Examination of herpes simplex virus protease mutant capsids to define the role of the protease in capsid assembly and maturation
Date of Completion
January 2000
Keywords
Biology, Molecular|Biology, Microbiology
Degree
Ph.D.
Abstract
Herpes simplex virus type 1 (HSV-1) capsids are assembled with an internal protein scaffold. Scaffold proteins, encoded by overlapping in-frame UL26 and UL26.5 transcripts, are essential for formation and efficient maturation of capsids. UL26 encodes an N-terminal protease domain and shares identical C-terminal oligomerization and capsid protein-binding domains with UL26.5. The UL26 protease cleaves itself (releasing VP24 and VP21) and the more abundant UL26.5 protein (releasing VP22a). Unlike VP21 and VP22a, which are removed from capsids upon DNA packaging, we demonstrate that VP24 is quantitatively retained. To investigate factors controlling UL26 capsid incorporation and retention we used a virus that fails to express UL26.5 (ΔICP35 virus). Purified ΔICP35 B-capsids showed altered sucrose gradient sedimentation and lacked the dense scaffold core seen in micrographs of wild-type B-capsids. ΔICP35 capsids did not contain compensatory high levels of UL26 proteins. In contrast to UL26.5, increased expression of UL26 did not complement Δ ICP35 virus growth. These findings suggest that despite identical C-termini, UL26 products are restricted from occupying UL26.5 binding sites within the capsid. This restriction is not controlled by expression levels. Furthermore, capsids can be maintained and/or assembled on a minimal scaffold containing only wild-type levels of UL26 proteins. ^ Internal scaffold release appears tightly linked to viral DNA packaging since no mutations have separated these events. However, the mechanisms underlying this linkage are unclear. Stable intracellular capsids are known to contain several proteins required for cleavage and packaging of viral DNA. To test whether scaffold cleavage and accompanying changes in the capsid shell affected association of packaging proteins, we isolated unstable precursor procapsids from protease mutant virus infected cells. Procapsids contained unprocessed scaffold and lacked one capsid shell protein, VP26. The complement of packaging proteins associated with procapsids resembled those of B-capsids containing processed scaffold. Notably, however, amounts of the UL25 protein, essential for stable packaging of DNA, were reduced relative to B-capsids. In contrast, the ΔICP35 capsids contained increased levels of UL25. DNA-containing C-capsids lacking scaffold had the most UL25. We suggest a model whereby the UL25 protein associates with and seals capsids in response to cleavage and loss of internal scaffold proteins. ^
Recommended Citation
Sheaffer, Amy K, "Examination of herpes simplex virus protease mutant capsids to define the role of the protease in capsid assembly and maturation" (2000). Doctoral Dissertations. AAI9963292.
https://digitalcommons.lib.uconn.edu/dissertations/AAI9963292