The catalytic subunit of the herpes simplex virus 1 DNA polymerase (HSV-1 Pol) has been extensively studied; however, its full complement of functional domains has yet to be characterized. The previously uncharacterized pre-NH2-terminal domain (residues 1-140) within HSV-1 Pol is unique to the herpesvirus Pol family. We sought to investigate the importance of this domain for viral replication in cell culture and an animal model of infection. We evaluated the enzymatic activity of purified pre-NH2-terminal Pol mutant proteins in which conserved residues had been deleted or substituted. Subsequently, the corresponding pol mutant viruses were engineered for viral genetic analyses. We found that the extreme N-terminal 51 residues were not required for wild type 5’-3’ polymerase activity in vitro. Interestingly, the extreme N-terminal 42 residues were dispensable for viral replication in cell culture while a conserved motif at residues 44-49 was necessary for efficient viral DNA synthesis and production of infectious virus. Viral replication proteins have proven to be particularly important in the context of acute and latent infections in animals. Characterization of pol mutant virus replication in a mouse ocular model of infection revealed that the extreme N-terminal 42 residues were not required for viral replication and reactivation from latency. The conserved motif, however, was shown to be required for robust acute ganglionic replication and efficient latency establishment. We hypothesized that the conserved motif at residues 44-49 mediates a protein- protein interaction that positively impacts viral DNA synthesis during infection. Specific protein candidates were evaluated using purified proteins in vitro, and proteins that coprecipitated with wild type and mutant polymerases from infected cell lysates were analyzed. To date, we have yet to identify a protein whose binding was disrupted as a result of the mutation. Ultimately, we have established a role for the pre-NH2-terminal domain of HSV-1 Pol during viral replication that is distinct from 5’-3’ polymerase activity. The conserved motif mediates a function that is required for efficient viral DNA synthesis in cell culture and is of even greater importance for acute ganglionic replication in mice. The mechanism of action more than likely reflects a conserved mechanism for herpesvirus replication.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/10416140 |
| Date | 06 February 2014 |
| Creators | Terrell, Shariya Louise |
| Contributors | Coen, Donald Mark |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | open |
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