Isolation of herpes simplex virus procapsids from cells infected with a protease-deficient mutant virus.
Herpes simplex virus type 1 (HSV-1) capsid proteins assemble in vitro into spherical procapsids that differ markedly in structure and stability from mature polyhedral capsids but can be converted to the mature form. Circumstantial evidence suggests that assembly in vivo follows a similar pathway of procapsid assembly and maturation, a pathway that resembles those of double-stranded DNA bacteriophages. We have confirmed the above pathway by isolating procapsids from HSV-1-infected cells and characterizing their morphology, thermal sensitivity, and protein composition. Experiments were carried out with an HSV-1 mutant (m100) deficient in the maturational protease for which it was expected that procapsids-normally, short-lived intermediates-would accumulate in infected cells. Particles isolated from m100-infected cells were found to share the defining properties of procapsids assembled in vitro. For example, by electron microscopy, they were found to be spherical rather than polyhedral in shape, and they disassembled at 0 degrees C, unlike mature capsids, which are stable at this temperature. A three-dimensional reconstruction computed at 18-A resolution from cryoelectron micrographs showed m100 procapsids to be structurally indistinguishable from procapsids assembled in vitro. In both cases, their predominant components are the four essential capsid proteins: the major capsid protein (VP5), the scaffolding protein (pre-VP22a), and the triplex proteins (VP19C and VP23). VP26, a small, abundant but dispensable capsid protein, was not found associated with m100 procapsids, suggesting that it binds to capsids only after they have matured into the polyhedral form. Procapsids were also isolated from cells infected at the nonpermissive temperature with the HSV-1 mutant tsProt.A (a mutant with a thermoreversible lesion in the protease), and their identity as procapsids was confirmed by cryoelectron microscopy. This analysis revealed density on the inner surface of the procapsid scaffolding core that may correspond to the location of the maturational protease. Upon incubation at the permissive temperature, tsProt.A procapsids transformed into polyhedral, mature capsids, providing further confirmation of their status as precursors.
Newcomb
W W
WW
Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
Trus
B L
BL
Cheng
N
N
Steven
A C
AC
Sheaffer
A K
AK
Tenney
D J
DJ
Weller
S K
SK
Brown
J C
JC
D000818:Animals; D002213:Capsid / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D017353:Gene Deletion; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D011498:Protein Precursors / Q000096:biosynthesis* / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D014709:Vero Cells; D014764:Viral Proteins / Q000378:metabolism; D019065:Virus Assembly / Q000502:physiology
D011498:Protein Precursors; D014764:Viral Proteins; C079287:scaffold protein, Herpes simplex virus-1; D012697:Serine Endopeptidases