PACKAGING DETERMINANTS OF THE HERPES SIMPLEX VIRUS TYPE 1 TEGUMENT PROTEIN pUL46
Open Access
- Author:
- Murphy, Michael Allen
- Graduate Program:
- Microbiology and Immunology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 05, 2008
- Committee Members:
- Richard James Courtney, Dissertation Advisor/Co-Advisor
Richard James Courtney, Committee Chair/Co-Chair
Craig Matthew Meyers, Committee Member
David Joseph Spector, Committee Member
John Warren Wills, Committee Member
Vincent Chau, Committee Member - Keywords:
- HSV
Tegument
Virus Assembly
UL46
VP11/12 - Abstract:
- ABSTRACT All herpesviruses are composed of three prototypical structures: a DNA-filled capsid, a glycoprotein-containing envelope, and a complex proteinaceous layer located between the capsid and envelope known as the tegument. The process of virion assembly that gives rise to these structures occurs within multiple cellular sites. Herpesvirus assembly initiates within the nucleus, where the capsid assembles and incorporates the viral genomic DNA. Upon completion of capsid assembly, the initial layer of tegument is added to the capsid within the nucleus. Additional tegument proteins are added to the capsid after it exits the nucleus and travels through the cytoplasm. Although the addition of tegument proteins to the capsid (tegumentation) occurs in both the nucleus and the cytoplasm, the majority of the tegument proteins are incorporated into the virion during secondary envelopment at trans-Golgi network (TGN)-derived vesicles. Packaging of the individual tegument proteins, during secondary envelopment, is dependent upon complexes of tegument proteins and glycoproteins, which assemble on the TGN-derived vesicles. The molecular mechanisms that are responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To gain a better understanding of the tegumentation process, we initiated studies that focused on the herpes simplex virus type 1 (HSV-1) tegument protein pUL46. The UL46 gene encodes two proteins that are packaged into the virion; therefore, pUL46 is also designated as virion polypeptides 11 and 12 (VP11/12). In addition to the structural role they play, several tegument proteins also perform regulatory functions once the virion is uncoated within the cytoplasm. pUL46 enhances the trans-activation of immediate-early (IE) promoters, an event primarily orchestrated by VP16. Despite reports of an interaction between pUL46 and VP16, the mechanism for enhancement of VP16 activity is currently unknown. Since deletion of UL46 from alphaherpesvirus homologues does not inhibit viral replication, this function of pUL46 is not required for completion of the viral life cycle. In addition to being non-essential for replication, pUL46 is packaged into the virion at relatively high levels and because of these two attributes; we chose pUL46 as the centerpiece of our studies. Inclusion of a tegument protein into the virion is dependent upon the protein trafficking to the site of virus assembly; therefore, experiments were designed to compare the cellular localization of pUL46 in the absence and presence of HSV-1 infection. Regardless of the conditions used, the majority of pUL46 localizes within the cytoplasm as punctate structures, which is similar in appearance to the membrane-associated tegument protein VP22. To determine if pUL46 also associates with membranes, we performed membrane flotation experiment, which showed that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. The membrane flotation results also showed that expression of pUL46 during infection results in hyperphosphorylation of pUL46, which is infection-specific. In an effort to identify the residues that facilitate association of pUL46 with membranes, we constructed plasmids encoding pUL46 truncations and then analyzed these proteins for their ability to associate with membranes. The results indicate that the amino terminus of pUL46 (residues 1-446) associates with membranes at levels equivalent to that of the full-length protein (residues 1-720). In comparison to full-length pUL46, the carboxy terminus (residues 447-720) associates with membranes at decreased levels, which suggests the amino terminus contains the domains that facilitate membrane association. Because pUL46 associates with cellular membranes, we expected the protein to associate with the inner surface of the virus envelope. Despite the propensity for pUL46 to associate with membranes, the majority of pUL46 associates with capsids upon removal of the envelope from extracellular virions. This ability of pUL46 to bind capsids was subsequently confirmed by an in vitro capsid pulldown assay, in which purified pUL46-GST specifically interacts with capsids purified from the nuclear fraction of HSV-1 infected cells. Because of these results, we hypothesize that pUL46 displays a dynamic interaction between cellular membranes and capsids. Since the amino terminus of pUL46 facilitates its association with membranes, we proposed that the same region of the protein is critical for its packaging into the virion. Initial studies, using plasmid-based expression of truncated pUL46, showed that the carboxy terminus of pUL46 is dispensable for virion incorporation. However, the carboxy terminus (residues 447-720), when expressed from a plasmid, is packaged into the virion at levels comparable to full-length pUL46. In order to pursue these studies in a more relevant context, we constructed multiple viruses that encode various truncations of pUL46. Analysis of these recombinant viruses showed that residues 1-446 are sufficient to facilitate packaging of this truncated protein at levels comparable with full-length pUL46. In contrast to the plasmid-based assay, residues 447-720 are not packaged into virions, despite their relative abundance within infected cells. Confocal microscopy of both transfected and infected cells showed that residues 447-720 accumulate within the nucleus, while residues 1-446 accumulate within the cytoplasm. Surprisingly, the use of these recombinant viruses also revealed that infection with a virus that encodes full-length pUL46 results in the release of virions that contain both full-length pUL46 and a form that is truncated from the amino terminus. Based upon these results, we propose that a population of pUL46 is truncated during virus assembly and that removal of the amino terminus targets the truncated protein to the nucleus, where the protein enhances the activity of VP16. In summary, the studies described within this dissertation provide novel insights into the packaging of tegument proteins that occurs during HSV-1 assembly. Analysis of pUL46 has revealed the protein’s use of packaging mechanisms that are common among other tegument proteins, as well as, mechanisms that may be unique to pUL46. The identification of these unique mechanisms provides a basis for future dissection of the pathways that result in packaging and truncation of pUL46.