INTRACELLULAR MANIPULATION OF DISULFIDE BOND FORMATION IN ROTAVIRUS PROTEINS DURING ASSEMBLY

Rotavirus undergoes a unique mode of assembly in the rough endoplasmic reticulum (RER) of infected cells. Luminal RER proteins undergo signi ficant cotranslational and posttranslational modifications, including disulfide bond formation. Addition of a reducing agent (dithiothreitol [DTT]) to rotavirus-infected cells did not significantly inhibit tran slation or disrupt established disulfide bonds in rotavirus proteins b ut prevented the formation of new disulfide bonds and infectious viral progeny. In DTT-treated, rotavirus-infected cells, all vp4, vp6, and ns28 epitopes but no vp7 epitopes were detected by immunohistochemical staining with a panel of monoclonal antibodies. When oxidizing condit ions were reestablished in DTT-treated cells, intramolecular disulfide bonds in vp7 were rapidly and correctly established with the restorat ion of antigenicity, although prolonged DTT treatment led to the accum ulation of permanently misfolded vp7. Electron microscopy revealed tha t cytosolic assembly of single-shelled particles and budding into the ER was not affected by DTT treatment but that outer capsid assembly wa s blocked, leading to the accumulation of single-shelled and enveloped intermediate subviral particles in the RER lumen.