title: Single amino acid insertions at the junction of the sindbis virus E2 transmembrane domain and endodomain disrupt virus envelopment and alter infectivity.
abstract: The final steps in the envelopment of Sindbis virus involve specific interactions of the E2 endodomain with the virus nucleocapsid. Deleting E2 K at position 391 (E2 DeltaK391) resulted in the disruption of virus assembly in mammalian cells but not insect cells (host range mutant). This suggested unique interactions of the E2 DeltaK391 endodomain with the different biochemical environments of the mammalian and insect cell lipid bilayers. To further investigate the role of the amino acid residues located at or around position E2 391 and constraints on the length of the endodomain on virus assembly, amino acid insertions/substitutions at the transmembrane/endodomain junction were constructed. An additional K was inserted at amino acid position 392 (KK391/392), a K-->F substitution at position 391 was constructed (F391), and an additional F was inserted at 392 (FF391/392). These changes should lengthen the endodomain in the KK391/392 insertion mutant or shorten the endodomain in the FF391/392 mutant. The mutant FF391/392 grown in BHK cells formed virus particles containing extruded material not found on wild-type virus. This characteristic was not seen in FF391/392 virus grown in insect cells. The mutant KK391/392 grown in BHK cells was defective in the final membrane fission reaction, producing multicored or conjoined virus particles. The production of these aberrant particles was ameliorated when the KK391/392 mutant was grown in insect cells. These data indicate that there is a critical minimal spanning distance from the E2 membrane proximal amino acid at position 391 and the conserved E2 Y400 residue. The observed phenotypes of these mutants also invoke an important role of the specific host membrane lipid composition on virus architecture and infectivity.
authors.affiliation: Department of Molecular and Structural Biochemistry, Campus Box 7622, North Carolina State University, Raleigh, NC 27695-7622, USA.
authors.forename: Raquel
authors.initials: R
authors.lastname: Hernandez
authors.forename: Davis
authors.initials: D
authors.lastname: Ferreira
authors.forename: Christine
authors.initials: C
authors.lastname: Sinodis
authors.forename: Katherine
authors.initials: K
authors.lastname: Litton
authors.forename: Dennis T
authors.initials: DT
authors.lastname: Brown
mesh_terms: D000330:Aedes; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D017353:Gene Deletion; D008854:Microscopy, Electron; D009154:Mutation; D012845:Sindbis Virus / Q000378:metabolism / Q000472:pathogenicity* / Q000648:ultrastructure*; D013045:Species Specificity; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010948:Viral Plaque Assay; D014771:Virion / Q000378:metabolism; D019065:Virus Assembly
chemical_list: D014759:Viral Envelope Proteins; C064950:glycoprotein E2, Sindbis virus