Poliovirus, a well-studied member of the family Picornaviridae, uses the small viral protein VPg as a primer for viral RNA synthesis, which results in nascent viral RNAs containing a 5&feet; tyrosyl-RNA linkage. During the early stages of poliovirus replication, VPg is unlinked from the viral genome by an endogenous activity in HeLa cells, termed VPg unlinkase. Viral RNAs that are packaged into virions, however, are still linked to VPg, which suggests that the unlinking of VPg may have a regulatory role during the picornavirus replication process. Therefore, we hypothesize that the VPg unlinkase-picornavirus interaction is a novel source of targets for the development of general anti-picornavirus therapeutics.
To test our hypothesis, the cellular identity of VPg unlinkase had to be determined in order to characterize its role(s) during infection. Using our facile VPg unlinkase activity assay, we carried out a two-part approach to identify VPg unlinkase. First, we developed a biochemical/enzymatic model of VPg unlinkase to generate an unbiased profile for filtering candidates. We also used the information obtained from this work to identify ligands for affinity purification of VPg unlinkase. Second, we developed a purification scheme to isolate VPg unlinkase from HeLa cells.
Our biochemical studies showed that at least three different species of VPg unlinkase activity exist, ranging from 25 to 50 kDa in apparent molecular weight. Furthermore, we found that partially purified VPg unlinkase activity has an apparent molecular weight of ∼39 kDa. The search for VPg unlinkase inhibitors found that increasing concentrations of single-stranded DNA (ssDNA) inhibits VPg unlinkase, suggesting that this enzyme binds ssDNA.
To isolate VPg unlinkase from HeLa cells, we tested several different biochemical methods such as ion exchange, size exclusion, and heparin sepharose chromatography. Although these methods were successful in generating highly enriched preparations of VPg unlinkase, we were unable to correlate activity with a specific protein. Upon discovering that VPg unlinkase binds ssDNA, we included a ssDNA cellulose step into our purification protocol. We subjected HeLa cell extracts to heparin sepharose, ssDNA cellulose, anion exchange, size exclusion, and cation exchange chromatography, to generate a nearly homogeneous preparation of VPg unlinkase, enriched by greater than 10,000-fold. This preparation of VPg unlinkase contained a predominant 38 kDa protein (p38), which correlated with VPg unlinkase activity. Following in-gel trypsin digestion and mass spectrometry analysis, we identified p38 as 5' tyrosyl-DNA phosphodiesterase (TDP2).
TDP2 is a DNA repair enzyme that cleaves 5'-tyrosyl-DNA adducts that occur by topoisomerase II DNA damage, which is similar to the bond hydrolyzed by VPg unlinkase. We then proceeded to express and purify recombinant TDP2 from bacterial extracts. With our VPg unlinkase activity assay, we determined that recombinant TDP2 has authentic VPg unlinkase activity.
Using immunofluorescence, we investigated the VPg unlinkase-picornavirus interaction in vivo. In mock-infected cells the subcellular localization of TDP2 was predominantly nuclear, with some TDP2 detected in the cytoplasm. We observed a similar pattern for the subcellular localization of TDP2 in poliovirus-infected cells at early time points of infection. However, at a late time after infection, we observed a dramatic re-localization of TDP2. TDP2 was located at the periphery of the cytoplasm, in regions distinct from those containing makers for viral RNA synthesis (the viral 3A protein) and encapsidation (the viral capsid proteins). From these results, we propose that TDP2 is excluded from sites of viral RNA synthesis and encapsidation, at late times during infection, to promote virion formation.
Although it is unclear how TDP2 is re-localized during poliovirus infection, we predict that disruption of this process by a small molecule may interfere with viral replication, resulting in reduced viral titers. If true, these compounds may lead to the development of drugs for treating illnesses caused by picornaviruses, such as, aseptic meningitis, encephalitis, hepatitis, and the common cold.
|Advisor:||Semler, Bert L.|
|Commitee:||Afasizhev, Ruslan D., Hertel, Klemens, Sandri-Goldin, Rozanne M.|
|School:||University of California, Irvine|
|Department:||Biomedical Sciences - Ph.D.|
|School Location:||United States -- California|
|Source:||DAI-B 74/05(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry, Virology|
|Keywords:||DNA repair, Picornavirus, Poliovirus, RNA phosphodiesterase, Rhinovirus|
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