Vaccinia virus (VV) encodes at least six proteins that are modified by the
addition of a 14-carbon saturated fatty acid through an amide linkage and at least
eight proteins that are modified post-translationally by the addition of a 16-carbon
saturated fatty acid through linkage to cysteine residues. These post-translational
modifications are referred to as myristylation and palmitylation, respectively. The
purpose of this work was to further characterize the known myristylproteins and to
define a consensus motif for the palmitylation of a protein so that we could identify
and begin the characterization of new palmitylproteins.
Through this work we have identified a loosely conserved consensus motif
that directs the palmitylation of a protein. Using the VV palmitylprotein p37, we
characterized this motif and then used it in the identification three new VV
palmitylproteins. We have also determined the membrane orientation of the VV
myristylproteins, L1R, within the intracellular mature virus (IMV) particle.
Hydrophobicity plot analysis identified two possible membrane orientations based
on two putative transmembrane domains. Through transient expression data, L1R
was determined to span the IMV membrane twice, with both the amino and
carboxy termini being on the lumen side.
Three lac recombinant viruses which are inducible for the A16L, E7R, and
G9R open reading frames were created and analyzed using a newly developed
vector system that fuses the green fluorescent protein to the neomycin resistance
gene. Propagation of these viruses in the absence of the inducer IPTG determined
that these genes are essential to VV replication.
We have identified and characterized the primary structural determinants
specifying the modification of a protein by palmitate, and have identified three new
VV palmitylproteins. In addition, the membrane orientation of the VV
myristylprotein L1R was deduced, which can enable the construction of better
recombinant vaccines through efficient antigen presentation. Lastly, we have
developed a technique and vector system to easily create and isolate VV
recombinants for characterization. This system enabled us to further characterize
the A16L, E7R, and G9R myristylproteins. / Graduation date: 2002
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/32372 |
| Date | 05 September 2001 |
| Creators | Hansen, Scott G. |
| Contributors | Hruby, Dennis E. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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