Reconstitution of herpes simplex virus type 1 nuclear capsid egress in vitro.
Newly assembled herpesvirus capsids travel from the nucleus to the plasma membrane by a mechanism that is poorly understood. Furthermore, the contribution of cellular proteins to this egress has yet to be clarified. To address these issues, an in vitro nuclear egress assay that reproduces the exit of herpes simplex virus type 1 (HSV-1) capsids from nuclei isolated from infected cells was established. As expected, the assay has all the hallmarks of intracellular transport assays, namely, a dependence on time, energy, and temperature. Surprisingly, it is also dependent on cytosol and was slightly enhanced by infected cytosol, suggesting an implication of both host and viral proteins in the process. The capsids escaped these nuclei by budding through the inner nuclear membrane, accumulated as enveloped capsids between the two nuclear membranes, and were released in cytosol exclusively as naked capsids, exactly as in intact cells. This is most consistent with the view that the virus escapes by crossing the two nuclear membranes rather than through nuclear pores. Unexpectedly, nuclei isolated at the nonpermissive temperature from cells infected with a U(L)26 thermosensitive protease mutant (V701) supported capsid egress. Although electron microscopy, biochemical, and PCR analyses hinted at a likely reconstitution of capsid maturation, DNA encapsidation could not be confirmed by a traditional SQ test. This assay should prove very useful for identification of the molecular players involved in HSV-1 nuclear egress.
Rémillard-Labrosse
Gaudeline
G
Department of Pathology and Cell Biology, University of Montreal, P.O. Box 6128, Succursale Centre-Ville, Montreal, Quebec, Canada H3C 3J7.
Guay
Ginette
G
Lippé
Roger
R
D036022:Capsid Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002459:Cell Fusion; D002467:Cell Nucleus / Q000235:genetics; D003600:Cytosol / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D018259:Herpesvirus 1, Human / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D046529:Microscopy, Electron, Transmission; D013696:Temperature; D019065:Virus Assembly
D036022:Capsid Proteins; D004279:DNA, Viral