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ICP34.5 influences herpes simplex virus type 1 maturation and egress from infected cells in vitro
S M Brown1, A R MacLean, J D Aitken
1MRC Virology Unit, Glasgow, U.K.
Abstract:
We have previously demonstrated that efficient replication of mutant herpes simplex virus which fails to synthesize the polypeptide ICP34.5 is cell type and cell state dependent. ICP34.5 negative viruses do not grow in stationary state mouse embryo fibroblast 3T6 cells whereas the growth kinetics in BHK cells are indistinguishable from those of wild-type. We now demonstrate that this defect is not due to an inability of mutant virus to adsorb to 3T6 cells but rather to an inability to spread from the initially infected cells. Electron microscopic studies with wild-type HSV in both BHK and 3T6 cells revealed virus particles equally distributed between nucleus and cytoplasm, and additionally in the extracellular matrix. In BHK cells infected with the ICP34.5 negative mutant 1716, virus is likewise distributed between nucleus and cytoplasm but in 50% of the infected cells there is marked delamination and swelling of the nuclear membrane. In addition there is evidence of a significant number of particles trapped between the nuclear lamellae. When 1716 is used to infect 3T6 cells, over 90% of the virus particles are confined to the nuclei and the number of infected cells remains constant between 24 and 48 h with no increase in the proportion of extracellular virus. Failure to express ICP34.5 appears therefore to result in a defect in virus maturation and egress from the nuclei of infected cells. Egress of HSV from the nuclei to the extracellular space is thought to occur via two pathways. We postulate that lack of expression of ICP34.5 results in one of these pathways being blocked. In BHK cells this leads to overloading of the alternative pathway with a buildup of particles in the nuclear lamellae and associated endoplasmic reticulum. In stationary state 3T6 cells, it appears that there is no functional alternative pathway. We conclude that ICP34.5 exerts an effect on HSV maturation by controlling the passage of virus through infected cells.
Insights
The herpes simplex virus (HSV) ICP34.5 protein is crucial for efficient virus replication and cell-to-cell spread. Its absence impairs HSV maturation and egress from infected cells, particularly in stationary cells.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Herpes simplex virus (HSV) replication is influenced by the viral protein ICP34.5.
- ICP34.5-deficient HSV exhibits cell type and cell state-dependent growth defects.
- Previous studies showed ICP34.5-negative viruses do not grow in stationary 3T6 cells but replicate normally in BHK cells.
Purpose of the Study:
- To investigate the mechanism behind the cell type and cell state-dependent replication defect of ICP34.5-negative HSV.
- To determine if the defect in ICP34.5-negative HSV replication is due to impaired adsorption or cell-to-cell spread.
- To elucidate the role of ICP34.5 in HSV maturation and egress from infected cells.
Main Methods:
- Comparative analysis of wild-type and ICP34.5-negative HSV (1716) replication in BHK and 3T6 cells.
- Electron microscopy to visualize virus particle distribution and cellular alterations.
- Assessment of virus adsorption, spread, and intracellular localization.
Main Results:
- ICP34.5-negative HSV adsorbs normally to 3T6 cells, indicating the defect lies in spread.
- In BHK cells, ICP34.5-negative HSV shows nuclear/cytoplasmic distribution with some nuclear membrane abnormalities.
- In 3T6 cells, >90% of ICP34.5-negative HSV particles are nuclear-restricted, with no increase in extracellular virus, suggesting impaired nuclear egress and maturation.
Conclusions:
- Failure to express ICP34.5 results in a defect in HSV maturation and nuclear egress.
- ICP34.5 likely controls virus passage through infected cells, potentially by regulating two egress pathways.
- In BHK cells, an alternative pathway is overloaded; in 3T6 cells, this pathway appears non-functional, blocking virus release.