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Viruses 2015, 7(12), 6182-6199; doi:10.3390/v7122926

Fluorescent Protein-Tagged Sindbis Virus E2 Glycoprotein Allows Single Particle Analysis of Virus Budding from Live Cells

1
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
2
Department of Chemistry and Biochemistry and Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA
3
Department of Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA
Present Address: Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA
*
Author to whom correspondence should be addressed.
Academic Editor: Andrew Mehle
Received: 27 August 2015 / Revised: 7 November 2015 / Accepted: 13 November 2015 / Published: 27 November 2015
(This article belongs to the Special Issue Replication-Competent Reporter-Expressing Viruses)
View Full-Text   |   Download PDF [10733 KB, uploaded 27 November 2015]   |  

Abstract

Sindbis virus (SINV) is an enveloped, mosquito-borne alphavirus. Here we generated and characterized a fluorescent protein-tagged (FP-tagged) SINV and found that the presence of the FP-tag (mCherry) affected glycoprotein transport to the plasma membrane whereas the specific infectivity of the virus was not affected. We examined the virions by transmission electron cryo-microscopy and determined the arrangement of the FP-tag on the surface of the virion. The fluorescent proteins are arranged icosahedrally on the virus surface in a stable manner that did not adversely affect receptor binding or fusion functions of E2 and E1, respectively. The delay in surface expression of the viral glycoproteins, as demonstrated by flow cytometry analysis, contributed to a 10-fold reduction in mCherry-E2 virus titer. There is a 1:1 ratio of mCherry to E2 incorporated into the virion, which leads to a strong fluorescence signal and thus facilitates single-particle tracking experiments. We used the FP-tagged virus for high-resolution live-cell imaging to study the spatial and temporal aspects of alphavirus assembly and budding from mammalian cells. These processes were further analyzed by thin section microscopy. The results demonstrate that SINV buds from the plasma membrane of infected cells and is dispersed into the surrounding media or spread to neighboring cells facilitated by its close association with filopodial extensions. View Full-Text
Keywords: alphavirus; live imaging; virus assembly; filopodia; virus egress alphavirus; live imaging; virus assembly; filopodia; virus egress
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Jose, J.; Tang, J.; Taylor, A.B.; Baker, T.S.; Kuhn, R.J. Fluorescent Protein-Tagged Sindbis Virus E2 Glycoprotein Allows Single Particle Analysis of Virus Budding from Live Cells. Viruses 2015, 7, 6182-6199.

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