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Journal of Virology, February 2001, p. 1236-1251, Vol. 75, No. 3
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.3.1236-1251.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Characterization of Herpes Simplex Virus-Containing Organelles by Subcellular Fractionation: Role for Organelle Acidification in Assembly of Infectious Particles

Carol A. Harley, Anindya Dasgupta, and Duncan W. Wilson*

Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461

Received 26 September 2000/Accepted 7 November 2000

The cytoplasmic compartments occupied by exocytosing herpes simplex virus (HSV) are poorly defined. It is unclear which organelles contain the majority of trafficking virions and which are occupied by virions on a productive rather than defective assembly pathway. These problems are compounded by the fact that HSV-infected cells produce virus continuously over many hours. All stages in viral assembly and export therefore coexist, making it impossible to determine the sequence of events and their kinetics. To address these problems, we have established assays to monitor the presence of capsids and enveloped virions in cell extracts and prepared HSV-containing organelles from normally infected cells and from cells undergoing a single synchronized wave of viral egress. We find that, in both cases, HSV particles exit the nucleus and accumulate in organelles which cofractionate with the trans-Golgi network (TGN) and endosomes. In addition to carrying enveloped infectious virions in their lumen, HSV-bearing organelles also displayed nonenveloped capsids attached to their cytoplasmic surface. Neutralization of organellar pH by chloroquine or bafilomycin A resulted in the accumulation of noninfectious enveloped particles. We conclude that the organelles of the TGN/endocytic network play a key role in the assembly and trafficking of infectious HSV.

* Corresponding author. Mailing address: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: (718) 430 2305. Fax: (718) 430 8567. E-mail: wilson{at}aecom.yu.edu

Journal of Virology, February 2001, p. 1236-1251, Vol. 75, No. 3
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.3.1236-1251.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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