title: Fluorescent Protein-Tagged Sindbis Virus E2 Glycoprotein Allows Single Particle Analysis of Virus Budding from Live Cells.
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.
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA. jjose@purdue.edu.
authors.forename: Joyce
authors.initials: J
authors.lastname: Jose
authors.affiliation: Department of Chemistry and Biochemistry and Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA. jinghuat@gmail.com.
authors.forename: Jinghua
authors.initials: J
authors.lastname: Tang
authors.affiliation: Department of Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA. taylora10@janelia.hhmi.org.
authors.forename: Aaron B
authors.initials: AB
authors.lastname: Taylor
authors.affiliation: Department of Chemistry and Biochemistry and Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA. tsb@ucsd.edu.
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Baker
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA. kuhnr@purdue.edu.|Department of Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA. kuhnr@purdue.edu.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Kuhn
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012845:Sindbis Virus / Q000235:genetics / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods*; D014759:Viral Envelope Proteins / Q000235:genetics*; D019065:Virus Assembly; D053585:Virus Attachment; D053586:Virus Internalization; D057074:Virus Release; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D000097573:Red Fluorescent Protein; C064950:glycoprotein E2, Sindbis virus
keywords: alphavirus; filopodia; live imaging; virus assembly; virus egress