Vaccinia virus (VV) is a large double-stranded DNA virus that is a prototypic
member of the orthopoxvirus family. Previous works has showed that three of the major
structural proteins found within the mature VV virion core 4a, 4b, and 25K are produced
from higher molecular weight precursors at late times during infection and processed via
a common morphogenic cleavage pathway that is intimately linked with virion assembly
and maturation. The enzyme that carries out these cleavage reactions is unknown.
A transient expression assay was used to demonstrate that the 17L gene product
and its encoded cysteine proteinase activity is responsible for cleavage of each of the
three major core protein precursors. Cleavage was demonstrated to occur at the authentic
Ala-Gly-Xaa cleavage sites and require active enzyme. A truncated 17L protein lost the
ability to cleave the core protein precursors.
A conditional-lethal recombinant virus was constructed in which the expression of
the 17L gene is under the control of the tetracycline operator/repressor system. In the
absence of 17L expression, processing of the major VV core proteins is inhibited and
electron microscopy revealed defects in virion morphogenesis prior to complete core
condensation. Plasmid-borne 17L is capable of rescuing the growth of this virus.
A structural model of 17L was developed and a unique chemical library was
assayed for both cell toxicity and the ability to inhibit the growth of VV in tissue culture
cells. A novel class of inhibitors was discovered that is capable of inhibiting VV.
An in-vitro cleavage assay was developed to further characterize the activity of
17L. This assay is based on producing the major core protein precursors in a coupled
transcription and translation assay and then mixing them with 17L enzyme extracts. Using
this assay, 17L is shown to be capable of cleavage of each substrate. 17L is further
characterized as a cysteine proteinase due to the inhibitory effects of known cysteine
proteinase inhibitors such as NEM and iodoacetic acid, as well as through the use of
specific small molecule inhibitors in this in-vitro assay. / Graduation date: 2005
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/29927 |
| Date | 08 April 2005 |
| Creators | Byrd, Chelsea M. |
| Contributors | Hruby, Dennis E. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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