| Popis: |
Studies presented in this thesis involve the identification and/or further characterization of cis-acting and trans-acting genetic elements required for minute virus of mice (MVM) DNA replication and gene expression. Plasmids with mutations introduced into the viral genome within the full length infectious plasmid pMM984 were transfected into murine A9 and primate COS-7 cells and viral DNA replication and gene expression were assessed using in vivo transient DNA replication and chloramphenicol acetyl transferase expression assays.;Results indicated that the nonstructural gene product NS-1 is a trans-acting protein that is required for the excision and replication of the viral genome and for capsid gene expression. Furthermore, studies using a NS-1 frameshift mutant expected to synthesize a truncated form of NS-1 consisting of 258 N-terminal wild type amino acids was severely inhibited for both functions whereas an in-frame NS-1 mutant with 6 amino acids inserted at pro-258 was defective for only viral DNA replication. These experiments suggested that the replication and trans-activation functional domains of NS-1 can be separated, consistent with the observations first made by M. Skiadopoulos (unpublished data). Similar genetic analysis of the capsid genes demonstrated that the capsid gene products VP-1/VP-2 were needed for the synthesis of progeny single stranded DNA and the production of infectious virus particles.;Studies with subgenomic viral plasmids containing all the necessary cis-acting elements indicated that both viral termini must be present on the same DNA molecule and that NS-1 must be provided for replication to occur. Furthermore, two distinct cis-acting elements near the 5{dollar}\sp\prime{dollar} end of the viral genome were defined. Firstly, the terminal hairpin sequences were absolutely essential for the excision and replication of the viral genome. Evidence suggested that the formation of a simple hairpin that would act as a primer was not sufficient and that secondary structure plays an integral role. Secondly, a 65 bp tandemly repeated sequence located inboard of the terminal palindrome was identified that was capable of modulating MVM DNA replication in a species-specific manner. |
