ROTAVIRUS PROTEIN EXPRESSION IS IMPORTANT FOR VIRUS ASSEMBLY AND PATHOGENESIS

Rotaviruses have a unique morphogenesis in which particles obtain a tr ansient membrane-envelope as newly made subviral particles bud into th e endoplasmic reticulum (ER). This process is mediated by a viral nons tructural glycoprotein, NSP4. We have found that NSP4 has pleiotropic properties that became evident following expression of this protein in eukaryotic cells. NSP4 expressed in insect cells bound double-layered rotavirus particles in a manner similar to receptor-ligand interactio ns and this interaction is thought to trigger the particle budding pro cess. Expression of NSP4 in insect cells also increases intracellular calcium ([Ca2+]i) levels and this effect may explain the toxicity of t his protein in eukaryotic cells. Increases in [Ca2+]i levels in insect cells also are observed following exogenous addition to cells of puri fied NSP4 or of a synthetic peptide of NSP4. Experiments to determine the mechanism by which NSP4 causes an increase in [Ca2+]i showed that Ca2+ is released from a subset of the thapsigargin-sensitive store [en doplasmic reticulum (ER)]. However, exogenously added and endogenously expressed NSP4 use different mechanisms to alter the Ca2+ permeabilit y of the ER membrane. We hypothesize that NSP4-mediated changes in ER membrane permeability trigger viral budding into the lumen of the ER, and eventually induce cell death and release of virus particles from i nfected cells. We also propose that release of NSP4 following cell lys is and the concomitant stimulation of a Ca2+ signal transduction pathw ay in neighboring cells contributes to altered ion transport in intest inal epithelium resulting in diarrheal disease.