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Journal of Virology, August 2004, p. 8002-8014, Vol. 78, No. 15
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.15.8002-8014.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Compartmentalization of VP16 in Cells Infected with Recombinant Herpes Simplex Virus Expressing VP16-Green Fluorescent Protein Fusion Proteins

Sylvie La Boissière, Ander Izeta, Sophie Malcomber, and Peter O'Hare*

Marie Curie Research Institute, Oxted, Surrey RH8 OTL, United Kingdom

Received 7 January 2004/ Accepted 19 April 2004

VP16 is an essential structural protein of herpes simplex virus. It plays important roles in immediate-early transcriptional regulation, in the modulation of the activities of other viral components, and in the pathway of assembly and egress of infectious virions. To gain further insight into the compartmentalization of this multifunctional protein we constructed and characterized recombinant viruses expressing VP16 linked to the green fluorescent protein (GFP). These viruses replicate with virtually normal kinetics and yields and incorporate the fusion protein into the virion, resulting in autofluorescent particles. De novo-synthesized VP16-GFP was first detected in a diffuse pattern within the nucleus. Nuclear VP16-GFP was progressively recruited to replication compartments, which coalesced into large globular domains. By 10 to 12 h after infection additional distinct foci containing VP16-GFP could be seen, almost exclusively located at the periphery of the replication compartments. At the same time pronounced accumulation was observed in the cytoplasm, first in a diffuse pattern and then accumulating in vesicle-like compartments which were concentrated in an asymmetric fashion reminiscent of the Golgi. Inhibition of DNA replication resulted in prolonged diffuse nuclear distribution with minimal cytoplasmic accumulation. Treatment with brefeldin disrupted the cytoplasm vesicular pattern, resulting in redistributed large foci. Time-lapse microscopy demonstrated various dynamic features of infection, including the active induction of very long cellular projections (up to 100 µM). Vesicular clusters containing VP16 were transported within projections to the termini, which developed bulbous ends and appeared to embed into the membranes of adjacent uninfected cells.

* Corresponding author. Mailing address: Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 OTL, United Kingdom. Phone: 44 1 883722306. Fax: 44 1 883714375. E-mail: p.ohare{at}mcri.ac.uk.

Journal of Virology, August 2004, p. 8002-8014, Vol. 78, No. 15
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.15.8002-8014.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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