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Journal of Virology, March 2009, p. 2611-2622, Vol. 83, No. 6
0022-538X/09/$08.00+0     doi:10.1128/JVI.01668-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells

Subash C. Das,^, Debasis Panda, Debasis Nayak, and Asit K. Pattnaik^*

Department of Veterinary and Biomedical Sciences and the Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, Nebraska 68583

Received 5 August 2008/ Accepted 19 December 2008

A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-M-Mtc and VSV-M-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-M-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-M-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.

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* Corresponding author. Mailing address: University of Nebraska-Lincoln, 109 Morrison Life Science Research Center, 4240 Fair Street, East Campus, Lincoln, NE 68583-0900. Phone: (402) 472-1067. Fax: (402) 472-3323. E-mail: apattnaik2{at}unl.edu

Published ahead of print on 19 January 2009.

S.C.D. and D.P. contributed equally to this work.

Present address: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706.

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Journal of Virology, March 2009, p. 2611-2622, Vol. 83, No. 6
0022-538X/09/$08.00+0     doi:10.1128/JVI.01668-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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