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In order to produce infectious virus progeny, vaccinia virus (VV)
undergoes morphogenic proteolysis to regulate the structural rearrangements of
virus particles. Several of the major structural precursor proteins of VV are
cleaved at a conserved Ala-Gly-X (where X is any amino acid) motif by the VV
I7L core protein proteinase at a step, which is necessary for formation of mature
virus particles. VV A12L encodes a 25kDa core protein, which is cleaved at an
AG/A site, yielding a 17kDa cleavage product. Both A12L precursor and the
cleavage product are localized to mature virions. The open reading frame (ORF)
of A12L contains two more AG/X (AG/K) sites, however, cleavage at these sites
has not been analyzed. Therefore, the aim of this study is to characterize the in
vivo processing of A12L proteolysis and elucidate the biological function of A12L.
The result of these studies would provide more details on the regulation and
participation of VV proteolysis during the morphogenic transitions.
Proteolytic processing of A12L produces multiple peptides, which do not
appear to utilize AG/K sites, but rather cleavages occur at both the N- and Cterminus.
Of the three AG/X motifs in A12L, cleavage has only been
demonstrated at the AG/A site. The enzyme responsible for this cleavage has
been shown to be I7L. Immunoprecipitation studies have shown that A12L
associates with VV core and membrane proteins. A conditional mutant virus of
A12L was constructed and determined the essentiality of A12L in virus replication
and helped to elucidate its functions in the assembly of virus particles. An AG/A
site mutation abrogated the ability of the transfected A12L gene to rescue the
conditional mutant under non-permissive conditions, indicating that its proteolysis
at the AG/A site is required during viral replication. Next, we compared the protein
expression of A12L with D13L, an internal scaffolding protein, to investigate the
role of A12L in virus assembly. We showed that A12L is stably synthesized only in
the presence of D13L. Consequently, we established that A12L protein and its
proteolysis participate in viral assembly subsequent to D13L involvement. |
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