THE PROCESSING OF THE ROTAVIRUS SA11 GLYCOPROTEINS IN THE ENDOPLASMIC RETICULUM

Abstract

Current research has focused on the elucidation of the mechanisms governing the targeting of membrane glycoproteins to their correct intracellular location. Due to the difficulties inherent in studying the complex array of proteins normally present in membranes, membrane maturing viruses have often been utilized as model systems because of the high abundance of a small number of glycoprotein species in infected cells. The simian rotavirus SA11 provides such a system for the examination of endoplasmic reticulum (ER) glycoproteins.;Rotavirus assembly is initiated as viral cores, composed of double-stranded RNA and three inner capsid proteins bud into the lumen of ER resulting in the formation of membrane-enveloped particles. Maturation is completed with the loss of this membrane and the addition of the outer capsid proteins VP3 and VP7, which was demonstrated to occur with a lag time of 15 minutes. The virus encodes two membrane-associated glycoproteins: VP7, the neutralizing antigen, and NCVP5, a non-structural protein. Only a fraction or the NP7 molecules synthesized were incorporated into infectious virions, the rest remained in the ER membrane and could be separated from virus using fractionation techniques, of two VP7-specific antisera. Virus VP7 and membrane VP7 contain unique epitopes, suggesting that a conformational alteration occurs during virus maturation.;In rotavirus infected cells, the N-linked carbohydrate modifying VP7 was processed to Mannose{dollar}\sb 6{dollar}N-acetylglucosamine{dollar}\sb 2{dollar} (Man{dollar}\sb 6{dollar}GlcNAc{dollar}\sb 2{dollar}), while NCVP5 was trimmed to Man{dollar}\sb 8{dollar}GlcNAc{dollar}\sb 2{dollar} confirming the existence of multiple {dollar}\alpha{dollar}-1,2-mannosidase activities within the ER. In the presence of the energy inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP), trimming of VP7 was blocked at Man{dollar}\sb 8{dollar}GlcNAc{dollar}\sb 2{dollar}. Since the carbohydrate processing reactions are not energy requiring, the block is presumably in the movement of the protein to an ER subcompartment containing the appropriate enzymes. The barrier may also represent the point of departure of membrane proteins, such as vesicular stomatitis virus G protein, which continue along the secretory pathway. G protein leaves the ER with Man{dollar}\sb 8{dollar}GlcNAc{dollar}\sb 2{dollar} to be additionally processed by the Golgi apparatus {dollar}\alpha{dollar}-mannosidases. These observations are consistent with the existence of an energy requiring process which sorts resident proteins from those destined for export.