INTERACTIONS BETWEEN THE TEGUMENT PROTEINS, UL11 AND UL16, AND THE GLYCOPROTEIN E OF HERPES SIMPLEX VIRUS
Open Access
- Author:
- Yeh, Pei-Chun
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 21, 2009
- Committee Members:
- John Warren Wills, Dissertation Advisor/Co-Advisor
John Warren Wills, Committee Chair/Co-Chair
Richard James Courtney, Committee Member
Rebecca C Craven, Committee Member
Sarah Bronson, Committee Member - Keywords:
- Herpes simplex virus
tegument proteins - Abstract:
- The herpesvirus tegument region present between the virion envelope and the nucleocapsid contains more than 20 different virus-encoded proteins. The process of tegument assembly and final envelopment has been unfoldeding over the past few decades. It is thought that a few tegument proteins are added to the capsid in the nucleus, whereas most of them are acquired after entering the cytoplasm or traveling to the site of final envelopment at the trans-Golgi network. Overall, the research presented in this dissertation provides insights into the molecular mechanism of protein-protein interactions that may be involved in (or contribute to) assembly and maturation of herpes simplex virus type 1 (HSV-1). The UL11 (membrane-bound) and UL16 (capsid-associated) tegument proteins are conserved among all herpesviruses, and interaction between the two has been implicated in linking the viral capsid, tegument, and membrane during final envelopment process. Both in transfected and virus-infected cells, a subpopulation of the UL11 protein was found associated with detergent-resistant membranes via modifications with two fatty- acid chains (myristate and palmitate). UL11 can directly interact with UL16 in a manner dependent on the acidic cluster and leucine-isoleucine motifs of UL11. And, N-ethylmaleimide-modified UL16 was found to be incapable of binding UL11, suggesting that free cysteines in UL16 somehow play a role in the interaction. UL16 is stably associated with cytoplasmic capsids isolated from infected cells. In response to initial attachment of virus to the cell surface, an ‘outside-in’ signal is transmitted across the virion membrane, and as a result, UL16 is dissociated from the capsid. The mechanism by which the signal is sent to UL16 remains unclear but seems likely to be mediated by the glycoproteins on the virion envelope. A GST chimera bearing the cytoplasmic tail of glycoprotein E (gE.CT) was found to be capable of binding UL16 expressed in mammalian or insect cells. To better understand the molecular mechanism of this signaling process, the interaction network emanating from UL16 was investigated. In addition, previously using a GST pull-down assay, it was observed that UL16 interacts with virus-specific proteins from HSV- and PRV-infected cell lysates, providing evidence that UL16 is present in protein complexes. To characterize native complexes that contain UL16, a recombinant virus was constructed expressing a tagged derivative. Using the combination of tandem affinity purification and mass spectrometry analysis, we identified gE to be present in the complexes isolated from infected cells. The UL16-gE interaction was confirmed in co-immunoprecipitation assays with infected cell lysates. Moreover, mutational analyses of gE.CT have suggested that in infected cells UL16 may interact with gE.CT in both UL11-dependent and -independent manners. Based on all available data, we hypothesize that UL11, UL16, and gE may form a tripartite complex which plays a role in multiple aspects of the virus life cycle, including signaling events during virus attachment, virion maturation, or cell-to-cell spread. Collectively, our research focused on the protein-protein network has built a foundation for future studies, and also advanced our current knowledge of herpesvirus replication.