THE DYNAMIC HERPESVIRUS TEGUMENT: RECEPTOR BINDING INDUCED RELEASE OF UL16 FROM THE HERPES SIMPLEX VIRUS CAPSID
Open Access
- Author:
- Meckes, David
- Graduate Program:
- Microbiology and Immunology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 12, 2008
- Committee Members:
- John Warren Wills, Committee Chair/Co-Chair
Richard James Courtney, Committee Member
Jianming Hu, Committee Member
Leslie Joan Parent, Committee Member
John Michael Flanagan Jr., Committee Member - Keywords:
- glycoprotein
NEM
HSV
capsid
tegument
assembly
herpes
entry
virus
cyteines
signaling - Abstract:
- The complex and multistep process of herpesvirus assembly occurs in numerous cellular compartments, and ultimately concludes with the construction of the mature virion. Herpes simplex virus (HSV) particles are composed of more than 40 different virally-encoded proteins that comprise three morphologically distinct structures: the icosahedral nucleocapsid containing the viral DNA; the host-derived lipid envelope with virus-encoded glycoproteins; and the tegument, an assortment of proteins between the nucleocapsid and the envelope. The specific mechanisms by which these various viral proteins come together to drive the assembly and budding processes are poorly understood. The focus of this dissertation is UL16, a conserved tegument protein whose function remains a mystery for any herpesvirus. It has been reported to be capsid-associated, possibly through an interaction with UL21, and may be involved in budding by linking capsids to the membrane-bound tegument protein UL11. UL16 has been shown to be present in all the major locations that capsids are found (i.e., the nucleus, cytoplasm, and virions), but whether it is actually capsid-associated in each of these has not been reported. Therefore, capsids were purified from detergent lysates of each compartment, and it was found that UL16 is present on cytoplasmic, not nuclear, capsids. This cytoplasmic capsid binding did not require an interaction with previously identified binding partners UL21 and UL11. Additionally, the population of UL16 found on cytoplasmic capsids was added prior to reaching the site of budding. This is due to the fact that unenveloped capsids still contained UL16 in membrane floatation assays, and in experiments with mutant viruses (∆UL36, ∆UL37, and gE-/gD-) that are unable to perform final envelopment. When extracellular capsids were analyzed using similar lysis conditions, the majority of UL16 (87%) was surprisingly not capsid associated. These data suggests that the interaction of UL16 with the capsid is transient during egress, and may represent the first example of a herpesvirus maturation event. Because HSV buds into the acidic compartment of the trans-Golgi network (TGN), the effect of pH on the interaction was examined. The amount of capsid-associated UL16 dramatically increased when extracellular virions were exposed to mildly-acidic media (pH 5.0-5.5), and this association was fully reversible. After budding into the TGN, capsid and tegument proteins also encounter an oxidizing environment which is conducive to disulfide bond formation. UL16 contains 20 cysteines, including two that are conserved within a putative CxxC motif, which are commonly found in protein disulfide isomerases. Pretreatment with N-ethylmaleimide (NEM), a small membrane-permeable compound that covalently modifies free cysteines, restabilizes the UL16-capsid interaction, thereby permitting the complex to be isolated following disruption of virions with NP-40. The natural trigger for releasing UL16 from extracellular capsids was found to be the binding of HSV to host cells, such that NEM was unable to preserve the interaction of UL16 with the capsid. This internal change was also observed upon binding to immobilized attachment receptors (heparin) in a manner that requires glycoprotein C. Therefore, the interaction of HSV with its initial cell-attachment proteins induces a rapid and highly efficient structural change in the tegument. This represents the first evidence for transmission of a signal—from the “outside-in”—across a virus envelope as a result of receptor binding. Based on all the available data, UL16 is stably added to capsids prior to their arrival at the site of budding in the cytoplasm. Subsequently, something destabilizes this capsid interaction during egress (e.g., low pH and oxidizing conditions), so that UL16 is efficiently released from the capsid upon gC-heparan sulfate binding during early entry events. Where the tegument was once viewed as static layers of proteins, the studies described in this dissertation reveal that this region is more dynamic than previously recognized.