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Journal of Virology, July 2006, p. 6368-6377, Vol. 80, No. 13
0022-538X/06/$08.00+0     doi:10.1128/JVI.00211-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Visualization of Intracellular Transport of Vesicular Stomatitis Virus Nucleocapsids in Living Cells

Subash C. Das, Debasis Nayak, You Zhou, and Asit K. Pattnaik*

Department of Veterinary and Biomedical Sciences and Nebraska Center for Virology, University of Nebraska—Lincoln, Lincoln, Nebraska 68588

Received 29 January 2006/ Accepted 7 April 2006

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the viral RNA polymerase. In previous studies, we demonstrated that insertion of 19 amino acids in the hinge region of the protein had no significant effect on P protein function. In the present study, we inserted full-length enhanced green fluorescent protein (eGFP) in frame into the hinge region of P and show that the fusion protein (PeGFP) is functional in viral genome transcription and replication, albeit with reduced activity. A recombinant vesicular stomatitis virus encoding PeGFP in place of the P protein (VSV-PeGFP), which possessed reduced growth kinetics compared to the wild-type VSV, was recovered. Using the recombinant VSV-PeGFP, we show that the viral replication proteins and the de novo-synthesized RNA colocalize to sites throughout the cytoplasm, indicating that replication and transcription are not confined to any particular region of the cytoplasm. Real-time imaging of the cells infected with the eGFP-tagged virus revealed that, following synthesis, the nucleocapsids are transported toward the cell periphery via a microtubule (MT)-mediated process, and the nucleocapsids were seen to be closely associated with mitochondria. Treatment of cells with nocodazole or Colcemid, drugs known to inhibit MT polymerization, resulted in accumulation of the nucleocapsids around the nucleus and also led to inhibition of infectious-virus production. These findings are compatible with a model in which the progeny viral nucleocapsids are transported toward the cell periphery by MT and the transport may be facilitated by mitochondria.

* Corresponding author. Mailing address: E126 Beadle Center, 1901 Vine Street, University of Nebraska—Lincoln, Lincoln, NE 68588. Phone: (402) 472-1067. Fax: (402) 472-8722. E-mail: apattnaik2{at}unl.edu.

Journal of Virology, July 2006, p. 6368-6377, Vol. 80, No. 13
0022-538X/06/$08.00+0     doi:10.1128/JVI.00211-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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