Mutations in the endo domain of Sindbis virus glycoprotein E2 block phosphorylation, reorientation of the endo domain, and nucleocapsid binding.

Exposure of the carboxyl terminus (endo domain) of Sindbis virus membrane glycoprotein E2 to the cell cytoplasm is critical for the interaction of the nucleocapsid with viral envelope proteins in modified cell membranes. We have shown that the endo domain of PE2/E2 is initially translocated into membranes of the endoplasmic reticulum and subsequently drawn back into the cell cytoplasm during virus assembly. We suggested that phosphorylation of PE2/E2 might be responsible for the reorganization of the PE2/E2 carboxyl terminus. To test this hypothesis, two potential phosphorylation sites in the endo domain of E2, Thr398 and Tyr400, were changed by site-directed mutagenesis. Virus structural proteins are produced at normal levels in BHK-21 cells transfected with RNA containing the double mutation, nucleocapsids are formed, and the envelope proteins are exported from the endoplasmic reticulum; however, no virus is produced. The double mutation prevents phosphorylation of PE2/E2, and electron microscopy of cells transfected with the double mutant RNA reveals no attachment of nucleocapsids to cell membranes. The double mutation blocks exposure of the carboxyl terminus of E2 to the cytoplasm. Revertants of the double mutant to virus production all restored tyrosine at position 400 and restored the ability of the E2 protein to be phosphorylated. Although the threonine at position 398 is conserved among the alphaviruses, no revertant restored threonine at this position.