| dc.contributor.author | Skiadopoulos, Mario H. | en_US |
| dc.date.accessioned | 2014-10-21T12:33:20Z | |
| dc.date.available | 1993 | |
| dc.date.issued | 1993 | en_US |
| dc.identifier.other | AAINN93739 | en_US |
| dc.identifier.uri | http://hdl.handle.net/10222/55396 | |
| dc.description | The major non-structural gene (NS-1) of the minute virus of mice (MVM) parvovirus encodes a multifunctional protein that is absolutely required for replication of the viral genome and for transcriptional regulation of the two MVM promoters. A shuttle vector encoding the NS-1 protein was constructed and a replication complementation assay was developed, to study the effects of a series of insertion and point mutations in the NS-1 coding sequence, and the localization of activities required for viral DNA replication and transcriptional activation. Mutant NS-1 genes were transiently expressed in transfected COS-7 cells by using an SV40 promoter driven NS-1 expression vector. The ability of the mutant proteins to complement a replication defective NS-1 mutant of the infectious MVM plasmid pMM984 and to activate transcription from the MVM capsid gene promoter in chloramphenicol acetyl transferase (CAT) expression assays was determined. | en_US |
| dc.description | Six independent insertions ranging in size from 2 to 12 amino acids inhibited the DNA replication activity of NS-1 between 20 and at least 100-fold. There was no apparent correlation between the extent of inhibition of parvoviral DNA replication and the location of the mutations. The transcriptional activation function of NS-1 was inhibited between 1.5 and at least 20-fold and was therefore overall relatively less sensitive to mutagenesis than was its DNA replication function. An exception to this was a 5 amino acid insertion between Tyr543 and Gln544 that abolished transactivation as well as the ability of NS-1 to complement viral DNA replication. Two point mutations Ser249 to Ala and Lys250 to Gln and a one amino acid insertion between Asp606 and Leu607 had no effect on viral DNA replication and transactivation activities. | en_US |
| dc.description | NS-1 has previously been shown to be covalently linked to the 5$\sp\prime$ ends of single stranded and replicative forms of the viral DNA. The linkage is stable under alkaline conditions and is therefore presumed to occur through a tyrosine residue. Tyrosine to phenylalanine mutations were constructed at the 11 conserved tyrosines of MVM NS-1. The ability of the mutant NS-1 proteins to complement the replication defective target plasmid in transient replication assays and to stimulate transcription from the capsid gene promoter of MVM in CAT assays was determined. F47, F227, and F543 replicated viral DNA and transactivated the capsid gene promoter at approximately wild type levels. Mutagenesis of Tyr6 to Phe and a double mutation Tyr550 to Phe and Ser445 to Thr exhibited increased levels of viral replication activity, while a Tyr to Phe conversion at position 209 replicated viral DNA at approximately 50% of wild type. F6 and F209 were wild type for transactivation, while F550/T445 transactivated the capsid gene promoter at approximately 50% of WT NS-1. Three mutations in the N-terminal portion of NS-1 (F188, F197 and F210) blocked the replication activity. F188 and F197 were defective for the transactivation function and F210 was wild type. Mutagenesis of Tyr310 and Tyr422 inhibited the replication activity but not transactivation. These mutations begin to define domains that are required for replication and suggest that the transactivation function may require more than one functional domain. | en_US |
| dc.description | Thesis (Ph.D.)--Dalhousie University (Canada), 1993. | en_US |
| dc.language | eng | en_US |
| dc.publisher | Dalhousie University | en_US |
| dc.publisher | | en_US |
| dc.subject | Biology, Molecular. | en_US |
| dc.title | Mutational analysis of the NS-1 protein of the MVM parvovirus. | en_US |
| dc.type | text | en_US |
| dc.contributor.degree | Ph.D. | en_US |
