CIS-acting sequences found within the MVM genome required for DNA replication

• Main Author: Tam, Patrick
• Format: Others
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/7128

Minute virus of mice (MVM) belongs to the Parvovirus genera of the Parvoviridae family of eukaryotic viruses. Its genome consists of approximately 5 kb of primarily single stranded negative sense DNA. Since the primary sequences at the genomic termini are palindromic, duplex hairpin structures are found at the termini. It is clear that parvoviral terminal hairpins are important for viral DNA replication. In order to determine the sequence requirements for MVM DNA replication, an in vivo DNA replication assay was developed. In this assay, two plasmids were required. First, the plasmid vector, pPTLR, was constructed such that it encoded a minigenome of MVM containing 411 nt and 807 nt of the left and right termini, respectively. This minigenome was designed to contain only the terminal sequences since the analysis of DNAs found within defective interfering (DI) particles suggested that the cis-acting sequences required for viral DNA synthesis are found at or near the termini (Faust and Ward, 1979). When pPTLR was co transfected into mouse LA9 cells or COS-7 cells with a second plasmid which expresses MVM NS-1, the major viral non-structural protein, the viral minigenome was rescued from the plasmid sequences and replicated in the host cell. The replicated DNA exhibited heterogeneous termini suggesting that both terminal hairpins were functional during viral DNA replication. Deletion analysis of the pPTLR minigenome suggested that in addition to previous studies which partially defined the requirements of the right end palindrome, two additional regions of the MVM genome are important for viral DNA replication. First, analysis of the left terminal hairpin suggested that an element(s) between MVM nucleotide position 11 and 25 (or 31) is important.. Although the nature of the left terminal hairpin allows minor deletion mutants to regenerate wild type terminal hairpins, it is hypothesized that the bubble sequence which, results from nucleotide positions 25-26 being mispaired with nucleotide positions 91-89 in the duplex hairpin, is altered during DNA replication. The potential effect of this change in the DNA template on viral DNA replication is discussed. Second, deletion analysis of the region internal of the right hairpin suggested that two adjacent elements, A (nucleotide positions 4489-4636) and B (nucleotide positions 4636-4695) are important for minigenome DNA replication. Two Rsa I restriction fragments which partially span elements A and B, Rsa A (nucleotide positions 4431-4579) and Rsa B (nucleotide positions 4579-4662), were used to probe nuclear extracts for sequence specific DNA binding proteins in electrophoretic mobility shift assays. A number of DNA-protein interactions were discovered. The binding site of one relatively abundant cellular factor, MVM DNA replication factor B5 or MRF B5, was determined. MRF B5 protected two regions of the Rsa B probe, site I (—nucleotide positions 4589-4610) and site II (—nucleotide positions 4616-4646), in DNase I footprinting assays. Although the function of MRF B5 in viral DNA replication is unknown, it is speculated that it might activate DNA replication at the right terminus. Analysis of the replication of identical end minigenome mutants showed that viral DNA can also be replicated using either two left termini (LL) or two right termini (RR). Replication of the RR minigenome was observed to be substantially greater that of the LL minigenome. However, it was demonstrated that the Xba I (nucleotide position 4342)/Sau3a (nucleotide position 4741) fragment, which contained elements A and B, could stimulate DNA replication of LL type minigenomes. These data confirm the finding that sequences at both termini encode origins of DNA replication. === Medicine, Faculty of === Biochemistry and Molecular Biology, Department of === Graduate