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McNab 1999; Newcomb et al. 2000) and constituted or closely approximated in
vitro (Newcomb et al. 1999, 2000), are less angular than late-stage capsids and
contain scaffolding proteins but no viral DNA. An involvement of the pAP amino-
conserved domain during this nuclear stage of capsid formation was discovered
with the L47A mutant virus, which showed a dramatically altered distribution of
pAP within the nucleus (Fig. 5) and gave rise to overall fewer and aberrant capsids
(Loveland et al. 2007).
Absence of detected procapsid formation in the cytoplasm, where all of the nec-
essary proteins are present, may be explained by the comparatively higher protein
concentrations in the nucleus or by the presence of specific nuclear initiating or
enhancing factors. Alternatively or additionally, there may be assembly-enhancing
changes in the complexes that signal or result from nuclear translocation. In HSV,
where it has been possible to examine capsid assembly in isolation from other viral
proteins, the homologs of MCP, pAP, mCP, and mCBP (i.e., HSV VP5, preVP22a,
VP23, VP19c) are all necessary and collectively sufficient to assemble the capsid
shell (Tatman et al. 1994; Thomsen et al. 1994). Similar attempts to make CMV
capsids from their recombinantly cloned and expressed counterpart genes have not
yet succeeded and it is unresolved whether this is due to technical factors (e.g.,
CMV protein expression levels too low) or perhaps to differences in the minimal
compliment of proteins required.
Fig. 5
Distribution of UL80 proteins in nuclei of HCMV-infected cells. An HCMV-AD169 bac-
mid was mutated to block self-interaction of the UL80 proteins (i.e., point mutation L47A in pAP
sequence) (Loveland et al. 2007). Both the HCMV-AD169 bacmid and the parental wild-type
bacmid were also modified to incorporate a tetra-cysteine tag (CCPGCC) into the UL80 proteins.
Virus derived from each bacmid was used to infect human foreskin fibroblasts, which were stained
with the biarsenical dye FIAsH when strong cytopathic effects were observed. Shown here are
images of the stained, living cells taken by confocal fluorescence microscopy. The FIAsH-stained
UL80 proteins are organized in tubular and rod-shaped structures in nuclei of cells infected with
the L47A mutant (
first three panels from the left
), but in an entirely different pattern reminiscent
of the intranuclear inclusions typically observed in nuclei of cells infected with wild type virus
(
right-hand panel
). (Modified from Loveland et al. 2007)
