ACYL DETERMINANTS OF THE HERPES SIMPLEX VIRUS TYPE 1 UL11 TEGUMENT PROTEIN REQUIRED FOR MEMBRANE TRAFFICKING AND VIRION ENVELOPMENT
Open Access
- Author:
- Baird, Nicholas Lindsay
- Graduate Program:
- Microbiology and Immunology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 03, 2009
- Committee Members:
- John Warren Wills, Dissertation Advisor/Co-Advisor
John Warren Wills, Committee Chair/Co-Chair
Richard James Courtney, Committee Member
David Joseph Spector, Committee Member
Leslie Joan Parent, Committee Member
Ira Joseph Ropson, Committee Member - Keywords:
- Lipid Rafts
Myristylation
Palmitylation
Tegument
UL11
Herpes - Abstract:
- Herpes simplex virus type 1 (HSV-1) is a complex virus composed of over 50 virally encoded proteins that form 3 distinct physical structures. The innermost structure, the nucleocapsid, contains the viral genome. The outermost structure is a host-derived lipid envelope coated with virally-encoded membrane proteins, many of which are glycosylated. Located between the two regions is the tegument, of which UL11 is a member. UL11 is a small (96 amino acid) protein that is membrane bound via two covalently attached fatty acids. Myristate is 14-carbons long and is added co-translationally to UL11 via a non-reversible amide bond, whereas palmitate is 16-carbons in length and is added post-translationally via a reversible thioester bond. Myristylation is a prerequisite to palmitylation; however, the former provides only weak membrane binding strength whereas the latter penetrates deeper into the lipid bilayer to form a more stable protein/membrane interaction. The addition of both fatty acids to UL11 is required for membrane trafficking and the subsequent accumulation at the site of virus assembly, where UL11 is predicted to function during the envelopment step, albeit via an unknown mechanism. The studies in this dissertation were designed to better understand what role UL11 plays during virus envelopment, and specifically to what extent the acylations of UL11 are required for such functions. As a first step, the ability of UL11 to localize to lipid rafts, or detergent resistant membranes (DRMs), was analyzed. Dual acylation of proteins with myristate and palmitate is a classical DRM targeting signal, and UL11 was found to be no exception. These studies also demonstrated the importance of two additional motifs within UL11 for DRM accumulation. The “di-leucine” (LI, leucine-isoleucine) and acidic cluster (AC) motifs are involved in targeting UL11 from the plasma membrane (PM) to the site of virus assembly, the trans-Golgi network (TGN). Mutants of UL11 that lack the palmitate moiety were reduced for DRM association and mutants that lack both myristate and palmitate were further reduced to background levels. Removal of only a single trafficking motif (LI or AC) did not alter the ability of UL11 to associate with DRMs, but mutants of UL11 that lack both the LI and AC had increased DRM association. Taken together, these data suggest that UL11 has highly dynamic membrane-trafficking properties, and that UL11 may require trafficking through DRMs prior to accumulation at the TGN to ensure proper function during virus envelopment. To directly examine the hypothesis that UL11 requires both myristate and palmitate for function during virus envelopment, recombinant viruses were created to express variants of UL11 with different acylation patterns. The mutants included a non-acylated UL11 (no myristate or palmitate), a partially-acylated UL11 (myristate only), or one of two UL11 chimeras that contain acylation signals from non-HSV proteins (myristylated only or myristylated and palmitylated). The results showed that only WT UL11 (with both myristate and palmitate moieties) fully rescued the growth properties of a UL11-null virus. However, it was very surprising that the non-acylated UL11 (no myristate or palmitate) rescued some growth defects of the UL11-nulll virus. These experiments demonstrated that although not essential, the acylations are important for the function of UL11 during virus envelopment. The studies detailed within this dissertation provide new insights into how the UL11 protein functions during virus assembly and envelopment. With further studies, it should be possible to elucidate the exact mechanism(s) by which UL11 functions during the assembly and envelopment of HSV-1.