Lytic herpes simplex virus (HSV) infections in neonates and immunocompromised patients result in high morbidity an mortality. Protein nucleotidylation is a novel posttranslational modification which may act as a means to regulate lytic HSV infection. Eight HSV proteins were previously shown to be nucleotidylated in vitro in isolated nuclei from infected cells, including the four alpha regulatory proteins and four late gene products.
The specific aims of this project are: (i) to determine whether HSV protein nucleotidylation occurs inside cells during viral infection and (ii) to determine what function this modification plays during th HSV replicative cycle. The hypothesis to be tested is that nucleotidylation occurs at distinct sites on the target viral protein and that it is essential for the function of theses proteins. To determine whether nucleotidylation occurs inside cells, cells will be metabolically labeled with tritiated nucleotide prior to infection with HSV. The viral proteins labeled in these cells will then be purified and their modification will be characterized biochemically. The viral protein modification sites will be identified by mapping the targets in fusion proteins that contain portions of the viral polypeptides. Site-directed mutations in the fusion proteins will be made to determine the specific amino acids required for modification. Initially, the focus will be on three representative viral proteins. Once the target modification sites are identified, point mutations will be made at these sites in the genes. The mutated genes will be placed back into the virus by recombination and the growth of the resulting viruses will be studied. Specifically, the levels of viral DNA, RNA and protein for each of the recombinants will be measured. Human casein kinase II (CKII) was implicated in the nucleotidylation of the viral alpha regulator ICP22. To determine the role that CKII plays in this process, it will be purified to homogeneity and its novel activity will be characterized biochemically. Nucleotidylation of the other alpha regulatory factors requires a viral component which is made late in infection. This component will be purified to the extent that a partial amino acid sequence can be obtained and the gene encoding the factor may be identified. These studies will help in determining whether nucleotidylation is a necessary process in the replication of HSV. In the long term, this research will help define the molecular basis for regulation of the life-cycle of this important human pathogen.
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