title: A novel system for visualizing alphavirus assembly.
abstract: Alphaviruses are small, enveloped RNA viruses that form infectious particles by budding through the cellular plasma membrane. To help visualize and understand the intracellular assembly of alphavirus virions we have developed a bimolecular fluorescence complementation-based system (BiFC) that allows visualization of capsid and E2 subcellular localization and association in live cells. In this system, N- or C-terminal Venus fluorescent protein fragments (VN- and VC-) are fused to the N-terminus of the capsid protein on the Sindbis virus structural polyprotein, which results in the formation of fluorescent capsid-like structures in the absence of viral genomes that associate with the plasma membrane of cells. Mutation of the capsid autoprotease active site blocks structural polyprotein processing and alters the subcellular distribution of capsid fluorescence. Incorporating mCherry into the extracellular domain of the E2 glycoprotein allows the visualization of E2 glycoprotein localization and showed a close association of the E2 and capsid proteins at the plasma membrane as expected. These results suggest that this system is a useful new tool to study alphavirus assembly in live cells and may be useful in identifying molecules that inhibit alphavirus virion formation.
authors.affiliation: Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: J Jordan
authors.initials: JJ
authors.lastname: Steel
authors.affiliation: Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA; Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA. Electronic address: Brian.Geiss@colostate.edu.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Geiss
mesh_terms: D000818:Animals; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000821:virology; D006224:Cricetinae; D004847:Epithelial Cells / Q000821:virology; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008856:Microscopy, Fluorescence; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012845:Sindbis Virus / Q000502:physiology*; D013194:Staining and Labeling; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C064950:glycoprotein E2, Sindbis virus
keywords: Alphavirus; Fluorescence microscopy; Sindbis; Virus budding