptide. Antibodies directed a gainst the amino- or carboxy-terminal regions of NS1 precipitated the NS1-origin complexes, but antibodies directed against NS1 amino acids 284 to 459 blocked complex formation. Using affinity-purified histidin e-tagged NS1 preparations, we have shown that the specific protein-DNA interaction is of moderate affinity, being stable in 0.1 M salt but r apidly lost at higher salt concentrations. In contrast, generalized (o r nonspecific) DNA binding by NS1 could be demonstrated only in low sa lt. Addition of ATP or gamma S-ATP enhanced specific DNA binding by wi ld-type NS1 severalfold, but binding was lost under conditions which f avored ATP hydrolysis. NS1 molecules with mutations in a critical lysi ne residue (amino acid 405) in the consensus ATP-binding site bound to the origin, but this binding could not be enhanced by ATP addition. D Nase I protection assays carried out with wild-type NS1 in the presenc e of gamma S-ATP gave footprints which extended over 43 nucleotides on both DNA strands, from the middle of the origin bubble sequence to a position some 14 bp beyond the nick site. The DNA-binding site for NS1 was mapped to a 22-bp fragment from the middle of the 3' replication origin which contains the sequence ACCAACCA. This conforms to a reiter ated motif (ACCA)(2-3), which occurs, in more or less degenerate form, at many sites throughout the minute virus of mice genome (J. W. Bodne r, Virus Genes 2:167-182, 1989). Insertion of a single copy of the seq uence (ACCA)(3) was shown to be sufficient to confer NS1 binding on an otherwise unrecognized plasmid fragment. The functions of NS1 in the viral life cycle are reevaluated in the light of this result.