Functional analysis of UL9, the origin-binding protein of herpes simplex virus-1
Date of Completion
January 2002
Keywords
Biology, Molecular|Biology, Microbiology
Degree
Ph.D.
Abstract
UL9 is an essential gene for HSV-1 replication. UL9 exhibits helicase and origin-binding activities. It is thought that UL9 binds and unwinds the HSV-1 origin of replication, creating a replication bubble and promoting the assembly of the replication machinery. Mutants in conserved residues of UL9 helicase motifs I-VI, which are unable to support the growth of UL9 null virus in vivo have been previously isolated. The overexpression of wild type UL9 results in moderate inhibition of HSV-1 infection; whereas, mutants in several motifs exert transdominant effect. In contrast, a mutant in motif V is potentiating and stimulates HSV-1 infection. The aims of this thesis were to better understand the molecular basis of the noncomplementing phenotype of UL9 helicase motif mutants and the mechanism of the transdominance/potentiation phenomenon. Mutant proteins carrying mutations in helicase motifs I, II, III, IV and VI exhibited decreased levels of ATPase activity and most had abolished helicase activity; whereas their abilities to dimerize and bind the HSV-1 origin of replication were comparable to wild type. ^ A bioinformatical approach was used to test the hypothesis that UL9 motif Ia is involved in the ssDNA-binding activity of the protein. Rationally designed mutations in residues expected to bind ssDNA were found to affect the ssDNA binding activity of UL9 and to abolish its helicase activity. Furthermore, this study confirms the conserved nature of the helicase structure and the function of the helicase motifs. ^ Based on genetic and biochemical experiments, a model for transdominance/potentiation of the UL9 helicase motif mutants was proposed. It postulates that the inhibitory effect of wild type UL9 overexpression is mediated by the origin-binding function of the protein. Transdominance is mediated by origin binding, dimerization and lack of helicase activity. On the other hand potentiation is most likely mediated by protein instability and decrease of the wild type UL9 protein levels. Our results suggest that the levels of the UL9 protein are an important factor in the progression of HSV-1 infection and perhaps subject of regulation. ^
Recommended Citation
Marintcheva, Boriana Davidova, "Functional analysis of UL9, the origin-binding protein of herpes simplex virus-1" (2002). Doctoral Dissertations. AAI3076711.
https://digitalcommons.lib.uconn.edu/dissertations/AAI3076711