Herpes Simplex Virus Type 1 (HSV-1) establishes latent infections in neurons in vivo and lytic infections in epithelial cells and fibroblasts. During latent infections, HSV-1 transcription is restricted and the genomes are not replicated. Latent HSV-1 genomes are chromatinized, such that digestion with micrococcal nuclease (MCN) releases DNA fragments with sizes characteristic of nucleosomal DNA. During lytic infections, in contrast, all HSV-1 genes are expressed, the genomes are replicated, and their digestion produces primarily heterogeneously sized fragments. However, as evaluated by ChIP assays, HSV-1 DNA interacts with histones during lytic infections, although in most cases only a small percentage of HSV-1 DNA co-immunoprecipitates with histones (or is cleaved to nucleosome sizes following MCN digestion). Therefore, although current models propose that chromatin regulates HSV-1 transcription, it remains unclear how the association of histones with only a small percentage of HSV-1 DNA can globally regulate viral transcription. Moreover, the physical properties of the complexes containing histones and HSV-1 DNA are unknown. My objective was therefore to evaluate the biophysical properties of the HSV-1 DNA-containing complexes during lytic infection. Differing from pervious studies, however, I used classical chromatin purification techniques. I show that most HSV-1 DNA is in unstable nucleoprotein complexes and, consequently, more accessible to MCN than DNA in cellular chromatin. This HSV-1 DNA is protected from MCN redigestion only after crosslinking, similar to unstable cellular nucleosomes. HSV-1 DNA is in such complexes throughout lytic infection. Using unrelated small-molecule inhibitors, I further show that inhibition of HSV-1 transcription is associated with a decrease in MCN accessibility of HSV-1 DNA. Roscovitine, a cyclin-dependent kinase inhibitor, prevents activation but not elongation of IE, E, and L HSV-1 transcription. Consistent with a functional association between accessibility and transcription, roscovitine only decreases the accessibility of DNA templates of which it also inhibits transcription, independent of specific promoter sequences. In summary, I show that most HSV-1 DNA is in unstable nucleosome-like complexes during lytic infection and that accessibility to HSV-1 DNA likely plays a key role in regulating HSV-1 transcription.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:AEU.10048/1359 |
| Date | 11 1900 |
| Creators | Lacasse, Jonathan J |
| Contributors | Schang, Luis (Biochemistry and Medical Microbiology and Immunology), Schultz, Michael (Biochemistry), Hobman, Tom (Cell Biology), Hendzel, Michael (Oncology), Kristie, Thomas (Laboratory of Viral Disease, NIAID) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 4449244 bytes, application/pdf |
| Relation | Lacasse JJ, Schang LM; J Virol. 2010; 84(4):1920-33, Schang LM, St. Vincent, Lacasse JJ; 2006; 17(6): 293-320, Lacasse JJ, Provencher V, Urbanowski MD, Schang LM; Therapy. 2005; 2(1):77-90, Diwan P, Lacasse JJ, Schang LM; J Virol. 2004; 78(17):9352-65 |
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