Characterization of a metastable, partially replicated dimeric intermediate of minute virus of mice

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Mutations were introduced into plasmid pMM984, a full-length infectious clone of the fibrotropic strain of minute virus of mice, to identify cis-acting genetic elements required for the excision and replication of the viral genome. The replicative capacity of these mutants was measured directly, using an in vivo transient DNA replication assay following transfection of plasmids into murine A9 cells and primate COS-7 cells. Experiments with subgenomic constructs indicated that both viral termini must be present on the same DNA molecule for replication to occur and that the viral nonstructural protein NS-1 must be provided in trans. The necessary sequences were located within 1,084 and 807 nucleotides of the 3' and 5' ends of the minute virus of mice genome, respectively. The inhibitory effect of deletions within the 206-bp 5'-terminal palindrome demonstrated that these sequences comprise a cis-acting genetic element that is absolutely essential for the excision and replication of viral DNA. The results further indicated a requirement for a stem-plus-arms T structure as well as for the formation of a simple hairpin. In addition, the removal of one copy of a tandemly arranged 65-bp repeat found 94 nucleotides inboard of the 5'-terminal palindrome inhibited viral DNA replication in cis by 10and just greater than 100-fold in A9 and COS-7 cells, respectively. The latter results define a novel genetic element within the 65-bp repeated sequence, distinct from the terminal palindrome, that is capable of regulating minute virus of mice DNA replication in a species-specific manner.

Section: Resultssupporting

confidence: 63%

Two spatially distinct genetic elements constitute a bipartite DNA replication origin in the minute virus of mice genome

Salvino

Skiadopoulos

Faust

et al. 1991

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“…7) but terminate this synthesis prematurely near the 3'-terminal hairpin (12). Faust and Gloor suggest that these strong-stop intermediates may be due to the intervention of a site-specific telomeric nuclease (12). It is also possible that the DNA polymerase simply has difficulty in extending through the guanineand cytosine-rich 3'-terminal 5' end labeled with T4 polynucleotide kinase and digested with EcoRI, and the fragments were separated on a 1% agarose gel.…”

Section: Discussionmentioning

confidence: 99%

Sequence analysis of the termini of virion and replicative forms of minute virus of mice DNA suggests a modified rolling hairpin model for autonomous parvovirus DNA replication

Astell¹,

Chow²,

Ward³

1985

109

The nucleotide sequences of the terminal regions of monomer replicative form DNA, a pivotal intermediate species in the replication of minute virus of mice, were determined. The left (3') terminus had a unique sequence on both strands and in both 3'-hairpin configurations. In contrast, the right (5') terminus was sequence heterogenous and extended an additional 18 base pairs beyond that expected from the known sequence of the virion DNA. These data unambiguously establish the sequence complexity at the termini of both the single-stranded viral genome and the pool of replicative DNA. A comparison of the combined sequence information leads us to propose a modified rolling hairpin model for the replication of autonomous parvoviruses which is compatible with all available data.

“…1B and 2A). Unfortunately, in these preparations, other MVM DNA forms, such as tetramer RF and the 8.0-kilobase replicative intermediate (20), were not present in sufficient concentration to analyze. Because the characterization of any TP associated with these RF DNAs is crucial in understanding the role of the TP in MVM DNA replication, similar studies are necessary to characterize the TP on dimer RF, tetramer RF, and the 8.0-kilobase intermediate.…”

Section: Figmentioning

confidence: 99%

Identification and characterization of a protein covalently bound to DNA of minute virus of mice

Chow¹,

Bodnar²,

Polvino-Bodnar³

et al. 1986

We identified a protein which is covalently linked to a fraction of the DNA synthesized in cells infected with minute virus of mice. This protein is specifically bound to the 5' terminus of the extended terminal conformers of the minute virus of mice replicative-form DNA species and of a variable fraction of single-stranded viral DNA. The chemical stability of the protein-DNA linkage is characteristic of a phosphodiester bond between a tyrosine residue in the protein and the 5' end of the DNA. The terminal protein (TP) bound on all DNA forms has a relative molecular weight of 60,000; it is also seen free in extracts from infected cells. Immunologic comparison of the TP with the other known viral proteins suggests that the TP is not related to the capsid proteins or NS-1.