Role of glycoprotein PE2 in formation and maturation of the Sindbis virus spike.
Sindbis virus envelope assembly is a multistep process resulting in the maturation of a rigid, highly ordered T=4 icosahedral protein lattice containing 80 spikes composed of trimers of E1-E2 heterodimers. Intramolecular disulfide bonds within E1 stabilize E1-E1 associations required for envelope formation and maintenance of the envelope's structural integrity. The structural integrity of the envelope protein lattice is resistant to reduction by dithiothreitol (DTT), indicating that E1 disulfides which stabilize structural domains become inaccessible to DTT at some point during virus maturation. The development of E1 resistance to DTT occurs prior to the completion of E1 folding and is temporally correlated with spike assembly in the endoplasmic reticulum. From these data we have predicted that in the final stages of spike assembly, E1 intramolecular disulfides, which stabilize the structural integrity of the envelope protein lattice, are buried within the spike and become inaccessible to the reductive activity of DTT. The spike is formed prior to the completion of E1 folding, and we have suggested that PE2 (the precursor to E2) may play a critical role in E1 folding after PE2-E1 oligomer formation has occurred. In this study we have investigated the role of PE2 in E1 folding, oligomer formation, and development of E1 resistance to both protease digestion and reduction by DTT by using a Sindbis virus replicon (SINrep/E1) which allows for the expression of E1 in the presence of truncated PE2. Through pulse-chase analysis of both Sindbis virus- and SINrep/E1-infected cells, we have determined that the folding of E1 into a trypsin-resistant conformation and into its most compact and stable form is not dependent upon association of E1 with PE2. However, E1 association with PE2 is required for oligomer formation, the export of E1 from the endoplasmic reticulum, and E1 acquisition of resistance to DTT.
Carleton
M
M
Cell Research Institute and Department of Microbiology, University of Texas at Austin, 78713-7640, USA.
Lee
H
H
Mulvey
M
M
Brown
D T
DT
D004229:Dithiothreitol; D008562:Membrane Glycoproteins; D017510:Protein Folding; D011498:Protein Precursors; D012845:Sindbis Virus / Q000502:physiology*; D014764:Viral Proteins; D019065:Virus Assembly / Q000502:physiology*
D008562:Membrane Glycoproteins; D011498:Protein Precursors; D014764:Viral Proteins; C102373:glycoprotein PE2, Sindbis virus; D004229:Dithiothreitol