Examination of the mechanism of parvovirus B19 NS1 induced cellular toxicity.
Open Access
- Author:
- Kivovich, Violetta
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 31, 2009
- Committee Members:
- Mark Kester, Ph D, Dissertation Advisor/Co-Advisor
Mark Kester, Committee Chair/Co-Chair
Stanley J Naides, Committee Member
Robert Harold Bonneau, Committee Member
Robert G Levenson, Committee Member
Leslie Joan Parent, Committee Member - Keywords:
- homology modeling
B19
NS1
apoptosis
DNA damage
parvovirus
singla-strand breaks - Abstract:
- Human parvovirus B19 is a ubiquitous infectious agent known to cause erythema infectiosum in children. Numerous clinical observations have also linked the virus to diverse idiopathic conditions such as systemic lupus erythematosus (SLE)-like syndromes, a variety of rheumatic manifestations, liver and kidney disease. Tissues identified as non-permissive for B19 replication have been shown to harbor genomic evidence of B19 infection for extended periods of time. Although such non-permissive cells display an inability to produce viral structural proteins and replicate the viral genome, curiously these same tissues allow for transcription and translation of the B19 non-structural protein 1 (NS1). As the sole chaperone of all major steps in the viral life cycle and mediator of cytotoxicity in mammalian cells, the persistence of NS1 in replication non-permissive tissues may be responsible for many of the non-hematological conditions associated with parvovirus B19 infection. In this dissertation, I examine the mechanism of B19 NS1-induced cytotoxicity. I hypothesize that the endonuclease activity encoded in the B19 NS1 protein is responsible for B19 NS1-induced cytotoxicity in mammalian cells. To examine this hypothesis, I generated a homology model of B19 NS1, which demonstrated the spatial localization and key residues in the endonuclease active site of B19 NS1 as well as the ATP binding region of the protein. Residues in the putative metal coordination motif of the endonuclease active site were mutated in order to abrogate this catalytic function. Abrogation of the endonuclease active site was shown to disrupt B19 NS1-induced cell cycle arrest in the S phase, reduce B19 NS1-induced DNA damage and subsequent apoptotic cell death. The DNA damage induced by B19 NS1 was suggested to be single-strand breaks, as would be expected if the protein were nicking cellular DNA with the endonuclease active site. Together, these experiments showed that the endonuclease activity of B19 NS1 upon cellular DNA is partially responsible for B19 NS1-induced cytotoxicity. An additional amino acid substitution in B19 NS1 was examined for its effect on B19 NS1-induced cytotoxicity. B19 genotypes 1 and 3 have been associated with fulminant hepatitis, in contrast to genotype 2. Substitution I181M, only observed in B19 genotype 2 NS1 proteins, was introduced into a genotype 1 background of NS1. The NS1 protein with the I181M substitution was less toxic to mammalian cells in culture, suggesting that this substitution in genotype 2 NS1 proteins may reduce their ability to kill liver cells and thereby cause fulminant hepatitis. Together, these studies support the hypothesis that direct NS1-induced cellular damage contributes to non-hematological conditions associated with parvovirus B19 infection.