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1.
Virology ; 201(2): 251-62, 1994 Jun.
Article in English | MEDLINE | ID: mdl-8184536

ABSTRACT

Previous characterization of the terminal sequences of the minute virus of mice (MVM) genome demonstrated that the right hand palindrome contains two sequences, each the inverted complement of the other. However, the left hand palindrome was shown to exist as a unique sequence [Astell et al., J. Virol. 54: 179-185 (1985)]. The modified rolling hairpin (MRH) model for MVM replication provided an explanation of how the right hand palindrome could undergo hairpin transfer to generate two sequences, while the left end palindrome within the dimer bridge could undergo asymmetric resolution and retain the unique left end sequence. This report describes in vitro resolution of the wild-type dimer bridge sequence of MVM using recombinant (baculovirus) expressed NS-1 and a replication extract from LA9 cells. The resolution products are consistent with those predicted by the MRH model, providing support for this replication mechanism. In addition, mutant dimer bridge clones were constructed and used in the resolution assay. The mutant structures included removal of the asymmetry in the hairpin stem, inversion of the sequence at the initiating nick site, and a 2-bp deletion within one stem of the dimer bridge. In all cases, the mutant dimer bridge structures are resolved; however, the resolution pattern observed with the mutant dimer bridge compared with the wild-type dimer bridge is shifted toward symmetrical resolution. These results suggest that sequences within the left hand hairpin (and hence dimer bridge sequence) are responsible for asymmetric resolution and conservation of the unique sequence within the left hand palindrome of the MVM genome.


Subject(s)
DNA, Viral/genetics , Genome, Viral , Minute Virus of Mice/genetics , Virus Replication/genetics , Animals , Base Sequence , Blotting, Southern , Cloning, Molecular , DNA, Viral/biosynthesis , DNA, Viral/chemistry , Electrophoresis, Gel, Two-Dimensional , Mice , Minute Virus of Mice/chemistry , Models, Genetic , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Precipitin Tests , Repetitive Sequences, Nucleic Acid
2.
J Biol Chem ; 269(5): 3283-9, 1994 Feb 04.
Article in English | MEDLINE | ID: mdl-8106366

ABSTRACT

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.


Subject(s)
Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , DNA Helicases/metabolism , Minute Virus of Mice/enzymology , Trans-Activators , Viral Nonstructural Proteins/metabolism , Adenosine Triphosphate/analogs & derivatives , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Base Sequence , Binding Sites , Cell Line , DNA Replication , DNA-Binding Proteins/genetics , Gene Expression Regulation, Viral , Minute Virus of Mice/genetics , Molecular Sequence Data , Moths , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides , Parvoviridae/genetics , Protein Structure, Secondary , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , Sequence Homology, Amino Acid , Transcription, Genetic , Transfection , Viral Nonstructural Proteins/biosynthesis , Viral Nonstructural Proteins/genetics
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