Proteolytic maturation of vaccinia virus structural proteins Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/th83m150m

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  • 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.
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