title: A single deletion in the membrane-proximal region of the Sindbis virus glycoprotein E2 endodomain blocks virus assembly.
abstract: The envelopment of the Sindbis virus nucleocapsid in the modified cell plasma membrane involves a highly specific interaction between the capsid (C) protein and the endodomain of the E2 glycoprotein. We have previously identified a domain of the Sindbis virus C protein involved in binding to the E2 endodomain (H. Lee and D. T. Brown, Virology 202:390-400, 1994). The C-E2 binding domain resides in a hydrophobic cleft with C Y180 and W247 on opposing sides of the cleft. Structural modeling studies indicate that the E2 domain, which is proposed to bind the C protein (E2 398T, 399P, and 400Y), is located at a sufficient distance from the membrane to occupy the C protein binding cleft (S. Lee, K. E. Owen, H. K. Choi, H. Lee, G. Lu, G. Wengler, D. T. Brown, M. G. Rossmann, and R. J. Kuhn, Structure 4:531-541, 1996). To measure the critical spanning length of the E2 endodomain which positions the TPY domain into the putative C binding cleft, we have constructed a deletion mutant, DeltaK391, in which a nonconserved lysine (E2 K391) at the membrane-cytoplasm junction of the E2 tail has been deleted. This mutant was found to produce very low levels of virus from BHK-21 cells due to a defect in an unidentified step in nucleocapsid binding to the E2 endodomain. In contrast, DeltaK391 produced wild-type levels of virus from tissue-cultured mosquito cells. We propose that the phenotypic differences displayed by this mutant in the two diverse host cells arise from fundamental differences in the lipid composition of the insect cell membranes which affect the physical and structural properties of membranes and thereby virus assembly. The data suggest that these viruses have evolved properties adapted specifically for assembly in the diverse hosts in which they grow.
authors.affiliation: Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695, USA.
authors.forename: R
authors.initials: R
authors.lastname: Hernandez
authors.forename: H
authors.initials: H
authors.lastname: Lee
authors.forename: C
authors.initials: C
authors.lastname: Nelson
authors.forename: D T
authors.initials: DT
authors.lastname: Brown
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019251:Nucleocapsid / Q000378:metabolism; D012845:Sindbis Virus / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015678:Viral Structural Proteins / Q000378:metabolism; D019065:Virus Assembly / Q000502:physiology*
chemical_list: D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins; C064950:glycoprotein E2, Sindbis virus