title: Single-Site Glycoprotein Mutants Inhibit a Late Event in Sindbis Virus Assembly.
abstract: A panel of Sindbis virus mutants that were suspected to have deficiencies in one or more aspects of their replication cycles was examined in baby hamster kidney (BHK) cells. These included an amino acid deletion (ΔH230) and substitution (H230A) in the Sindbis glycoprotein E1_H230 and similar mutants in E2_G209 (G209A, G209D, and ΔG209). Neither H230 mutation produced a measurable titer, but repeated passaging of the H230A mutant in BHK cells produced a second-site compensatory mutant (V231I) that partially rescued both H230 mutants. Electron micrograph (EM) images of these mutants showed assembled viral nucleocapsids but no completed, mature virions. EM of the compensatory mutant strains showed complete virus particles, but these now formed paracrystalline arrays. None of the E2_G209 substitution mutants had any effect on virus production; however, the deletion mutant (ΔG209) showed a very low titer when grown at 37°C and no titer when grown at 28°C. When the deletion mutant grown at 28°C was examined by EM, partially budded virions were observed at the cell surface. (35)S labeling of this mutant confirmed the presence of mutant virus protein in the transfected BHK cell lysate. We conclude that H230 is essential for the assembly of complete infectious Sindbis virus virions and that the presence of an amino acid at E2 position 209 is required for complete budding of Sindbis virus particles although several different amino acids can be at this location without affecting the titer.
  
  
  
  Our data show the importance of single-site mutations at E1_H230 and E2_G209 in Sindbis virus glycoproteins. These sites have been shown to affect assembly and antibody binding in previous studies. Our data indicate that mutation of one histidine residue in E1 is detrimental to the assembly of Sindbis virus particles in baby hamster kidney cells. Repeated passaging leads to a second-site substitution that partially restores the titer although EM still shows an altered phenotype. Substitutions at position G209 in E2 have no effect on titer, but deletion of this residue greatly reduces titer and again prevents assembly. When this mutant is grown at a lower temperature, virus particles bud from the host cell, but budding arrests before the progeny virus escapes. These results allow us to conclude that these sites have essential roles in assembly, and E2_G209 shows us a new viral egress phenotype.
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Magliocca
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Vancini
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Raquel
authors.initials: R
authors.lastname: Hernandez
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina, USA dennis_brown@ncsu.edu.
authors.forename: Dennis T
authors.initials: DT
authors.lastname: Brown
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D017354:Point Mutation; D017384:Sequence Deletion; D012845:Sindbis Virus / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D013489:Suppression, Genetic; D013696:Temperature; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D019562:Viral Load; D014771:Virion / Q000648:ultrastructure; D019065:Virus Assembly; D014776:Virus Cultivation; D057074:Virus Release
chemical_list: D008562:Membrane Glycoproteins; D050505:Mutant Proteins; D014759:Viral Envelope Proteins; C022012:glycoprotein E1, Sindbis virus; C064950:glycoprotein E2, Sindbis virus