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Benjamin P. Steil, Ph.D.

Barton Lab


 

THESIS

Complete Title of Thesis:

"Protein Primers and a Telomerase-like Mechanism of Poliovirus RNA Replication Maintain the 3' End of the RNA Genome"


Prepared under the direction of: David J. Barton, Ph.D.

SUMMARY

Poliovirus (PV) RNA-dependent RNA polymerase is a primer-dependent enzyme that uses either the tyrosine hydroxyl of the PV protein VPg or the 3' hydroxyl of uridylylated VPg (VPgpUpU0H) to prime the initiation of RNA synthesis. A cis replication element (CRE) within PV RNA functions as the template for VPgpUpU0H synthesis. VPg and/or VPgpUpU0H prime the initiation of negative-strand RNA synthesis somewhere along the 3' poly(A) tail of PV RNA. VPgpUpUOH molecules reiteratively prime the initiation of positive-strand RNA synthesis. The mechanisms by which VPg and VPgpUpUOH prime the initiation of PV RNA replication are important because they maintain the integrity of the 5' and 3' ends of the viral RNA genome. My research investigated three unresolved aspects of PV RNA replication:

  1. When are VPg precursors converted into VPgpUpUOH products?
  2. Do VPg, VPgpUpUoH, or both prime negative-strand RNA synthesis?
  3. Where on PV 3' poly(A) templates do VPg and/or VPgpUpU0H prime RNA synthesis?

I used a cell-free system of HeLa cell extracts to study poliovirus RNA replication. I found that VPgpUpUOH synthesis was evident both before and after the initiation of negative-strand RNA synthesis. Thus, both VPgpUpUOH and VPg primers are present prior to the initiation of negative-strand RNA synthesis. CRE mutations which prevented VPgpUpUOH synthesis did not prevent VPg priming of negative-strand RNA synthesis; however, CRE-dependent VPg uridylylation lowered the Km of UTP required for negative-strand RNA synthesis. These results indicated that VPgpUpUOH primed the initiation of negative-strand RNA synthesis more efficiently than VPg, especially when UTP concentrations were low. The size ofVPg-poly(U) from the 5' end of PV negative-strand RNAs indicated that VPg and VPgpUpUOH both primed RNA synthesis at the same site on 3' poly(A) templates. Furthermore, poly(U) products at the 5' end of negative-strand RNAs and poly(A) products at the 3' end of positive strand RNA products were generally longer than the homopolymeric templates from which they were synthesized. These previously undiscovered aspects of PV RNA replication indicate that PV polymerase uses a stuttering mechanism on homopolymeric templates to maintain the integrity of the 3' end of PV RNA molecules. This telomerase-like mechanism may apply to all positive-strand RNA viruses with 3' poly(A) sequences.