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Journal of Virology, March 2005, p. 2754-2767, Vol. 79, No. 5
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.5.2754-2767.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Human Cytomegalovirus Labeled with Green Fluorescent Protein for Live Analysis of Intracellular Particle Movements

Kerstin Laib Sampaio,1 Yolaine Cavignac,1 York-Dieter Stierhof,2 and Christian Sinzger1*

Institute of Medical Virology,1 Center for Plant Molecular Biology, University of Tübingen, Tübingen, Germany2

Received 23 January 2004/ Accepted 12 October 2004

Human cytomegalovirus (HCMV) replicates in the nuclei of infected cells. Successful replication therefore depends on particle movements between the cell cortex and nucleus during entry and egress. To visualize HCMV particles in living cells, we have generated a recombinant HCMV expressing enhanced green fluorescent protein (EGFP) fused to the C terminus of the capsid-associated tegument protein pUL32 (pp150). The resulting UL32-EGFP-HCMV was analyzed by immunofluorescence, electron microscopy, immunoblotting, confocal microscopy, and time-lapse microscopy to evaluate the growth properties of this virus and the dynamics of particle movements. UL32-EGFP-HCMV replicated similarly to wild-type virus in fibroblast cultures. Green fluorescent virus particles were released from infected cells. The fluorescence stayed associated with particles during viral entry, and fluorescent progeny particles appeared in the nucleus at 44 h after infection. Surprisingly, strict colocalization of pUL32 and the major capsid protein pUL86 within nuclear inclusions indicated that incorporation of pUL32 into nascent HCMV particles occurred simultaneously with or immediately after assembly of the capsid. A slow transport of nuclear particles towards the nuclear margin was demonstrated. Within the cytoplasm, most particles performed irregular short-distance movements, while a smaller fraction of particles performed centripetal and centrifugal long-distance movements. Although numerous particles accumulated in the cytoplasm, release of particles from infected cells was a rare event, consistent with a release rate of about 1 infectious unit per h per cell in HCMV-infected fibroblasts as calculated from single-step growth curves. UL32-EGFP-HCMV will be useful for further investigations into the entry, maturation, and release of this virus.

* Corresponding author. Mailing address: Institut für Medizinische Virologie, Universität Tübingen, Elfriede-Aulhorn-Strasse 6, D-72076 Tübingen, Germany. Phone: 7071 2987459. Fax: 7071 295790. E-mail: christian.sinzger{at}med.uni-tuebingen.de.

Journal of Virology, March 2005, p. 2754-2767, Vol. 79, No. 5
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.5.2754-2767.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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