Biology Reference
In-Depth Information
Taking procapsids as the end point of capsid assembly, two major changes occur
during its maturation: elimination of the scaffolding proteins and incorporation of
viral DNA. The two processes appear to be coupled. Elimination of the internal scaf-
folding proteins is effected by proteolysis catalyzed by pPR (Fig. 4, step 6). Although
able to cleave itself at five sites, pPR activity appears to be modulated during infec-
tion such that its autoproteolysis is initiated following procapsid formation. It has
recently been determined using purified HCMV pPR that the active enzyme is a
trimer or tetramer whose primary subunit interaction is through the amino-conserved
domain of its scaffolding region (Brignole and Gibson 2007). This quaternary struc-
ture is very different from that of purified assemblin, whose monomer activates by
dimerizing through sequences located in its carboxyl end (Chen et al. 1996; Cole
1996; Darke et al. 1996; Margosiak et al. 1996; Qiu et al. 1996; Shieh et al. 1996;
Tong et al. 1996). Active pPR cleaves the R site to release the well-characterized
proteolytic domain, assemblin, and cleaves the M site to sever the tail sequence link-
ing itself and pAP to MCP in the capsid shell (Figs. 2, 3).
Although having comparable overall enzymatic rates (e.g., k
cat
/K
M
), pPR and
assemblin are distinguished in ways thought to reflect mechanistic differences
(Brignole and Gibson 2007). First, whereas imidazole can chemically rescue the
enzymatic activity of assemblin substituted with Gly at its catalytically critical
His63, it fails to restore activity to the same mutation in pPR (McCartney et al.
2005), indicating a difference in the catalytic sites of the two forms of the enzyme.
Second, the sequences driving assemblin dimerization have comparatively little
effect on pPR oligomerization, yet this interaction of assemblin is thought to induce
catalytic-site changes required for its activity (Tong et al. 1996; Buisson et al.
2002). And third, there is evidence that the enzymatic rates of pPR and assemblin
may be comparable because of offsetting differences in their k
cat
(higher for pPR)
and K
M
, again suggesting catalytic-site differences between pPR and assemblin that
may be important to regulating activity.
Proteolysis results in essentially all M and R sites being cleaved. HCMV pPR
makes three additional cleavages. Two are at the internal (I) and cryptic (C) sites
within assemblin and reduce production of infectious virus when blocked (Chan
et al. 2002; Loveland et al. 2005). These cleavages reduce the size and interac-
tions of the scaffolding proteins, facilitating their elimination from the capsid in
preparation for DNA packaging (Fig. 4, step 7). Unlike HSV, which retains
assemblin in its mature virion, all remnants of HCMV pAP and pPR, including
assemblin, are eliminated from the capsid. Absence of counterpart I and C sites
in the HSV assemblin homolog, and persistence of HSV assemblin in the mature
virion, may reflect a requirement for additional space within the CMV capsid to
accommodate its 51% longer DNA genome (Chan et al. 2002; Loveland et al.
2005). The significance of a fifth cleavage site just discovered in the carboxyl tail
(T site) of purified pPR is undetermined. Electrostatic repulsion by the incoming
viral DNA has been suggested to play a role in displacing the internal scaffolding
proteins (McClelland et al. 2002), and specific pAP phosphorylations that weaken
scaffolding protein self-interactions may by important to this process (Casaday et al.
2004; Gibson 2006).
