Vaccinia virus (VV) is a large DNA virus belonging to the Orthopoxvirus
family. The viral replicative life cycle takes place solely within the cytoplasm
of a mammalian host cell. The VV genome contains 196 open reading frames
which are expressed in a highly regulated and temporal fashion in order to
bring about the production of a mature virion. In the process of viral
replication many VV proteins are synthesized that require posttranslational
modifications to become functional. A few of these modifications include,
glycosylation, ADP-ribosylation, phosphorylation, fatty acid acylation, and
proteolytic processing. This last modification is especially important with
regard to the structural proteins of the virus in that they undergo prysis
for an infectious virus particle to be formed, a common theme in viral
systems. In order to understand these events in more detail, three abundant
virion protein constituents 4a, 4b, and 25K were chosen as models for study.
The three main questions we wanted to answer were: Is there a cleavage
consensus site within the precursors, what protease(s) and/or factors are
necessary for the process, and how are the events regulated in vivo? Our
approach included development of specific immunological reagents to identify
cleavage products as well as to show where these core proteins are located
during virion assembly. We have subsequently identified cleavage products
by N-terminal microsequence from each of the three structural proteins and
this information has elucidated a putative cleavage consensus site of Ala-Gly-
X, where cleavage is proposed to take place between the Gly and X and X is
usually an aliphatic residue. The immunological reagents were used in
conjunction with immunofluorescent and immunogold labeling analyses to
identify the location of these core proteins during virion assembly. Core
proteins were localized to the virosomes in VV infected cells, to the viroplasm
of immature virus particles, and to the center of mature virions. Precursor
specific antiserum indicated that the larger molecular weight precursors of
core proteins are within immature virions as well. From these results the
following conclusions can be made. Identification of a putative cleavage
consensus site suggests that proteolytic processing is an endoproteolytic
event. The observation that precursor structural proteins were found within
immature particles indicates that the proteinase responsible for cleavage is
also present. The fact that assembly has to occur before proteolytic
processing of VV structural proteins suggests that the cleavage events are
dependent upon a specific core protein conformation. However the nature of
this conformational requirement is not known. Further research is underway
to develop a full understanding of the proteolytic events during virion
morphogensis. / Graduation date: 1993
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/36129 |
| Date | 05 November 1992 |
| Creators | VanSlyke, Judy K. |
| Contributors | Hruby, Dennis E. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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