Functional analysis of the triplex proteins (VP19C and VP23) of herpes simplex virus type 1.
The triplex of herpesvirus capsids is a unique structural element. In herpes simplex virus type 1 (HSV-1), one molecule of VP19C and two of VP23 form a three-pronged structure that acts to stabilize the capsid shell through interactions with adjacent VP5 molecules. The interaction between VP19C and VP23 was inferred by yeast cryoelectron microscopy studies and subsequently confirmed by the two-hybrid assay. In order to define the functional domains of VP19C and VP23, a Tn7-based transposon was used to randomly insert 15 bp into the coding regions of these two proteins. The mutants were initially screened for interaction in the yeast two-hybrid assay to identify the domains important for triplex formation. Using genetic complementation assays in HSV-1-infected cells, the domains of each protein required for virus replication were similarly uncovered. The same mutations that abolish interaction between these two proteins in the yeast two-hybrid assay similarly failed to complement the growth of the VP23- and VP19C-null mutant viruses in the genetic complementation assay. Some of these mutants were transferred into recombinant baculoviruses to analyze the effect of the mutations on herpesvirus capsid assembly in insect cells. The mutations that abolished the interaction in the yeast two-hybrid assay also abolished capsid assembly in insect cells. The outcome of these experiments showed that insertions in at least four regions and especially the amino terminus of VP23 abolished function, whereas the amino terminus of VP19C can tolerate transposon insertions. A novel finding of these studies was the ability to assemble herpesvirus capsids in insect cells using VP5 and VP19C that contained a histidine handle at their amino terminus.
Okoye
Mercy E
ME
Molecular Virology Laboratories, Viral Oncology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 1650 Orleans Street, The Johns Hopkins University, Baltimore, MD 21231, USA.
Sexton
Gerry L
GL
Huang
Eugene
E
McCaffery
J Michael
JM
Desai
Prashant
P
D000595:Amino Acid Sequence; D000818:Animals; D036022:Capsid Proteins / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D018259:Herpesvirus 1, Human / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D018411:Spodoptera; D019065:Virus Assembly
D036022:Capsid Proteins; C082563:VP19 protein, Human herpesvirus 1; C079750:VP23 protein, Human herpesvirus 1