The formation of intramolecular disulfide bridges is required for induction of the Sindbis virus mutant ts23 phenotype.
The Sindbis virus envelope protein spike is a hetero-oligomeric complex composed of a trimer of glycoprotein E1-E2 heterodimers. Spike assembly is a multistep process which occurs in the endoplasmic reticulum (ER) and is required for the export of E1 from the ER. PE2 (precursor to E2), however, can transit through the secretory pathway and be expressed at the cell surface in the absence of E1. Although oligomer formation does not appear to be required for the export of PE2, there is evidence that defects in E1 folding can affect PE2 transit from the ER. Temperature-sensitive mutant ts23 of Sindbis virus contains two amino acid substitutions in E1, while PE2 and capsid protein have the wild-type sequence; however, at the nonpermissive temperature, both E1 and PE2 are retained within the ER and can be isolated in protein aggregates with the molecular chaperone GRP78-BiP. We previously demonstrated that the temperature sensitivity for ts23 was lost as oligomer formation took place at the permissive temperature, suggesting that temperature sensitivity is initiated early in the process of viral spike assembly (M. Carleton and D. T. Brown, J. Virol. 70:952-959, 1996). Experiments described herein investigated the defects in envelope protein maturation that occur in ts23-infected cells and which result in retention of both envelope proteins in the ER. The data demonstrate that in ts23-infected cells incubated at the nonpermissive temperature, E1 folding is disrupted early after synthesis, resulting in the rapid incorporation of both E1 and PE2 into disulfide-stabilized aggregates. Furthermore, the aberrant E1 conformation which is responsible for induction of the ts phenotype requires the formation of intramolecular disulfide bridges formed prior to E1 association with PE2 and the completion of E1 folding.
Carleton
M
M
Cell Research Institute and Department of Microbiology, The University of Texas at Austin, 78713-7640, USA.
Brown
D T
DT
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D007668:Kidney; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D010641:Phenotype; D012845:Sindbis Virus / Q000235:genetics / Q000502:physiology*; D013696:Temperature; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D004220:Disulfides; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; C022012:glycoprotein E1, Sindbis virus; C064950:glycoprotein E2, Sindbis virus; D004229:Dithiothreitol