title: Probing the early temporal and spatial interaction of the Sindbis virus capsid and E2 proteins with reverse genetics.
abstract: A 7-Å cryoelectron microscopy-based reconstruction of Sindbis virus (SINV) was recently generated. Fitting the crystal structure of the SINV capsid protein (Cp) into the density map revealed that the F2-G2 loop of the Cp was shifted away from cytoplasmic domain of E2 (cdE2) in the 7-Å reconstruction relative to its position in the Cp crystal structure. Furthermore, the reconstruction demonstrated that residue E395 in region I of the cytoplasmic domain of the E2 envelope protein (cdE2-RI) and K252 of Cp, part of the Cp F2-G2 loop, formed a putative salt bridge in the virion. We generated amino acid substitutions at residues K250 and K252 of the SINV Cp and explored the resulting phenotypes. In the context of cells infected with wild-type or mutant virus, reversing the charge of these two residues resulted in the appearance of Cp aggregates around cytopathic vacuole type I (CPV-I) structures, the absence of nucleocapsid (NC) formation, and a lack of virus particle release in the infected mammalian cell. However, expressing the same Cp mutants in the cell without the envelope proteins or expressing and purifying the mutants from an Escherichia coli expression system and assembling in vitro yielded NC assembly in all cases. In addition, second-site mutations within cdE2 restored NC assembly but not release of infectious particles. Our data suggest an early temporal and spatial interaction between cdE2-RI and the Cp F2-G2 loop that, when ablated, leads to the absence of NC assembly. This interaction also appears to be important for budding of virus particles.
authors.affiliation: Markey Center for Structural Biology, Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA.
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Snyder
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Berrios
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Edwards
authors.forename: Joyce
authors.initials: J
authors.lastname: Jose
authors.forename: Rushika
authors.initials: R
authors.lastname: Perera
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Kuhn
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002213:Capsid / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D020285:Cryoelectron Microscopy / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D008854:Microscopy, Electron / Q000379:methods; D009154:Mutation; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012845:Sindbis Virus / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism*
chemical_list: D014759:Viral Envelope Proteins; C064950:glycoprotein E2, Sindbis virus