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Two spatially distinct genetic elements constitute a bipartite DNA replication origin in the minute virus of mice
R Salvino1, M Skiadopoulos, E A Faust
1Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
Abstract:
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 10- and 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.
Insights
Minute virus of mice (MVM) DNA replication requires specific cis-acting elements, including the 5' terminal palindrome and a novel 65-bp repeat. These elements are essential for viral genome excision and replication, with the repeat showing species-specific regulation.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Minute virus of mice (MVM) is a parvovirus with a single-stranded DNA genome.
- Understanding the cis-acting elements governing MVM replication is crucial for deciphering parvovirus life cycles.
Purpose of the Study:
- To identify and characterize cis-acting genetic elements essential for MVM genome excision and replication.
- To investigate the role of the 5' terminal palindrome and adjacent sequences in MVM DNA replication.
Main Methods:
- Site-directed mutagenesis of an MVM infectious clone (pMM984).
- In vivo transient DNA replication assays in murine A9 and primate COS-7 cells.
- Analysis of subgenomic constructs and deletion mutants.
Main Results:
- Both viral termini are required on the same molecule for replication; MVM nonstructural protein NS-1 is needed in trans.
- The 5' terminal 206-bp palindrome is essential for MVM DNA excision and replication.
- A novel 65-bp repeat element, located 94 nucleotides from the 5' palindrome, regulates MVM DNA replication in a species-specific manner.
Conclusions:
- The 5' terminal palindrome and the 65-bp repeat are critical cis-acting elements for MVM DNA replication.
- The 65-bp repeat represents a novel regulatory element influencing MVM replication across different cell types.